Sample records for dynamic cofilin phosphorylation

  1. Signaling mechanisms and functional roles of cofilin phosphorylation and dephosphorylation. (United States)

    Mizuno, Kensaku


    Cofilin and actin-depolymerizing factor (ADF) are actin-binding proteins that play an essential role in regulating actin filament dynamics and reorganization by stimulating the severance and depolymerization of actin filaments. Cofilin/ADF are inactivated by phosphorylation at the serine residue at position 3 by LIM-kinases (LIMKs) and testicular protein kinases (TESKs) and are reactivated by dephosphorylation by the slingshot (SSH) family of protein phosphatases and chronophin. This review describes recent advances in our understanding of the signaling mechanisms regulating LIMKs and SSHs and the functional roles of cofilin phospho-regulation in cell migration, tumor invasion, mitosis, neuronal development, and synaptic plasticity. Accumulating evidence demonstrates that the phospho-regulation of cofilin/ADF is a key convergence point of cell signaling networks that link extracellular stimuli to actin cytoskeletal dynamics and that spatiotemporal control of cofilin/ADF activity by LIMKs and SSHs plays a crucial role in a diverse array of cellular and physiological processes. Perturbations in the normal control of cofilin/ADF activity underlie many pathological conditions, including cancer metastasis and neurological and cardiovascular disorders.

  2. Aurora A kinase modulates actin cytoskeleton through phosphorylation of Cofilin: Implication in the mitotic process. (United States)

    Ritchey, Lisa; Chakrabarti, Ratna


    Aurora A kinase regulates early mitotic events through phosphorylation and activation of a variety of proteins. Specifically, Aur-A is involved in centrosomal separation and formation of mitotic spindles in early prophase. The effect of Aur-A on mitotic spindles is mediated by the modulation of microtubule dynamics and association with microtubule binding proteins. In this study we show that Aur-A exerts its effects on spindle organization through the regulation of the actin cytoskeleton. Aurora A phosphorylates Cofilin at multiple sites including S(3) resulting in the inactivation of its actin depolymerizing function. Aur-A interacts with Cofilin in early mitotic phases and regulates its phosphorylation status. Cofilin phosphorylation follows a dynamic pattern during the progression of prophase to metaphase. Inhibition of Aur-A activity induced a delay in the progression of prophase to metaphase. Aur-A inhibitor also disturbed the pattern of Cofilin phosphorylation, which correlated with the mitotic delay. Our results establish a novel function of Aur-A in the regulation of actin cytoskeleton reorganization, through Cofilin phosphorylation during early mitotic stages.

  3. Phosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin. (United States)

    Zhou, Guo-Lei; Zhang, Haitao; Wu, Huhehasi; Ghai, Pooja; Field, Jeffrey


    Cell signaling can control the dynamic balance between filamentous and monomeric actin by modulating actin regulatory proteins. One family of actin regulating proteins that controls actin dynamics comprises cyclase-associated proteins 1 and 2 (CAP1 and 2, respectively). However, cell signals that regulate CAPs remained unknown. We mapped phosphorylation sites on mouse CAP1 and found S307 and S309 to be regulatory sites. We further identified glycogen synthase kinase 3 as a kinase phosphorylating S309. The phosphomimetic mutant S307D/S309D lost binding to its partner cofilin and, when expressed in cells, caused accumulation of actin stress fibers similar to that in cells with reduced CAP expression. In contrast, the non-phosphorylatable S307A/S309A mutant showed drastically increased cofilin binding and reduced binding to actin. These results suggest that the phosphorylation serves to facilitate release of cofilin for a subsequent cycle of actin filament severing. Moreover, our results suggest that S307 and S309 function in tandem; neither the alterations in binding cofilin and/or actin, nor the defects in rescuing the phenotype of the enlarged cell size in CAP1 knockdown cells was observed in point mutants of either S307 or S309. In summary, we identify a novel regulatory mechanism of CAP1 through phosphorylation.

  4. Cofilin/Twinstar phosphorylation levels increase in response to impaired coenzyme a metabolism.

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    Katarzyna Siudeja

    Full Text Available Coenzyme A (CoA is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK, which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. Here we use Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture--a process that is strongly dependent on actin remodeling. Our results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics.

  5. Cofilin phosphorylation is elevated after F-actin disassembly induced by Rac1 depletion

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    Liu, Linna; Li, Jing; Zhang, Liwang


    that actin filaments disassembled. In the epidermis of mice in which Rac1 was knocked out only in keratinocytes, cofilin phosphorylation was aberrantly elevated, corresponding to repression of the phosphatase slingshot1 (SSH1). These effects were independent of the signaling pathways for p21-activated kinase....../LIM kinase (Pak/LIMK), protein kinase C, or protein kinase D or generation of reactive oxygen species. Similarly, when actin polymerization was specifically inhibited or Rac1 was knocked down, cofilin phosphorylation was enhanced and SSH1 was repressed. Repression of SSH1 partially blocked actin...

  6. Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels (United States)

    Delorme-Walker, Violaine; Seo, Ji-Yeon; Gohla, Antje; Fowler, Bruce; Bohl, Ben; DerMardirossian, Céline


    Cofilin, a critical player of actin dynamics, is spatially and temporally regulated to control the direction and force of membrane extension required for cell locomotion. In carcinoma cells, although the signaling pathways regulating cofilin activity to control cell direction have been established, the molecular machinery required to generate the force of the protrusion remains unclear. We show that the cofilin phosphatase chronophin (CIN) spatiotemporally regulates cofilin activity at the cell edge to generate persistent membrane extension. We show that CIN translocates to the leading edge in a PI3-kinase–, Rac1-, and cofilin-dependent manner after EGF stimulation to activate cofilin, promotes actin free barbed end formation, accelerates actin turnover, and enhances membrane protrusion. In addition, we establish that CIN is crucial for the balance of protrusion/retraction events during cell migration. Thus, CIN coordinates the leading edge dynamics by controlling active cofilin levels to promote MTLn3 cell protrusion. PMID:26324884

  7. Rho-GTPase effector ROCK phosphorylates cofilin in actin-meditated cytokinesis during mouse oocyte meiosis. (United States)

    Duan, Xing; Liu, Jun; Dai, Xiao-Xin; Liu, Hong-Lin; Cui, Xiang-Shun; Kim, Nam-Hyung; Wang, Zhen-Bo; Wang, Qiang; Sun, Shao-Chen


    During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

  8. Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin-phosphorylation

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    Podlisny Marcia


    Full Text Available Abstract Background Previously we reported 1 μM synthetic human amyloid beta1-42 oligomers induced cofilin dephosphorylation (activation and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus. Results Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aβ dimers and trimers (Aβd/t induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aβ oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aβ1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC50 in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aβd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state, lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species. Aβd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aβd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation. Conclusions Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

  9. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development. (United States)

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong


    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated.

  10. The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation. (United States)

    Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung


    Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation.

  11. Roles of cofilin in development and its mechanisms of regulation. (United States)

    Ohashi, Kazumasa


    Reorganization of the actin cytoskeleton is essential for cellular processes during animal development. Cofilin and actin depolymerizing factor (ADF) are potent actin-binding proteins that sever and depolymerize actin filaments, acting to generate the dynamics of the actin cytoskeleton. The activity of cofilin is spatially and temporally regulated by a variety of intracellular molecular mechanisms. Cofilin is regulated by cofilin binding molecules, is phosphorylated at Ser-3 (inactivation) by LIM-kinases (LIMKs) and testicular protein kinases (TESKs), and is dephosphorylated (reactivation) by slingshot protein phosphatases (SSHs). Although studies of the molecular mechanisms of cofilin-induced reorganization of the actin cytoskeleton have been ongoing for decades, the multicellular functions of cofilin and its regulation in development are just becoming apparent. This review describes the molecular mechanisms of generating actin dynamics by cofilin and the intracellular signaling pathways for regulating cofilin activity. Furthermore, recent findings of the roles of cofilin in the development of several tissues and organs, especially neural tissues and cells, in model animals are described. Recent developmental studies have indicated that cofilin and its regulatory mechanisms are involved in cellular proliferation and migration, the establishment of cellular polarity, and the dynamic regulation of organ morphology.

  12. Cofilin takes the lead. (United States)

    DesMarais, Vera; Ghosh, Mousumi; Eddy, Robert; Condeelis, John


    Cofilin has emerged as a key regulator of actin dynamics at the leading edge of motile cells. Through its actin-severing activity, it creates new actin barbed ends for polymerization and also depolymerizes old actin filaments. Its function is tightly regulated in the cell. Spatially, its activity is restricted by other actin-binding proteins, such as tropomyosin, which compete for accessibility of actin filament populations in different regions of the cell. At the molecular level, it is regulated by phosphorylation, pH and phosphatidylinositol (4,5)-bisphosphate binding downstream of signaling cascades. In addition, it also appears to be regulated by interactions with 14-3-3zeta and cyclase-associated protein. In vivo, cofilin acts synergistically with the Arp2/3 complex to amplify local actin polymerization responses upon cell stimulation, which gives it a central role in setting the direction of motility in crawling cells.

  13. AIP1 acts with cofilin to control actin dynamics during epithelial morphogenesis. (United States)

    Chu, Dandan; Pan, Hanshuang; Wan, Ping; Wu, Jing; Luo, Jun; Zhu, Hong; Chen, Jiong


    During epithelial morphogenesis, cells not only maintain tight adhesion for epithelial integrity but also allow dynamic intercellular movement to take place within cell sheets. How these seemingly opposing processes are coordinated is not well understood. Here, we report that the actin disassembly factors AIP1 and cofilin are required for remodeling of adherens junctions (AJs) during ommatidial precluster formation in Drosophila eye epithelium, a highly stereotyped cell rearrangement process which we describe in detail in our live imaging study. AIP1 is enriched together with F-actin in the apical region of preclusters, whereas cofilin displays a diffuse and uniform localization pattern. Cofilin overexpression completely rescues AJ remodeling defects caused by AIP1 loss of function, and cofilin physically interacts with AIP1. Pharmacological reduction of actin turnover results in similar AJ remodeling defects and decreased turnover of E-cadherin, which also results from AIP1 deficiency, whereas an F-actin-destabilizing drug affects AJ maintenance and epithelial integrity. Together with other data on actin polymerization, our results suggest that AIP1 enhances cofilin-mediated actin disassembly in the apical region of precluster cells to promote remodeling of AJs and thus intercellular movement, but also that robust actin polymerization promotes AJ general adhesion and integrity during the remodeling process.

  14. Dephosphorylated cofilin expression is associated with poor prognosis in cases of human breast cancer: a tissue microarray analysis

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    Maimaiti Y


    Full Text Available Yusufu Maimaiti,1,2,* Zeming Liu,1,* Jie Tan,1 Kelimu Abudureyimu,2 Bangxing Huang,3 Chunping Liu,1 Yawen Guo,1 Changwen Wang,1 Xiu Nie,3 Jing Zhou,1 Tao Huang1 1Department of Breast and Thyroid Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 2Department of General Surgery, Research Institute of Minimally Invasive, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi, 3Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China *These authors contributed equally to this work Background: Proteins in the cofilin pathway regulate actin dynamics and may be involved in cancer cell migration and invasion. However, there are no direct data that suggest that dephosphorylated cofilin can affect breast cancer prognosis.Methods: We assessed the expressions of cofilin and phosphorylated cofilin (P-cofilin in breast cancer tissue microarrays (290 patients, mean follow-up: 95.7±2.49 months to evaluate dephosphorylated cofilin and its relationship with breast cancer prognosis. The associations of pathological characteristics with cumulative survival were evaluated using Kaplan–Meier analysis.Results: Univariate analyses revealed that overall survival was associated with cofilin levels, N category, TNM stage, estrogen receptor status, progesterone receptor status, and molecular subtypes. Cofilin status and TNM stage independently affected overall survival, although P-cofilin expression was not associated with patient survival. In the P-cofilin-negative subgroup, cofilin expression was significantly associated with patient survival, although cofilin expression was not significantly associated with patient survival in the P-cofilin-positive subgroup. We further analyzed the P-cofilin-negative cases and found that Ki-67 expression was significantly elevated in the subgroup that was strongly positive for

  15. Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

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    Thirone, Ana C P; Speight, Pam; Zulys, Matthew


    Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. While de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this proble...... in the hyperosmotic stress-induced F-actin increase. Key words: cytoskeleton, hypertonicity, cell volume, small GTPases.......Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. While de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem...... we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, whose activity is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes, and can regulate cofilin phosphorylation, we also asked if they might link osmostress...

  16. Cofilin activation during podosome belt formation in osteoclasts.

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    Anne Blangy

    Full Text Available Podosomes are dynamic actin-based structures found constitutively in cells of monocytic origin such as macrophages, dendritic cells and osteoclasts. They have been involved in osteoclast cell adhesion, motility and matrix degradation, and all these functions rely on the ability of podosomes to form supra-molecular structures called podosome belts or sealing zones on mineralized substrates. Podosomes contain two distinct domains, an actin-rich core enriched in actin polymerization regulators, surrounded by a ring of signaling and plaque molecules. The organization of podosome arrays into belts is linked to actin dynamics. Cofilin is an actin-severing protein that is known to regulate cytoskeleton architecture and cell migration. Cofilin is present in lamellipodia and invadopodia where it regulates actin polymerization. In this report, we show that cofilin is a novel component of the podosome belt, the mature osteoclast adhesion structure. Time-course analysis demonstrated that cofilin is activated during primary osteoclast differentiation, at the time of podosome belt assembly. Immunofluorescence studies reveal a localization of active cofilin in the podosome core structure, whereas phosphorylated, inactive cofilin is concentrated in the podosome cloud. Pharmacological studies unraveled the role of a specific cofilin phosphatase to achieve cofilin activation during osteoclast differentiation. We ruled out the implication of PP1/PP2A and PTEN in this process, and rather provided evidence for the involvement of SSH1. In summary, our data involve cofilin as a regulator of podosome organization that is activated during osteoclast differentiation by a RANKL-mediated signaling pathway targeting the SSH1 phosphatase.

  17. Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells. (United States)

    Bertling, Enni; Hotulainen, Pirta; Mattila, Pieta K; Matilainen, Tanja; Salminen, Marjo; Lappalainen, Pekka


    Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.

  18. Immunological responses and actin dynamics in macrophages are controlled by N-cofilin but are independent from ADF.

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    Friederike Jönsson

    Full Text Available Dynamic changes in the actin cytoskeleton are essential for immune cell function and a number of immune deficiencies have been linked to mutations, which disturb the actin cytoskeleton. In macrophages and dendritic cells, actin remodelling is critical for motility, phagocytosis and antigen presentation, however the actin binding proteins, which control antigen presentation have been poorly characterized. Here we dissect the specific roles of the family of ADF/cofilin F-actin depolymerizing factors in macrophages and in local immune responses. Macrophage migration, cell polarization and antigen presentation to T-cells require n-cofilin mediated F-actin remodelling. Using a conditional mouse model, we show that n-cofilin also controls MHC class II-dependent antigen presentation. Other cellular processes such as phagocytosis and antigen processing were found to be independent of n-cofilin. Our data identify n-cofilin as a novel regulator of antigen presentation, while ADF on the other hand is dispensable for macrophage motility and antigen presentation.

  19. Rho GTPases RhoA and Rac1 mediate effects of dietary folate on metastatic potential of A549 cancer cells through the control of cofilin phosphorylation. (United States)

    Oleinik, Natalia V; Helke, Kristi L; Kistner-Griffin, Emily; Krupenko, Natalia I; Krupenko, Sergey A


    Folate, an important nutrient in the human diet, has been implicated in cancer, but its role in metastasis is not established. We have shown previously that the withdrawal of medium folate leads to the inhibition of migration and invasion of A549 lung carcinoma cells. Here we have demonstrated that medium folate regulates the function of Rho GTPases by enabling their carboxyl methylation and translocation to plasma membrane. Conversely, the lack of folate leads to the retention of these proteins in endoplasmic reticulum. Folate also promoted the switch from inactive (GDP-bound) to active (GTP-bound) GTPases, resulting in the activation of downstream kinases p21-activated kinase and LIM kinase and phosphorylation of the actin-depolymerizing factor cofilin. We have further demonstrated that in A549 cells two GTPases, RhoA and Rac1, but not Cdc42, are immediate sensors of folate status: the siRNA silencing of RhoA or Rac1 blocked effects of folate on cofilin phosphorylation and cellular migration and invasion. The finding that folate modulates metastatic potential of cancer cells was confirmed in an animal model of lung cancer using tail vein injection of A549 cells in SCID mice. A folate-rich diet enhanced lung colonization and distant metastasis to lymph nodes and decreased overall survival (35 versus 63 days for mice on a folate-restricted diet). High folate also promoted epithelial-mesenchymal transition in cancer cells and experimental mouse tumors. Our study provides experimental evidence for a mechanism of metastasis promotion by dietary folate and highlights the interaction between nutrients and metastasis-related signaling.

  20. Phosphorylation of cofilin-1 by ERK confers HDAC inhibitor resistance in hepatocellular carcinoma cells via decreased ROS-mediated mitochondria injury. (United States)

    Liao, P-H; Hsu, H-H; Chen, T-S; Chen, M-C; Day, C-H; Tu, C-C; Lin, Y-M; Tsai, F-J; Kuo, W-W; Huang, C-Y


    Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Despite the availability of several treatment strategies, resistance to chemotherapeutic agents, which limits the effectiveness of anticancer drugs, is a major problem in cancer therapy. In this study, we used a histone deacetylases inhibitor (HDACi) to establish drug-resistant HCC cells and further analyzed the molecular mechanisms underlying the development of resistance in HCC cells. Compared with the parental cells, HDACi-resistant cells showed high metastatic and pro-survival abilities. Two-dimensional electrophoresis data showed that the cofilin-1 (CFL-1) protein was altered in HDACi-resistant cells and was highly expressed in resistant cells compared with parental cells. The molecular function of CFL-1 is actin depolymerization, and it is involved in tumor metastasis. In this study, we showed that CFL-1 inhibition decreased cell migration and increased cell apoptosis in HDACi-resistant cells. We observed that HDACi induced ROS accumulation in cells and apoptosis via promotion of the CFL-1 interaction with Bax and CFL-1 translocation to the mitochondria, resulting in cytochrome C release. Importantly, phosphorylation of CFL-1 by activated extracellular signal-regulated kinases 1 and 2 (ERK1/2) confers strong protection against HDAC inhibitor-induced cell injury. p-CFL-1 shows a loss of affinity with Bax and will not translocate to mitochondria, stably remaining in the cytoplasm. These results indicate that phosphorylation to inactivate CFL-1 decreased the chemosensitivity to HDAC inhibitors and resulting in drug resistance of HCC cells.

  1. NKCC1 Regulates Migration Ability of Glioblastoma Cells by Modulation of Actin Dynamics and Interacting with Cofilin

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    Paula Schiapparelli


    Full Text Available Glioblastoma (GBM is the most aggressive primary brain tumor in adults. The mechanisms that confer GBM cells their invasive behavior are poorly understood. The electroneutral Na+-K+-2Cl− co-transporter 1 (NKCC1 is an important cell volume regulator that participates in cell migration. We have shown that inhibition of NKCC1 in GBM cells leads to decreased cell migration, in vitro and in vivo. We now report on the role of NKCC1 on cytoskeletal dynamics. We show that GBM cells display a significant decrease in F-actin content upon NKCC1 knockdown (NKCC1-KD. To determine the potential actin-regulatory mechanisms affected by NKCC1 inhibition, we studied NKCC1 protein interactions. We found that NKCC1 interacts with the actin-regulating protein Cofilin-1 and can regulate its membrane localization. Finally, we analyzed whether NKCC1 could regulate the activity of the small Rho-GTPases RhoA and Rac1. We observed that the active forms of RhoA and Rac1 were decreased in NKCC1-KD cells. In summary, we report that NKCC1 regulates GBM cell migration by modulating the cytoskeleton through multiple targets including F-actin regulation through Cofilin-1 and RhoGTPase activity. Due to its essential role in cell migration NKCC1 may serve as a specific therapeutic target to decrease cell invasion in patients with primary brain cancer.

  2. Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics. (United States)

    Roybal, Kole T; Buck, Taráz E; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J; Ambler, Rachel; Tunbridge, Helen M; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F


    Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems. Copyright © 2016, American Association for the Advancement of Science.

  3. Taurine chloramine-induced inactivation of cofilin protein through methionine oxidation. (United States)

    Luo, Shen; Uehara, Hiroshi; Shacter, Emily


    Cofilin regulates reorganization of actin filaments (F-actin) in eukaryotes. A recent finding has demonstrated that oxidation of cofilin by taurine chloramine (TnCl), a physiological oxidant derived from neutrophils, causes cofilin to translocate to the mitochondria inducing apoptosis (F. Klamt et al. Nat. Cell Biol.11:1241-1246; 2009). Here we investigated the effect of TnCl on biological activities of cofilin in vitro. Our data show that TnCl-induced oxidation of recombinant human cofilin-1 inhibits its F-actin-binding and depolymerization activities. Native cofilin contains four free Cys and three Met residues. Incubation of oxidized cofilin with DTT does not lead to its reactivation. A double Cys to Ala mutation on the two C-terminal Cys shows similar biological activities as the wild type, but does not prevent the TnCl-induced inactivation. In contrast, incubation of oxidized cofilin with methionine sulfoxide reductases results in its reactivation. Phosphorylation is known to inhibit cofilin activities. We found that Met oxidation also prevents phosphorylation of cofilin, which is reversed by incubating oxidized cofilin with methionine sulfoxide reductases. Interestingly, intact protein mass spectrometry of the oxidized mutant indicated one major oxidation product with an additional mass of 16 Da, consistent with oxidation of one specific Met residue. This residue was identified as Met-115 by peptide mapping and tandem mass spectrometry. It is adjacent to Lys-114, a known residue on globular-actin-binding site, implying that oxidation of Met-115 disrupts the globular-actin-binding site of cofilin, which causes TnCl-induced inactivation. The findings identify Met-115 as a redox switch on cofilin that regulates its biological activity. Published by Elsevier Inc.

  4. Slingshot-3 dephosphorylates ADF/cofilin but is dispensable for mouse development. (United States)

    Kousaka, Kazuyoshi; Kiyonari, Hiroshi; Oshima, Naoko; Nagafuchi, Akira; Shima, Yasuyuki; Chisaka, Osamu; Uemura, Tadashi


    Actin-depolymerizing factor (ADF) and cofilin constitute a family of key regulators of actin filament dynamics. ADF/cofilin is inactivated by phosphorylation at Ser-3 by LIM-kinases and reactivated by dephosphorylation by Slingshot (SSH) family phosphatases. Defects in LIM kinases or ADF/cofilin have been implicated in morbidity in human or mice; however, the roles of mammalian SSH in vivo have not been addressed. In this study, we examined the endogenous expression of each mouse SSH member in various cell lines and tissues, and showed that SSH-3L protein was strongly expressed in epithelial cells. Our structure-function analysis of SSH-3L suggested the possibility that the C-tail unique to SSH-3L negatively regulates the catalytic activity of this phosphatase. Furthermore we made ssh-3 knockout mice to examine its potential in vivo roles. Unexpectedly, ssh-3 was not essential for viability, fertility, or development of epithelial tissues; and ssh-3 did not genetically modify the corneal disorder of the corn1/ADF/destrin mutant.

  5. Malignant progressive tumor cell clone exhibits significant up-regulation of cofilin-2 and 27-kDa modified form of cofilin-1 compared to regressive clone. (United States)

    Kuramitsu, Yasuhiro; Wang, Yufeng; Okada, Futoshi; Baron, Byron; Tokuda, Kazuhiro; Kitagawa, Takao; Akada, Junko; Nakamura, Kazuyuki


    QR-32 is a regressive murine fibrosarcoma cell clone which cannot grow when they are transplanted in mice; QRsP-11 is a progressive malignant tumor cell clone derived from QR-32 which shows strong tumorigenicity. A recent study showed there to be differentially expressed up-regulated and down-regulated proteins in these cells, which were identified by proteomic differential display analyses by using two-dimensional gel electrophoresis and mass spectrometry. Cofilins are small proteins of less than 20 kDa. Their function is the regulation of actin assembly. Cofilin-1 is a small ubiquitous protein, and regulates actin dynamics by means of binding to actin filaments. Cofilin-1 plays roles in cell migration, proliferation and phagocytosis. Cofilin-2 is also a small protein, but it is mainly expressed in skeletal and cardiac muscles. There are many reports showing the positive correlation between the level of cofilin-1 and cancer progression. We have also reported an increased expression of cofilin-1 in pancreatic cancer tissues compared to adjacent paired normal tissues. On the other hand, cofilin-2 was significantly less expressed in pancreatic cancer tissues. Therefore, the present study investigated the comparison of the levels of cofilin-1 and cofilin-2 in regressive QR-32 and progressive QRsP-11cells by western blotting. Cofilin-2 was significantly up-regulated in QRsP-11 compared to QR-32 cells (p<0.001). On the other hand, the difference of the intensities of the bands of cofilin-1 (18 kDa) in QR-32 and QRsP-11 was not significant. However, bands of 27 kDa showed a quite different intensity between QR-32 and QRsP-11, with much higher intensities in QRsP-11 compared to QR-32 (p<0.001). These results suggested that the 27-kDa protein recognized by the antibody against cofilin-1 is a possible biomarker for progressive tumor cells.

  6. Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin

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    Hadas Smadar


    Full Text Available Abstract Background Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3 is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin remodeling (i.e., disassembly and reassembly by shifting between active unphosphorylated and inactive phosphorylated states. Results Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia, which, as we also revealed, are instrumental in myelin phagocytosis. Conclusions CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive

  7. Cyclase-associated Protein 1 (CAP1) Promotes Cofilin-induced Actin Dynamics in Mammalian Nonmuscle CellsV⃞


    Bertling, Enni; Hotulainen, Pirta; Mattila, Pieta K.; Matilainen, Tanja; Salminen, Marjo; Lappalainen, Pekka


    Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B1...

  8. Phosphorylation site dynamics of early T-cell receptor signaling

    DEFF Research Database (Denmark)

    Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn


    a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...... that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites...

  9. A genetically encoded reporter for real-time imaging of cofilin-actin rods in living neurons.

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    Jianjie Mi

    Full Text Available Filament bundles (rods of cofilin and actin (1:1 form in neurites of stressed neurons where they inhibit synaptic function. Live-cell imaging of rod formation is hampered by the fact that overexpression of a chimera of wild type cofilin with a fluorescent protein causes formation of spontaneous and persistent rods, which is exacerbated by the photostress of imaging. The study of rod induction in living cells calls for a rod reporter that does not cause spontaneous rods. From a study in which single cofilin surface residues were mutated, we identified a mutant, cofilinR21Q, which when fused with monomeric Red Fluorescent Protein (mRFP and expressed several fold above endogenous cofilin, does not induce spontaneous rods even during the photostress of imaging. CofilinR21Q-mRFP only incorporates into rods when they form from endogenous proteins in stressed cells. In neurons, cofilinR21Q-mRFP reports on rods formed from endogenous cofilin and induced by all modes tested thus far. Rods have a half-life of 30-60 min upon removal of the inducer. Vesicle transport in neurites is arrested upon treatments that form rods and recovers as rods disappear. CofilinR21Q-mRFP is a genetically encoded rod reporter that is useful in live cell imaging studies of induced rod formation, including rod dynamics, and kinetics of rod elimination.

  10. The reversible increase in tight junction permeability induced by capsaicin is mediated via cofilin-actin cytoskeletal dynamics and decreased level of occludin.

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    Tomoko Shiobara

    Full Text Available Previous results demonstrated that capsaicin induces the reversible tight junctions (TJ opening via cofilin activation. The present study investigated the mechanisms underlying the reversible TJ opening and compared the effect to the irreversible opening induced by actin inhibitors. Capsaicin treatment induced the F-actin alteration unique to capsaicin compared to actin-interacting agents such as latrunculin A, which opens TJ irreversibly. Along with TJ opening, capsaicin decreased the level of F-actin at bicellular junctions but increased it at tricellular junctions accompanied with its concentration on the apical side of the lateral membrane. No change in TJ protein localization was observed upon exposure to capsaicin, but the amount of occludin was decreased significantly. In addition, cosedimentation analyses suggested a decrease in the interactions forming TJ, thereby weakening TJ tightness. Introduction of cofilin, LIMK and occludin into the cell monolayers confirmed their contribution to the transepithelial electrical resistance decrease. Finally, exposure of monolayers to capsaicin augmented the paracellular passage of both charged and uncharged compounds, as well as of insulin, indicating that capsaicin can be employed to modulate epithelial permeability. Our results demonstrate that capsaicin induces TJ opening through a unique mechanism, and suggest that it is a new type of paracellular permeability enhancer.

  11. Peptide Regulation of Cofilin Activity in the CNS: A Novel Therapeutic Approach for Treatment of Multiple Neurological Disorders. (United States)

    Shaw, Alisa E; Bamburg, James R


    Cofilin is a ubiquitous protein which cooperates with many other actin-binding proteins in regulating actin dynamics. Cofilin has essential functions in nervous system development including neuritogenesis, neurite elongation, growth cone pathfinding, dendritic spine formation, and the regulation of neurotransmission and spine function, components of synaptic plasticity essential for learning and memory. Cofilin's phosphoregulation is a downstream target of many transmembrane signaling processes, and its misregulation in neurons has been linked in rodent models to many different neurodegenerative and neurological disorders including Alzheimer disease (AD), aggression due to neonatal isolation, autism, manic/bipolar disorder, and sleep deprivation. Cognitive and behavioral deficits of these rodent models have been largely abrogated by modulation of cofilin activity using viral-mediated, genetic, and/or small molecule or peptide therapeutic approaches. Neuropathic pain in rats from sciatic nerve compression has also been reduced by modulating the cofilin pathway within neurons of the dorsal root ganglia. Neuroinflammation, which occurs following cerebral ischemia/reperfusion, but which also accompanies many other neurodegenerative syndromes, is markedly reduced by peptides targeting specific chemokine receptors, which also modulate cofilin activity. Thus, peptide therapeutics offer potential for cost-effective treatment of a wide variety of neurological disorders. Here we discuss some recent results from rodent models using therapeutic peptides with a surprising ability to cross the rodent blood brain barrier and alter cofilin activity in brain. We also offer suggestions as to how neuronal-specific cofilin regulation might be achieved.

  12. Mammalian adenylyl cyclase-associated protein 1 (CAP1) regulates cofilin function, the actin cytoskeleton, and cell adhesion. (United States)

    Zhang, Haitao; Ghai, Pooja; Wu, Huhehasi; Wang, Changhui; Field, Jeffrey; Zhou, Guo-Lei


    CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

  13. Dynamic phosphorylation patterns of RNA polymerase II CTD during transcription. (United States)

    Heidemann, Martin; Hintermair, Corinna; Voß, Kirsten; Eick, Dirk


    The eukaryotic RNA polymerase II (RNAPII) catalyzes the transcription of all protein encoding genes and is also responsible for the generation of small regulatory RNAs. RNAPII has evolved a unique domain composed of heptapeptide repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the C-terminus (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation patterns of serine residues in CTD during gene transcription coordinate the recruitment of factors to the elongating RNAPII and to the nascent transcript. Recent studies identified threonine 4 and tyrosine 1 as new CTD modifications and thereby expanded the "CTD code". In this review, we focus on CTD phosphorylation and its function in the RNAPII transcription cycle. We also discuss in detail the limitations of the phosphospecific CTD antibodies, which are used in all studies. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

  14. Loss of cofilin 1 disturbs actin dynamics, adhesion between enveloping and deep cell layers and cell movements during gastrulation in zebrafish.

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    Chun-Wei Lin

    Full Text Available During gastrulation, cohesive migration drives associated cell layers to the completion of epiboly in zebrafish. The association of different layers relies on E-cadherin based cellular junctions, whose stability can be affected by actin turnover. Here, we examined the effect of malfunctioning actin turnover on the epibolic movement by knocking down an actin depolymerizing factor, cofilin 1, using antisense morpholino oligos (MO. Knockdown of cfl1 interfered with epibolic movement of deep cell layer (DEL but not in the enveloping layer (EVL and the defect could be specifically rescued by overexpression of cfl1. It appeared that the uncoordinated movements of DEL and EVL were regulated by the differential expression of cfl1 in the DEL, but not EVL as shown by in situ hybridization. The dissociation of DEL and EVL was further evident by the loss of adhesion between layers by using transmission electronic and confocal microscopy analyses. cfl1 morphants also exhibited abnormal convergent extension, cellular migration and actin filaments, but not involution of hypoblast. The cfl1 MO-induced cell migration defect was found to be cell-autonomous in cell transplantation assays. These results suggest that proper actin turnover mediated by Cfl1 is essential for adhesion between DEL and EVL and cell movements during gastrulation in zebrafish.

  15. Deficiency of Cks1 Leads to Learning and Long-Term Memory Defects and p27 Dependent Formation of Neuronal Cofilin Aggregates. (United States)

    Kukalev, Alexander; Ng, Yiu-Ming; Ju, Limei; Saidi, Amal; Lane, Sophie; Mondragon, Angeles; Dormann, Dirk; Walker, Sophie E; Grey, William; Ho, Philip Wing-Lok; Stephens, David N; Carr, Antony M; Lamsa, Karri; Tse, Eric; Yu, Veronica P C C


    In mitotic cells, the cyclin-dependent kinase (CDK) subunit protein CKS1 regulates S phase entry by mediating degradation of the CDK inhibitor p27. Although mature neurons lack mitotic CDKs, we found that CKS1 was actively expressed in post-mitotic neurons of the adult hippocampus. Interestingly, Cks1 knockout (Cks1-/-) mice exhibited poor long-term memory, and diminished maintenance of long-term potentiation in the hippocampal circuits. Furthermore, there was neuronal accumulation of cofilin-actin rods or cofilin aggregates, which are associated with defective dendritic spine maturation and synaptic loss. We further demonstrated that it was the increased p27 level that activated cofilin by suppressing the RhoA kinase-mediated inhibitory phosphorylation of cofilin, resulting in the formation of cofilin aggregates in the Cks1-/- neuronal cells. Consistent with reports that the peptidyl-prolyl-isomerase PIN1 competes with CKS1 for p27 binding, we found that inhibition of PIN1 diminished the formation of cofilin aggregates through decreasing p27 levels, thereby activating RhoA and increasing cofilin phosphorylation. Our results revealed that CKS1 is involved in normal glutamatergic synapse development and dendritic spine maturation in adult hippocampus through modulating p27 stability. © The Author 2016. Published by Oxford University Press.

  16. Quantitative and dynamic analysis of PTEN phosphorylation by NMR. (United States)

    Cordier, Florence; Chaffotte, Alain; Wolff, Nicolas


    The dual lipid and protein phosphatase PTEN is a tumor suppressor controlling key biological processes, such as cell growth, proliferation and neuro-survival. Its activity and intracellular trafficking is finely regulated notably by multi-site phosphorylation of its C-terminal tail. The reversible and highly dynamic character of these regulatory events confers a temporal dimension to the cell for triggering crucial decisions. In this review, we describe how a recently developed time-resolved NMR spectroscopy approach unveils the dynamic establishment of the phosphorylation events of PTEN C-terminal tail controlled by CK2 and GSK3β kinases. Two cascades of reactions have been identified, in vitro and in extracts of human neuroblastoma cells. They are triggered independently on two nearby clusters of sites (S380-S385 and S361-S370) and occur on different timescales. In each cascade, the reactions follow an ordered model with a distributive kinetic mechanism. The vision of these cascades as two delay timers activating distinct or time-delayed regulatory responses gives a temporal dimension on PTEN regulation and is discussed in relation to the known functional roles of each cluster. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Molecular dynamics simulation of phosphorylated KID post-translational modification.

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    Hai-Feng Chen

    Full Text Available BACKGROUND: Kinase-inducible domain (KID as transcriptional activator can stimulate target gene expression in signal transduction by associating with KID interacting domain (KIX. NMR spectra suggest that apo-KID is an unstructured protein. After post-translational modification by phosphorylation, KID undergoes a transition from disordered to well folded protein upon binding to KIX. However, the mechanism of folding coupled to binding is poorly understood. METHODOLOGY: To get an insight into the mechanism, we have performed ten trajectories of explicit-solvent molecular dynamics (MD for both bound and apo phosphorylated KID (pKID. Ten MD simulations are sufficient to capture the average properties in the protein folding and unfolding. CONCLUSIONS: Room-temperature MD simulations suggest that pKID becomes more rigid and stable upon the KIX-binding. Kinetic analysis of high-temperature MD simulations shows that bound pKID and apo-pKID unfold via a three-state and a two-state process, respectively. Both kinetics and free energy landscape analyses indicate that bound pKID folds in the order of KIX access, initiation of pKID tertiary folding, folding of helix alpha(B, folding of helix alpha(A, completion of pKID tertiary folding, and finalization of pKID-KIX binding. Our data show that the folding pathways of apo-pKID are different from the bound state: the foldings of helices alpha(A and alpha(B are swapped. Here we also show that Asn139, Asp140 and Leu141 with large Phi-values are key residues in the folding of bound pKID. Our results are in good agreement with NMR experimental observations and provide significant insight into the general mechanisms of binding induced protein folding and other conformational adjustment in post-translational modification.

  18. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin. (United States)

    Chaudhry, Faisal; Breitsprecher, Dennis; Little, Kristin; Sharov, Grigory; Sokolova, Olga; Goode, Bruce L


    Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

  19. S-nitrosylation of cofilin-1 mediates estradiol-17β-stimulated endothelial cytoskeleton remodeling. (United States)

    Zhang, Hong-hai; Lechuga, Thomas J; Tith, Tevy; Wang, Wen; Wing, Deborah A; Chen, Dong-bao


    Rapid nitric oxide (NO) production via endothelial NO synthase (eNOS) activation represents a major signaling pathway for the cardiovascular protective effects of estrogens; however, the pathways after NO biosynthesis that estrogens use to function remain largely unknown. Covalent adduction of a NO moiety to cysteines, termed S-nitrosylation (SNO), has emerged as a key route for NO to directly regulate protein function. Cofilin-1 (CFL1) is a small actin-binding protein essential for actin dynamics and cytoskeleton remodeling. Despite being identified as a major SNO protein in endothelial cells, whether SNO regulates CFL-1 function is unknown. We hypothesized that estradiol-17β (E2β) stimulates SNO of CFL1 via eNOS-derived NO and that E2β-induced SNO-CFL1 mediates cytoskeleton remodeling in endothelial cells. Point mutation studies determined Cys80 as the primary SNO site among the 4 cysteines (Cys39/80/139/147) in CFL1. Substitutions of Cys80 with Ala or Ser were used to prepare the SNO-mimetic/deficient (C80A/S) CFL1 mutants. Recombinant wild-type (wt) and mutant CFL1 proteins were prepared; their actin-severing activity was determined by real-time fluorescence imaging analysis. The activity of C80A CFL1 was enhanced to that of the constitutively active S3/A CFL1, whereas the other mutants had no effects. C80A/S mutations lowered Ser3 phosphorylation. Treatment with E2β increased filamentous (F)-actin and filopodium formation in endothelial cells, which were significantly reduced in cells overexpressing wt-CFL. Overexpression of C80A, but not C80S, CFL1 decreased basal F-actin and further suppressed E2β-induced F-actin and filopodium formation compared with wt-CFL1 overexpression. Thus, SNO(Cys80) of cofilin-1 via eNOS-derived NO provides a novel pathway for mediating estrogen-induced endothelial cell cytoskeleton remodeling.

  20. Neuronal actin dynamics, spine density and neuronal dendritic complexity are regulated by CAP2

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    Atul Kumar


    Full Text Available Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2gt/gt mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2gt/gt with increased number of excitatory synapse. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2gt/gt neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics.

  1. Towards the systematic discovery of signal transduction networks using phosphorylation dynamics data

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    Yachie Nozomu


    Full Text Available Abstract Background Phosphorylation is a ubiquitous and fundamental regulatory mechanism that controls signal transduction in living cells. The number of identified phosphoproteins and their phosphosites is rapidly increasing as a result of recent mass spectrometry-based approaches. Results We analyzed time-course phosphoproteome data obtained previously by liquid chromatography mass spectrometry with the stable isotope labeling using amino acids in cell culture (SILAC method. This provides the relative phosphorylation activities of digested peptides at each of five time points after stimulating HeLa cells with epidermal growth factor (EGF. We initially calculated the correlations between the phosphorylation dynamics patterns of every pair of peptides and connected the strongly correlated pairs to construct a network. We found that peptides extracted from the same intracellular fraction (nucleus vs. cytoplasm tended to be close together within this phosphorylation dynamics-based network. The network was then analyzed using graph theory and compared with five known signal-transduction pathways. The dynamics-based network was correlated with known signaling pathways in the NetPath and Phospho.ELM databases, and especially with the EGF receptor (EGFR signaling pathway. Although the phosphorylation patterns of many proteins were drastically changed by the EGF stimulation, our results suggest that only EGFR signaling transduction was both strongly activated and precisely controlled. Conclusions The construction of a phosphorylation dynamics-based network provides a useful overview of condition-specific intracellular signal transduction using quantitative time-course phosphoproteome data under specific experimental conditions. Detailed prediction of signal transduction based on phosphoproteome dynamics remains challenging. However, since the phosphorylation profiles of kinase-substrate pairs on the specific pathway were localized in the dynamics

  2. Cyclase-associated protein (CAP) acts directly on F-actin to accelerate cofilin-mediated actin severing across the range of physiological pH. (United States)

    Normoyle, Kieran P M; Brieher, William M


    Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization.

  3. Cyclase-associated Protein (CAP) Acts Directly on F-actin to Accelerate Cofilin-mediated Actin Severing across the Range of Physiological pH* (United States)

    Normoyle, Kieran P. M.; Brieher, William M.


    Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization. PMID:22904322


    Samuel, Filsy; Reddy, Jairus; Kaimal, Radhika; Segovia, Vianey; Mo, Huanbiao; Hynds, DiAnna L.


    Inhibitors of the mevalonate pathway, including the highly prescribed statins, reduce the production of cholesterol and isoprenoids such as geranylgeranyl pyrophosphates. The Rho family of small guanine triphosphatases (GTPases) requires isoprenylation, specifically geranylgeranylation, for activation. Because Rho GTPases are primary regulators of actin filament rearrangements required for process extension, neurite arborization and synaptic plasticity, statins may affect cognition or recovery from nervous system injury. Here, we assessed how manipulating geranylgeranylation affects neurite initiation, elongation and branching in neuroblastoma growth cones. Treatment with the statin, lovastatin (20 μM) decreased measures of neurite initiation by 17.0% to 19.0% when a source of cholesterol was present and increased neurite branching by 4.03 to 9.54 fold (regardless of exogenous cholesterol). Neurite elongation was increased by treatment with lovastatin only in cholesterol-free culture conditions. Treatment with lovastatin decreased growth cone actin filament content by up to 24.3%. In all cases, co-treatment with the prenylation precursor, geranylgeraniol (10 μM), reversed the effect of lovastatin. In prior work, statin effects on outgrowth were linked to modulating the actin depolymerizing factor, cofilin. In our assays, treatment with lovastatin or geranylgeraniol decreased cofilin phosphorylation in whole cell lysates. However, lovastatin increased cofilin phosphorylation in cell bodies and decreased it in growth cones, indicating differential regulation in specific cell regions. Together, we interpret these data to suggest that protein geranylgeranylation likely regulates growth cone actin filament content and subsequent neurite outgrowth through mechanisms that also affect actin nucleation and polymerization. PMID:24515839

  5. Molecular dynamics simulation on HP1 protein binding by histone H3 tail methylation and phosphorylation (United States)

    Jiang, Yan-Ke; Zou, Jian-Wei; Wu, Yu-Qian; Zhang, Na; Yu, Qing-Sen; Jiang, Yong-Jun

    Trimethylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging, and heterochromatin formation. Phosphorylation of histone H3 has been linked with mitotic chromatin condensation. During mitosis in vivo, H3 lysine 9 methylation and serine 10 phosphorylation can occur concomitantly on the same histone tail, whereas the influence of phosphorylation to trimethylation H3 tail recruiting HP1 remains controversial. In this work, molecular dynamics simulation of HP1 complexed with both trimethylated and phosphorylated H3 tail were performed and compared with the results from the previous methylated H3-HP1 trajectory. It is clear from the 10-ns dynamics simulation that two adjacent posttranslational modifications directly increase the flexibility of the H3 tail and weaken HP1 binding to chromatin. A combinatorial readout of two adjacent posttranslational modifications-a stable methylation and a dynamic phosphorylation mark-establish a regulatory mechanism of protein-protein interactions.

  6. Phosphorylated filamin A regulates actin-linked caveolae dynamics. (United States)

    Muriel, Olivia; Echarri, Asier; Hellriegel, Christian; Pavón, Dácil M; Beccari, Leonardo; Del Pozo, Miguel A


    Caveolae are relatively stable membrane invaginations that compartmentalize signaling, regulate lipid metabolism and mediate viral entry. Caveolae are closely associated with actin fibers and internalize in response to diverse stimuli. Loss of cell adhesion is known to induce rapid and robust caveolae internalization and trafficking toward a Rab11-positive recycling endosome; however, pathways governing this process are poorly understood. Here, we report that filamin A is required to maintain the F-actin-dependent linear distribution of caveolin-1. High spatiotemporal resolution particle tracking of caveolin-1-GFP vesicles by total internal reflection fluorescence (TIRF) microscopy revealed that FLNa is required for the F-actin-dependent arrest of caveolin-1 vesicles in a confined area and their stable anchorage to the plasma membrane. The linear distribution and anchorage of caveolin-1 vesicles are both required for proper caveolin-1 inwards trafficking. De-adhesion-triggered caveolae inward trafficking towards a recycling endosome is impaired in FLNa-depleted HeLa and FLNa-deficient M2-melanoma cells. Inwards trafficking of caveolin-1 requires both the ability of FLNa to bind actin and cycling PKCα-dependent phosphorylation of FLNa on Ser2152 after cell detachment. © 2011. Published by The Company of Biologists Ltd

  7. Cellular functions of the ADF/cofilin family at a glance. (United States)

    Kanellos, Georgios; Frame, Margaret C


    The actin depolymerizing factor (ADF)/cofilin family comprises small actin-binding proteins with crucial roles in development, tissue homeostasis and disease. They are best known for their roles in regulating actin dynamics by promoting actin treadmilling and thereby driving membrane protrusion and cell motility. However, recent discoveries have increased our understanding of the functions of these proteins beyond their well-characterized roles. This Cell Science at a Glance article and the accompanying poster serve as an introduction to the diverse roles of the ADF/cofilin family in cells. The first part of the article summarizes their actions in actin treadmilling and the main mechanisms for their intracellular regulation; the second part aims to provide an outline of the emerging cellular roles attributed to the ADF/cofilin family, besides their actions in actin turnover. The latter part discusses an array of diverse processes, which include regulation of intracellular contractility, maintenance of nuclear integrity, transcriptional regulation, nuclear actin monomer transfer, apoptosis and lipid metabolism. Some of these could, of course, be indirect consequences of actin treadmilling functions, and this is discussed.

  8. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators. (United States)

    Dopie, Joseph; Rajakylä, Eeva K; Joensuu, Merja S; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K


    Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes.

  9. Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy (United States)

    Yehl, Jenna; Kudryashova, Elena; Reisler, Emil; Kudryashov, Dmitri; Polenova, Tatyana


    Cellular actin dynamics is an essential element of numerous cellular processes, such as cell motility, cell division and endocytosis. Actin’s involvement in these processes is mediated by many actin-binding proteins, among which the cofilin family plays unique and essential role in accelerating actin treadmilling in filamentous actin (F-actin) in a nucleotide-state dependent manner. Cofilin preferentially interacts with older filaments by recognizing time-dependent changes in F-actin structure associated with the hydrolysis of ATP and release of inorganic phosphate (Pi) from the nucleotide cleft of actin. The structure of cofilin on F-actin and the details of the intermolecular interface remain poorly understood at atomic resolution. Here we report atomic-level characterization by magic angle spinning (MAS) NMR of the muscle isoform of human cofilin 2 (CFL2) bound to F-actin. We demonstrate that resonance assignments for the majority of atoms are readily accomplished and we derive the intermolecular interface between CFL2 and F-actin. The MAS NMR approach reported here establishes the foundation for atomic-resolution characterization of a broad range of actin-associated proteins bound to F-actin. PMID:28303963

  10. Sensitive targeted quantification of ERK phosphorylation dynamics and stoichiometry in human cells without affinity enrichment. (United States)

    Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J; Nicora, Carrie D; Fillmore, Thomas L; Chrisler, William B; Gritsenko, Marina A; Wu, Chaochao; He, Jintang; Bloodsworth, Kent J; Zhao, Rui; Camp, David G; Liu, Tao; Rodland, Karin D; Smith, Richard D; Wiley, H Steven; Qian, Wei-Jun


    Targeted mass spectrometry is a promising technology for site-specific quantification of posttranslational modifications. However, a major constraint is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents for enrichment. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometry using a sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection, and multiplexing (PRISM). PRISM provides effective enrichment of target peptides into a given fraction from complex mixture, followed by selected reaction monitoring quantification. Direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) was demonstrated from as little as 25 μg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided ∼10-fold higher signal intensities, presumably due to the better peptide recovery of PRISM. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of epidermal growth factor at both the peak activation (10 min) and steady state (2 h). The maximal ERK activation was observed with 0.3 and 3 ng/mL doses for 10 min and 2 h time points, respectively. The dose-response profiles of individual phosphorylated isoforms showed that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than distributed model of ERK phosphorylation. The PRISM-SRM quantification of protein phosphorylation illustrates the potential for simultaneous quantification of multiple PTMs.

  11. Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations. (United States)

    Velazquez, Hector A; Hamelberg, Donald


    Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.

  12. LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B (United States)

    Poirault-Chassac, Sonia; Adam, Frédéric; Muczynski, Vincent; Aymé, Gabriel; Casari, Caterina; Bordet, Jean-Claude; Soukaseum, Christelle; Rothschild, Chantal; Proulle, Valérie; Pietrzyk-Nivau, Audrey; Berrou, Eliane; Christophe, Olivier D.; Rosa, Jean-Philippe; Lenting, Peter J.; Bryckaert, Marijke; Baruch, Dominique


    von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein’s multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine Vwf gene and in a patient bearing this mutation. We provide evidence of a profound defect in megakaryocyte (MK) function since: (a) the extent of proplatelet formation was drastically decreased in 2B MKs, with thick proplatelet extensions and large swellings; and (b) 2B MKs presented actin disorganization that was controlled by upregulation of the RhoA/LIM kinase (LIMK)/cofilin pathway. In vitro and in vivo inhibition of the LIMK/cofilin signaling pathway rescued actin turnover and restored normal proplatelet formation, platelet count, and platelet size. These data indicate, to our knowledge for the first time, that the severe macrothrombocytopenia in VWD-type 2B p.V1316M is due to an MK dysfunction that originates from a constitutive activation of the RhoA/LIMK/cofilin pathway and actin disorganization. This suggests a potentially new function of vWF during platelet formation that involves regulation of actin dynamics. PMID:27734030

  13. Rapid actions of plasma membrane estrogen receptors regulate motility of mouse embryonic stem cells through a profilin-1/cofilin-1-directed kinase signaling pathway. (United States)

    Yun, Seung Pil; Ryu, Jung Min; Kim, Mi Ok; Park, Jae Hong; Han, Ho Jae


    Long-term estrogen actions are vital for driving cell growth, but more recent evidence suggests that estrogen mediates more rapid cellular effects. However, the function of estradiol-17β (E(2))-BSA in mouse embryonic stem cells has not been reported. Therefore, we examined the role of E(2)-BSA in mouse embryonic stem cell motility and its related signal pathways. E(2)-BSA (10(-8) m) significantly increased motility after 24 h incubation and increased filamentous (F)-actin expression; these effects were inhibited by the estrogen receptor antagonist ICI 182,780, indicating that E(2)-BSA bound membrane estrogen receptors and initiated a signal. E(2)-BSA increased c-Src and focal adhesion kinase (FAK) phosphorylation, which was attenuated by ICI 182,780. The E(2)-BSA-induced increase in epidermal growth factor receptor (EGFR) phosphorylation was inhibited by Src inhibitor PP2. As a downstream signal molecule, E(2)-BSA activated cdc42 and increased formation of a complex with the neural Wiskott-Aldrich syndrome protein (N-WASP)/cdc42/transducer of cdc42-dependent actin assembly-1 (TOCA-1), which was inhibited by FAK small interfering RNA (siRNA) and EGFR inhibitor AG 1478. In addition, E(2)-BSA increased profilin-1 expression and cofilin-1 phosphorylation, which was blocked by cdc42 siRNA. Subsequently, E(2)-BSA induced an increase in F-actin expression, and cell motility was inhibited by each signal pathway-related siRNA molecule or inhibitors but not by cofilin-1 siRNA. A combined treatment of cofilin-1 siRNA and E(2)-BSA increased F-actin expression and cell motility more than that of E(2)-BSA alone. These data demonstrate that E(2)-BSA stimulated motility by interacting with profilin-1/cofilin-1 and F-actin through FAK- and c-Src/EGFR transactivation-dependent N-WASP/cdc42/TOCA-1 complex.

  14. Influence of 63Ser phosphorylation and dephosphorylation on the structure of the stathmin helical nucleation sequence: a molecular dynamics study. (United States)

    Missimer, John H; Steinmetz, Michel O; van Gunsteren, Wilfred F; Dolenc, Jožica


    Phosphorylation is an important mechanism regulating protein-protein interactions involving intrinsically disordered protein regions. Stathmin, an archetypical example of an intrinsically disordered protein, is a key regulator of microtubule dynamics in which phosphorylation of 63Ser within the helical nucleation sequence strongly down-regulates the tubulin binding and microtubule destabilizing activities of the protein. Experimental studies on a peptide encompassing the 19-residue helical nucleation sequence of stathmin (residues 55-73) indicate that phosphorylation of 63Ser destabilizes the peptide's secondary structure by disrupting the salt bridges supporting its helical conformation. In order to investigate this hypothesis at atomic resolution, we performed molecular dynamics simulations of nonphosphorylated and phosphorylated stathmin-[55-73] at room temperature and pressure, neutral pH, and explicit solvation using the recently released GROMOS force field 54A7. In the simulations of nonphosphorylated stathmin-[55-73] emerged salt bridges associated with helical configurations. In the simulations of 63Ser phosphorylated stathmin-[55-73] these configurations dispersed and were replaced by a proliferation of salt bridges yielding disordered configurations. The transformation of the salt bridges was accompanied by emergence of numerous interactions between main and side chains, involving notably the oxygen atoms of the phosphorylated 63Ser. The loss of helical structure induced by phosphorylation is reversible, however, as a final simulation showed. The results extend the hypothesis of salt bridge derangement suggested by experimental observations of the stathmin nucleation sequence, providing new insights into regulation of intrinsically disordered protein systems mediated by phosphorylation.

  15. Cofilin Inhibition Restores Neuronal Cell Death in Oxygen-Glucose Deprivation Model of Ischemia. (United States)

    Madineni, Anusha; Alhadidi, Qasim; Shah, Zahoor A


    Ischemia is a condition associated with decreased blood supply to the brain, eventually leading to death of neurons. It is associated with a diverse cascade of responses involving both degenerative and regenerative mechanisms. At the cellular level, the changes are initiated prominently in the neuronal cytoskeleton. Cofilin, a cytoskeletal actin severing protein, is known to be involved in the early stages of apoptotic cell death. Evidence supports its intervention in the progression of disease states like Alzheimer's and ischemic kidney disease. In the present study, we have hypothesized the possible involvement of cofilin in ischemia. Using PC12 cells and mouse primary cultures of cortical neurons, we investigated the potential role of cofilin in ischemia in two different in vitro ischemic models: chemical induced oxidative stress and oxygen-glucose deprivation/reperfusion (OGD/R). The expression profile studies demonstrated a decrease in phosphocofilin levels in all models of ischemia, implying stress-induced cofilin activation. Furthermore, calcineurin and slingshot 1L (SSH) phosphatases were found to be the signaling mediators of the cofilin activation. In primary cultures of cortical neurons, cofilin was found to be significantly activated after 1 h of OGD. To delineate the role of activated cofilin in ischemia, we knocked down cofilin by small interfering RNA (siRNA) technique and tested the impact of cofilin silencing on neuronal viability. Cofilin siRNA-treated neurons showed a significant reduction of cofilin levels in all treatment groups (control, OGD, and OGD/R). Additionally, cofilin siRNA-reduced cofilin mitochondrial translocation and caspase 3 cleavage, with a concomitant increase in neuronal viability. These results strongly support the active role of cofilin in ischemia-induced neuronal degeneration and apoptosis. We believe that targeting this protein mediator has a potential for therapeutic intervention in ischemic brain injury and stroke.

  16. Structural dynamics and topology of phosphorylated phospholamban homopentamer reveal its role in the regulation of calcium transport in sarcoplasmic reticulum (United States)

    Vostrikov, Vitaly V.; Mote, Kaustubh R.; Verardi, Raffaello; Veglia, Gianluigi


    Phospholamban (PLN) inhibits the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), thereby regulating cardiac diastole. In membranes, PLN assembles into homopentamers that in both the phosphorylated and non-phosphorylated states have been proposed to form ion-selective channels. Here, we determined the structure of the phosphorylated pentamer using a combination of solution and solid-state nuclear magnetic resonance methods. We found that the pinwheel architecture of the homopentamer is preserved upon phosphorylation, with each monomer having an L-shaped conformation of each monomer. The TM domains form a hydrophobic pore of approximately 24 Å long, and 2 Å in diameter, which is inconsistent with canonical Ca2+ selective channels. Phosphorylation, however, enhances the conformational dynamics of the cytoplasmic region of PLN, causing the partial unwinding of the amphipathic helix. We propose that PLN oligomers act as storage for active monomers, keeping SERCA function within a physiological window. PMID:24207128

  17. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner. (United States)

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro


    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Phospholamban phosphorylation, mutation, and structural dynamics: a biophysical approach to understanding and treating cardiomyopathy (United States)

    Ablorh, Naa-Adjeley D.; Thomas, David D.


    We review the recent development of novel biochemical and spectroscopic methods to determine the site-specific phosphorylation, expression, mutation, and structural dynamics of phospholamban (PLB), in relation to its function (inhibition of the cardiac calcium pump, SERCA2a), with specific focus on cardiac physiology, pathology, and therapy. In the cardiomyocyte, SERCA2a actively transports Ca2+ into the sarcoplasmic reticulum (SR) during relaxation (diastole) to create the concentration gradient that drives the passive efflux of Ca2+ required for cardiac contraction (systole). Unphosphorylated PLB (U-PLB) inhibits SERCA2a, but phosphorylation at S16 and/or T17 (producing P-PLB) changes the structure of PLB to relieve SERCA2a inhibition. Because insufficient SERCA2a activity is a hallmark of heart failure, SERCA2a activation (by gene therapy (Andino et al. 2008; Fish et al. 2013; Hoshijima et al. 2002; Jessup et al. 2011) or drug therapy (Ferrandi et al. 2013; Huang 2013; Khan et al. 2009; Rocchetti et al. 2008; Zhang et al. 2012)) is a widely sought goal for treatment of heart failure. This review describes rational approaches to this goal. Novel biophysical assays, using site-directed labeling and high-resolution spectroscopy, have been developed to resolve the structural states of SERCA2a-PLB complexes in vitro and in living cells. Novel biochemical assays, using synthetic standards and multidimensional immunofluorescence, have been developed to quantitate PLB expression and phosphorylation states in cells and human tissues. The biochemical and biophysical properties of U-PLB, P-PLB, and mutant PLB will ultimately resolve the mechanisms of loss of inhibition and gain of inhibition to guide therapeutic development. These assays will be powerful tools for investigating human tissue samples from the Sydney Heart Bank, for the purpose of analyzing and diagnosing specific disorders. PMID:25774229

  19. Cdc42 regulates cofilin during the establishment of neuronal polarity

    DEFF Research Database (Denmark)

    Garvalov, Boyan K; Flynn, Kevin C; Neukirchen, Dorothee


    The establishment of polarity is an essential process in early neuronal development. Although a number of molecules controlling neuronal polarity have been identified, genetic evidence about their physiological roles in this process is mostly lacking. We analyzed the consequences of loss of Cdc42......, a central regulator of polarity in multiple systems, on the polarization of mammalian neurons. Genetic ablation of Cdc42 in the brain led to multiple abnormalities, including striking defects in the formation of axonal tracts. Neurons from the Cdc42 null animals sprouted neurites but had a strongly......-type, but not of mutant, neurons. Importantly, cofilin knockdown resulted in polarity defects quantitatively analogous to the ones seen after Cdc42 ablation. We conclude that Cdc42 is a key regulator of axon specification, and that cofilin is a physiological downstream effector of Cdc42 in this process....

  20. Dynamic Lipid-dependent Modulation of Protein Topology by Post-translational Phosphorylation. (United States)

    Vitrac, Heidi; MacLean, David M; Karlstaedt, Anja; Taegtmeyer, Heinrich; Jayaraman, Vasanthi; Bogdanov, Mikhail; Dowhan, William


    Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding. We previously demonstrated that the lipid environment is also a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition. However, the effect on protein topology of post-assembly phosphorylation of amino acids localized within initially cytoplasmically oriented extramembrane domains has never been investigated. Here, we show in a controlled in vitro system that phosphorylation of a membrane protein can trigger a change in topological arrangement. The rate of change occurred on a scale of seconds, comparable with the rates observed upon changes in the protein lipid environment. The rate and extent of topological rearrangement were dependent on the charges of extramembrane domains and the lipid bilayer surface. Using model membranes mimicking the lipid compositions of eukaryotic organelles, we determined that anionic lipids, cholesterol, sphingomyelin, and membrane fluidity play critical roles in these processes. Our results demonstrate how post-translational modifications may influence membrane protein topology in a lipid-dependent manner, both along the organelle trafficking pathway and at their final destination. The results provide further evidence that membrane protein topology is dynamic, integrating for the first time the effect of changes in lipid composition and regulators of cellular processes. The discovery of a new topology regulatory mechanism opens additional avenues for understanding unexplored structure-function relationships and the development of optimized topology prediction tools.

  1. Downregulation of LIMK1–ADF/cofilin by DADS inhibits the migration and invasion of colon cancer (United States)

    Su, Jian; Zhou, Yujuan; Pan, Zhibing; Shi, Ling; Yang, Jing; Liao, Aijun; Liao, Qianjin; Su, Qi


    This study aimed to explore whether the downregulation of LIM kinase 1 (LIMK1)-actin depolymerization factor (ADF, also known as destrin)/cofilin by diallyl disulfide (DADS) inhibited the migration and invasion of colon cancer. Previous studies have shown that silencing LIMK1 could significantly enhance the inhibitory effect of DADS on colon cancer cell migration and invasion, suggesting that LIMK1 was a target molecule of DADS, which needed further confirmation. This study reported that LIMK1 and destrin were highly expressed in colon cancer and associated with poor prognosis of patients with colon cancer. Also, the expression of LIMK1 was positively correlated with the expression of destrin. The overexpression of LIMK1 significantly promoted colon cancer cell migration and invasion. DADS obviously inhibited migration and invasion by suppressing the phosphorylation of ADF/cofilin via downregulation of LIMK1 in colon cancer cells. Furthermore, DADS-induced suppression of cell proliferation was enhanced and antagonized by the knockdown and overexpression of LIMK1 in vitro and in vivo, respectively. Similar results were observed for DADS-induced changes in the expression of vimentin, CD34, Ki-67, and E-cadherin in xenografted tumors. These results indicated that LIMK1 was a potential target molecule for the inhibitory effect of DADS on colon cancer cell migration and invasion. PMID:28358024

  2. Molecular dynamics simulation of phosphorylation-induced conformational transitions in the mycobacterium tuberculosis response regulator PrrA

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Guo [Los Alamos National Laboratory; Mcmahon, Benjamin H [Los Alamos National Laboratory; Tung, Chang - Shung [Los Alamos National Laboratory


    Phosphorylation-activated modulation of response regulators (RR) is predominantly used by bacteria as a strategy in regulating their two-component signaling (TCS) systems, the underlying molecular mechanisms are however far from fully understood. In this work we have conducted a molecular dynamics (MD) simulation of the phosphorylation-induced conformational transitions of RRs with the Mycobacterium Tuberculosis PrrA as a particular example. Starting from the full-length inactive structure of PrrA we introduced a local disturbance by phosphorylating the conserved aspartic acid residue, Asp-58, in the regulatory domain. A Go-model-type algorithm packaged with AMBER force fields was then applied to simulate the dynamics upon phosphorylation. The MD simulation shows that the phosphorylation of Asp-58 facilitates PrrA, whose inactive state has a compact conformation with a closed interdomain interface, to open up with its interdomain separation being increased by an average of about 1.5 {angstrom} for a simulation of 20 ns. The trans-activation loop, which is completely buried within the interdomain interface in the inactive PrrA, is found to become more exposed with the phosphorylated structure as well. These results provide more structural details of how the phosphorylation of a local aspartate activates PrrA to undergo a global conformational rearrangement toward its extended active state. This work also indicates that MD simulations can serve as a fast tool to unravel the regulation mechanisms of all RRs, which is especially valuable when the structures of full-length active RRs are currently unavailable.

  3. RNA Pol II Dynamics Modulate Co-transcriptional Chromatin Modification, CTD Phosphorylation, and Transcriptional Direction. (United States)

    Fong, Nova; Saldi, Tassa; Sheridan, Ryan M; Cortazar, Michael A; Bentley, David L


    Eukaryotic genes are marked by conserved post-translational modifications on the RNA pol II C-terminal domain (CTD) and the chromatin template. How the 5'-3' profiles of these marks are established is poorly understood. Using pol II mutants in human cells, we found that slow transcription repositioned specific co-transcriptionally deposited chromatin modifications; histone H3 lysine 36 trimethyl (H3K36me3) shifted within genes toward 5' ends, and histone H3 lysine 4 dimethyl (H3K4me2) extended farther upstream of start sites. Slow transcription also evoked a hyperphosphorylation of CTD Ser2 residues at 5' ends of genes that is conserved in yeast. We propose a "dwell time in the target zone" model to explain the effects of transcriptional dynamics on the establishment of co-transcriptionally deposited protein modifications. Promoter-proximal Ser2 phosphorylation is associated with a longer pol II dwell time at start sites and reduced transcriptional polarity because of strongly enhanced divergent antisense transcription at promoters. These results demonstrate that pol II dynamics help govern the decision between sense and divergent antisense transcription. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Dynamic modulation of the Kv2.1 channel by Src-dependent tyrosine phosphorylation


    Song, Min-Young; Hong, Chansik; Bae, Seong Han; So, Insuk; Park, Kang-Sik


    The voltage-gated K+ channel Kv2.1 is expressed as a highly phosphorylated protein in most central neurons, where it plays a key role in regulating neuronal membrane excitability. Previous studies have shown that Kv2.1 channel activity is upregulated by Src-mediated phosphorylation through an unknown mechanism. However, a systematic analysis of the molecular mechanism of Kv2.1 channel phosphorylation by Src is lacking. Here we show that tyrosine phosphorylation by Src plays a fundamental role...

  5. Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity (United States)

    Leu, Jyh-Der; Chiu, Yu-Wen; Lo, Chia-Chien; Chiang, Pei-Hsun; Chiu, Su-Jun; Tsai, Cheng-Han; Hwang, Jeng-Jong; Chen, Ran-Chou; Gorbunova, Vera; Lee, Yi-Jang


    Purpose A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores, the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity. Materials and Methods Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632. Results When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation induced double strand break (DSB) repair, systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1. Conclusion CFL-1 over-expression enhances radiosensitivity and this

  6. Aurora kinase A induces papillary thyroid cancer lymph node metastasis by promoting cofilin-1 activity. (United States)

    Maimaiti, Yusufu; Jie, Tan; Jing, Zhou; Changwen, Wang; Pan, Yu; Chen, Chen; Tao, Huang


    Aurora-A (Aur-A), a member of the serine/threonine Aurora kinase family, plays an important role in ensuring genetic stability during cell division. Previous studies indicated that Aur-A possesses oncogenic activity and may be a valuable therapeutic target in cancer therapy. However, the role of Aur-A in the most common thyroid cancer, papillary thyroid cancer (PTC), remains largely unknown. In patients with PTC, cancer cell migration and invasion account for most of the metastasis, recurrence, and cancer-related deaths. Cofilin-1 (CFL-1) is the most important effector of actin polymerization and depolymerization, determining the direction of cell migration. Here, we assessed the correlation between Aur-A and CFL-1 in PTC with lymph node metastasis. Tissue microarray data showed that simultaneous overexpression of Aur-A and CFL-1 correlated with lymph node metastasis in thyroid cancer tissue. Inhibition of Aur-A suppressed thyroid cancer cell migration in vitro and decreased lymph node metastasis in nude mice. Importantly, Aur-A increased the non-phosphorylated, active form of CFL-1 in TPC-1 cells, thus promoting cancer cell migration and thyroid cancer lymph node metastasis. Our findings indicate that the combination of Aur-A and CFL-1 may be useful as a molecular prediction model for lymph node metastasis in thyroid cancer and raise the possibility of targeting Aur-A and CFL-1 for more effective treatment of thyroid cancer.

  7. In vivo phosphorylation dynamics of the Bordetella pertussis virulence-controlling response regulator BvgA. (United States)

    Boulanger, Alice; Chen, Qing; Hinton, Deborah M; Stibitz, Scott


    We have used protein electrophoresis through polyacrylamide gels derivatized with the proprietary ligand Phos-tag™ to separate the response regulator BvgA from its phosphorylated counterpart BvgA∼P. This approach has allowed us to readily ascertain the degree of phosphorylation of BvgA in in vitro reactions, or in crude lysates of Bordetella pertussis grown under varying laboratory conditions. We have used this technique to examine the kinetics of BvgA phosphorylation after shift of B. pertussis cultures from non-permissive to permissive conditions, or of its dephosphorylation following a shift from permissive to non-permissive conditions. Our results provide the first direct evidence that levels of BvgA∼P in vivo correspond temporally to the expression of early and late BvgA-regulated virulence genes. We have also examined a number of other aspects of BvgA function predicted from previous studies and by analogy with other two-component response regulators. These include the site of BvgA phosphorylation, the exclusive role of the cognate BvgS sensor kinase in its phosphorylation in Bordetella pertussis, and the effect of the T194M mutation on phosphorylation. We also detected the phosphorylation of a small but consistent fraction of BvgA purified after expression in Escherichia coli.

  8. ATP-dependent regulation of actin monomer-filament equilibrium by cyclase-associated protein and ADF/cofilin. (United States)

    Nomura, Kazumi; Ono, Shoichiro


    CAP (cyclase-associated protein) is a conserved regulator of actin filament dynamics. In the nematode Caenorhabditis elegans, CAS-1 is an isoform of CAP that is expressed in striated muscle and regulates sarcomeric actin assembly. In the present study, we report that CAS-2, a second CAP isoform in C. elegans, attenuates the actin-monomer-sequestering effect of ADF (actin depolymerizing factor)/cofilin to increase the steady-state levels of actin filaments in an ATP-dependent manner. CAS-2 binds to actin monomers without a strong preference for either ATP- or ADP-actin. CAS-2 strongly enhances the exchange of actin-bound nucleotides even in the presence of UNC-60A, a C. elegans ADF/cofilin that inhibits nucleotide exchange. UNC-60A induces the depolymerization of actin filaments and sequesters actin monomers, whereas CAS-2 reverses the monomer-sequestering effect of UNC-60A in the presence of ATP, but not in the presence of only ADP or the absence of ATP or ADP. A 1:100 molar ratio of CAS-2 to UNC-60A is sufficient to increase actin filaments. CAS-2 has two independent actin-binding sites in its N- and C-terminal halves, and the C-terminal half is necessary and sufficient for the observed activities of the full-length CAS-2. These results suggest that CAS-2 (CAP) and UNC-60A (ADF/cofilin) are important in the ATP-dependent regulation of the actin monomer-filament equilibrium.

  9. Molecular Dynamics Simulation of Tau Peptides for the Investigation of Conformational Changes Induced by Specific Phosphorylation Patterns. (United States)

    Gandhi, Neha S; Kukic, Predrag; Lippens, Guy; Mancera, Ricardo L


    The Tau protein plays an important role due to its biomolecular interactions in neurodegenerative diseases. The lack of stable structure and various posttranslational modifications such as phosphorylation at various sites in the Tau protein pose a challenge for many experimental methods that are traditionally used to study protein folding and aggregation. Atomistic molecular dynamics (MD) simulations can help around deciphering relationship between phosphorylation and various intermediate and stable conformations of the Tau protein which occur on longer timescales. This chapter outlines protocols for the preparation, execution, and analysis of all-atom MD simulations of a 21-amino acid-long phosphorylated Tau peptide with the aim of generating biologically relevant structural and dynamic information. The simulations are done in explicit solvent and starting from nearly extended configurations of the peptide. The scaled MD method implemented in AMBER14 was chosen to achieve enhanced conformational sampling in addition to a conventional MD approach, thereby allowing the characterization of folding for such an intrinsically disordered peptide at 293 K. Emphasis is placed on the analysis of the simulation trajectories to establish correlations with NMR data (i.e., chemical shifts and NOEs). Finally, in-depth discussions are provided for commonly encountered problems.

  10. The distribution of cofilin and Dnase I in vivo

    Institute of Scientific and Technical Information of China (English)


    Actin is the principal component of the cytoskeleton, a structure that can be disassembled and reassem-bled in a matter of seconds in vivo. The state of assembly of actin in vivo is primarily regulated by one ormore actin binding proteins (ABPs). Typically, the actions of ABPs have been studied one by one, however,we propose that multiple ABPs, acting cooperatively, may be involved in the control of actin filament length.Cofilin and DNase I are two ABPs that have previously been demonstrated to form a ternary complex withactin in vitro. This is the first report to demonstrate their co-localisation in vivo, and differences in theirdistributions. Our observations strongly suggest a physiological role for higher order complexes of actin inregulation of cytoskeletal assembly during processes such as cell division.

  11. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development (United States)

    Loponte, Sara; Segré, Chiara V.; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A.; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna


    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  12. Jak3 enables chemokine-dependent actin cytoskeleton reorganization by regulating cofilin and Rac/Rhoa GTPases activation.

    Directory of Open Access Journals (Sweden)

    Xochitl Ambriz-Peña

    Full Text Available We have previously shown that Jak3 is involved in the signaling pathways of CCR7, CCR9 and CXCR4 in murine T lymphocytes and that Jak3⁻/⁻ lymphocytes display an intrinsic defect in homing to peripheral lymph nodes. However, the molecular mechanism underlying the defective migration observed in Jak3⁻/⁻ lymphocytes remains elusive. Here, it is demonstrated for the first time, that Jak3 is required for the actin cytoskeleton reorganization in T lymphocytes responding to chemokines. It was found that Jak3 regulates actin polymerization by controlling cofilin inactivation in response to CCL21 and CXCL12. Interestingly, cofilin inactivation was not precluded in PTX- treated cells despite their impaired actin polymerization. Additionally, Jak3 was required for small GTPases Rac1 and RhoA activation, which are indispensable for acquisition of the migratory cell phenotype and the generation of a functional leading edge and uropod, respectively. This defect correlates with data obtained by time-lapse video-microscopy showing an incompetent uropod formation and impaired motility in Jak3-pharmacologically inhibited T lymphocytes. Our data support a new model in which Jak3 and heterotrimeric G proteins can use independent, but complementary, signaling pathways to regulate actin cytoskeleton dynamics during cell migration in response to chemokines.

  13. Phagocytic receptors activate and immune inhibitory receptor SIRPalpha inhibits phagocytosis through paxillin and cofilin

    Directory of Open Access Journals (Sweden)

    Miri eGitik


    Full Text Available The innate-immune function of phagocytosis of apoptotic cells, tissue-debris, pathogens and cancer cells is essential for homeostasis, tissue repair, fighting infection and combating malignancy. Phagocytosis is carried out in the CNS by resident microglia and in both CNS and PNS by recruited macrophages. While phagocytosis proceeds, bystander healthy cells protect themselves by sending a do not eat me message to phagocytes as CD47 on their surface ligates immune inhibitory receptor SIRPα on the surface of phagocytes and SIRPα then produces the signaling which inhibits phagocytosis. This helpful mechanism becomes harmful when tissue-debris and unhealthy cells inhibit their own phagocytosis by employing the same mechanism. However, the inhibitory signaling that SIRPα produces has not been fully revealed. We focus here on how SIRPα inhibits the phagocytosis of the tissue-debris degenerated-myelin which hinders repair in axonal injury and neurodegenerative diseases. We tested whether SIRPα inhibits phagocytosis by regulating cytoskeleton function through paxillin and cofilin since (a the cytoskeleton generates the mechanical forces that drive phagocytosis and (b both paxillin and cofilin control cytoskeleton function. Paxillin and cofilin were transiently activated in microglia as phagocytosis was activated. In contrast, paxillin and cofilin were continuously activated and phagocytosis augmented in microglia in which SIRPα expression was knocked-down by SIRPα-shRNA. Further, levels of phagocytosis, paxillin activation and cofilin activation positively correlated with one another. Taken together, these observations suggest a novel mechanism whereby paxillin and cofilin are targeted to control phagocytosis by both the activating signaling that phagocytic receptors produce by promoting the activation of paxillin and cofilin and the inhibiting signaling that immune inhibitory SIRPα produces by promoting the inactivation of paxillin and cofilin.

  14. Phagocytic receptors activate and immune inhibitory receptor SIRPα inhibits phagocytosis through paxillin and cofilin. (United States)

    Gitik, Miri; Kleinhaus, Rachel; Hadas, Smadar; Reichert, Fanny; Rotshenker, Shlomo


    The innate immune function of phagocytosis of apoptotic cells, tissue debris, pathogens, and cancer cells is essential for homeostasis, tissue repair, fighting infection, and combating malignancy. Phagocytosis is carried out in the central nervous system (CNS) by resident microglia and in both CNS and peripheral nervous system by recruited macrophages. While phagocytosis proceeds, bystander healthy cells protect themselves by sending a "do not eat me" message to phagocytes as CD47 on their surface ligates immune inhibitory receptor SIRPα on the surface of phagocytes and SIRPα then produces the signaling which inhibits phagocytosis. This helpful mechanism becomes harmful when tissue debris and unhealthy cells inhibit their own phagocytosis by employing the same mechanism. However, the inhibitory signaling that SIRPα produces has not been fully revealed. We focus here on how SIRPα inhibits the phagocytosis of the tissue debris "degenerated myelin" which hinders repair in axonal injury and neurodegenerative diseases. We tested whether SIRPα inhibits phagocytosis by regulating cytoskeleton function through paxillin and cofilin since (a) the cytoskeleton generates the mechanical forces that drive phagocytosis and (b) both paxillin and cofilin control cytoskeleton function. Paxillin and cofilin were transiently activated in microglia as phagocytosis was activated. In contrast, paxillin and cofilin were continuously activated and phagocytosis augmented in microglia in which SIRPα expression was knocked-down by SIRPα-shRNA. Further, levels of phagocytosis, paxillin activation, and cofilin activation positively correlated with one another. Taken together, these observations suggest a novel mechanism whereby paxillin and cofilin are targeted to control phagocytosis by both the activating signaling that phagocytic receptors produce by promoting the activation of paxillin and cofilin and the inhibiting signaling that immune inhibitory SIRPα produces by promoting the

  15. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics. (United States)

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D


    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility.

  16. Cofilin/Twinstar Phosphorylation Levels Increase in Response to Impaired Coenzyme A Metabolism

    NARCIS (Netherlands)

    Siudeja, Katarzyna; Grzeschik, Nicola A.; Rana, Anil; de Jong, Jannie; Sibon, Ody C. M.


    Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neur

  17. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast. (United States)

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R; Drubin, David G


    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.

  18. Twinstar, the Drosophila homolog of cofilin/ADF, is required for planar cell polarity patterning. (United States)

    Blair, Adrienne; Tomlinson, Andrew; Pham, Hung; Gunsalus, Kristin C; Goldberg, Michael L; Laski, Frank A


    Planar cell polarity (PCP) is a level of tissue organization in which cells adopt a uniform orientation within the plane of an epithelium. The process of tissue polarization is likely to be initiated by an extracellular gradient. Thus, determining how cells decode and convert this graded information into subcellular asymmetries is key to determining how cells direct the reorganization of the cytoskeleton to produce uniformly oriented structures. Twinstar (Tsr), the Drosophila homolog of Cofilin/ADF (actin depolymerization factor), is a component of the cytoskeleton that regulates actin dynamics. We show here that various alleles of tsr produce PCP defects in the wing, eye and several other epithelia. In wings mutant for tsr, Frizzled (Fz) and Flamingo (Fmi) proteins do not properly localize to the proximodistal boundaries of cells. The correct asymmetric localization of these proteins instructs the actin cytoskeleton to produce one actin-rich wing hair at the distal-most vertex of each cell. These results argue that actin remodeling is not only required in the manufacture of wing hairs, but also in the PCP read-out that directs where a wing hair will be secreted.

  19. Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Xiao [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Whitten, Douglas A. [Research Technology Support Facility, Proteomics Core, Michigan State University, East Lansing, MI 48824 (United States); Wu, Ming [Department of Computer Science and Engineering, Michigan State University, East Lansing, MI 48824 (United States); Chan, Christina [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Department of Computer Science and Engineering, Michigan State University, East Lansing, MI 48824 (United States); Wilkerson, Curtis G. [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Research Technology Support Facility, Proteomics Core, Michigan State University, East Lansing, MI 48824 (United States); Pestka, James J., E-mail: [Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States); Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824 (United States)


    Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC phosphoproteomics using

  20. Dynamic distribution of Ser-10 phosphorylated histone H3 in cytoplasm of MCF-7 and CHO cells during mitosis

    Institute of Scientific and Technical Information of China (English)

    Deng Wen LI; Qin YANG; Jia Tong CHEN; Hao ZHOU; Ru Ming LIU; Xi Tai HUANG


    The dynamic distribution of phosphorylated Histone H3 on Ser10 (phospho-H3) in cells was investigated to determine its function during mitosis. Human breast adenocarcinoma cells MCF-7, and Chinese hamster cells CHO were analyzed by indirect immunofluorescence staining with an antibody against phospho-H3. We found that the phosphorylation begins at early prophase, and spreads throughout the chromosomes at late prophase. At metaphase, most of the phosphoH3 aggregates at the end of the condensed entity of chromosomes at equatorial plate. During anaphase and telophase,the fluorescent signal of phospho-H3 is detached from chromosomes into cytoplasm. At early anaphase, phospho-H3shows ladder bands between two sets of separated chromosome, and forms "sandwich-like structure" when the chromosomes condensed. With the cleavage progressing, the "ladders" of the histone contract into a bigger bright dot. Then the histone aggregates and some of compacted microtubules in the midbody region are composed into a "bar-like"complex to separate daughter cells. The daughter cells seal their plasma membrane along with the ends of the "bar",inside which locates microtubules and modified histones, to finish the cytokinesis and keep the "bar complex" out of the cells. The specific distribution and kinetics of phospho-H3 in cytoplasm suggest that the modified histones may take part in the formation of midbody and play a crucial role in cytokinesis.

  1. The differential DRP1 phosphorylation and mitochondrial dynamics in the regional specific astroglial death induced by status epilepticus

    Directory of Open Access Journals (Sweden)

    Ah-Reum eKo


    Full Text Available The response and susceptibility to astroglial degenerations are relevant to the distinctive properties of astrocytes in a hemodynamic-independent manner following status epilepticus (SE.Since impaired mitochondrial fission plays an important role in mitosis, apoptosis and programmed necrosis, we investigated whether the unique pattern of mitochondrial dynamics is involved in the characteristics of astroglial death induced by SE. In the present study, SE induced astroglial apoptosis in the molecular layer of the dentate gyrus, accompanied by decreased mitochondrial length. In contrast, clasmatodendritic (autophagic astrocytes in the CA1 region showed mitochondrial elongation induced by SE. Mdivi-1 (an inhibitor of mitochondrial fission effectively attenuated astroglial apoptosis, but WY14643 (an enhancer of mitochondrial fissionaggravated it. In addition, Mdivi-1accelerated clasmatodendritic changes in astrocytes. These regional specific mitochondrial dynamics in astrocytes were closely correlated with dynamin-related protein (DRP1, a mitochondrial fission protein phosphorylation, not optic atrophy 1 (a mitochondrial fusion protein expression. To the best of our knowledge, the present data demonstrate for the first time the novel role of DRP1-mediated mitochondrial fission in astroglial loss. Thus, the present findings suggest that the differential astroglial mitochondrial dynamics may participate in the distinct characteristics of astroglial death induced by SE.

  2. The mammalian circadian clock protein period counteracts cryptochrome in phosphorylation dynamics of circadian locomotor output cycles kaput (CLOCK). (United States)

    Matsumura, Ritsuko; Tsuchiya, Yoshiki; Tokuda, Isao; Matsuo, Takahiro; Sato, Miho; Node, Koichi; Nishida, Eisuke; Akashi, Makoto


    The circadian transcription factor CLOCK exhibits a circadian oscillation in its phosphorylation levels. Although it remains unclear whether this phosphorylation contributes to circadian rhythm generation, it has been suggested to be involved in transcriptional activity, intracellular localization, and degradative turnover of CLOCK. Here, we obtained direct evidence that CLOCK phosphorylation may be essential for autonomous circadian oscillation in clock gene expression. Importantly, we found that the circadian transcriptional repressors Cryptochrome (CRY) and Period (PER) showed an opposite effect on CLOCK phosphorylation; CRY impaired BMAL1-dependent CLOCK phosphorylation, whereas PER protected the phosphorylation against CRY. Interestingly, unlike PER1 and PER2, PER3 did not exert a protective action, which correlates with the phenotypic differences among mice lacking the Per genes. Further studies on the regulatory mechanism of CLOCK phosphorylation would thus lead to elucidation of the mechanism of CRY-mediated transcriptional repression and an understanding of the true role of PER in the negative feedback system.

  3. Chromosome segregation regulation in human zygotes: altered mitotic histone phosphorylation dynamics underlying centromeric targeting of the chromosomal passenger complex. (United States)

    van de Werken, C; Avo Santos, M; Laven, J S E; Eleveld, C; Fauser, B C J M; Lens, S M A; Baart, E B


    Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal phosphorylation dynamics of histone H2A at T120 (H2ApT120) by Bub1 kinase and subsequent recruitment of Shugoshin, but phosphorylation of histone H3 at threonine 3 (H3pT3) by Haspin failed to show the expected centromeric enrichment on metaphase chromosomes in the zygote. Human cleavage stage embryos show high levels of chromosomal instability. What causes this high error rate is unknown, as mechanisms used to ensure proper chromosome segregation in mammalian embryos are poorly described. In this study, we investigated the pathways regulating CPC targeting to the inner centromere in human embryos. We characterized the distribution of the CPC in relation to activity of its two main centromeric targeting pathways: the Bub1-H2ApT120-Sgo-CPC and Haspin-H3pT3-CPC pathways. The study was conducted between May 2012 and March 2014 on human surplus embryos resulting from in vitro fertilization treatment and donated for research. In zygotes, nuclear envelope breakdown was monitored by time-lapse imaging to allow timed incubations with specific inhibitors to arrest at prometaphase and metaphase, and to interfere with Haspin and Aurora B/C kinase activity. Functionality of the targeting pathways was assessed through characterization of histone phosphorylation dynamics by immunofluorescent analysis, combined with gene expression by RT-qPCR and immunofluorescent localization of key pathway proteins. Immunofluorescent analysis of the CPC subunit Inner Centromere Protein revealed the pool of stably bound CPC proteins was not strictly confined to the inner centromere of prometaphase chromosomes in human zygotes, as observed in later stages of preimplantation development and somatic cells. Investigation of the

  4. Dynamic fluorescence spectroscopy on single tryptophan mutants of EIImtl in detergent micelles : Effects of substrate binding and phosphorylation on the fluorescence and anisotropy decay

    NARCIS (Netherlands)

    Swaving Dijkstra, Dolf; Broos, J.; Visser, Antonie J.W.G.; van Hoek, A.; Robillard, George


    The effects of substrate and substrate analogue binding and phosphorylation on the conformational dynamics of the mannitol permease of Escherichia coli were investigated, using time-resolved fluorescence spectroscopy on mutants containing five single tryptophans situated in the membrane-embedded C d

  5. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin. (United States)

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q P; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q P


    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

  6. Organization of the Escherichia coli aerobic enzyme complexes of oxidative phosphorylation in dynamic domains within the cytoplasmic membrane. (United States)

    Erhardt, Heiko; Dempwolff, Felix; Pfreundschuh, Moritz; Riehle, Marc; Schäfer, Caspar; Pohl, Thomas; Graumann, Peter; Friedrich, Thorsten


    The Escherichia coli cytoplasmic membrane contains the enzyme complexes of oxidative phosphorylation (OXPHOS). Not much is known about their supramolecular organization and their dynamics within the membrane in this model organism. In mitochondria and other bacteria, it was demonstrated by nondenaturing electrophoretic methods and electron microscopy that the OXPHOS complexes are organized in so-called supercomplexes, stable assemblies with a defined number of the individual enzyme complexes. To investigate the organization of the E. coli enzyme complexes of aerobic OXPHOS in vivo, we established fluorescent protein fusions of the NADH:ubiquinone oxidoreductase, the succinate:ubiquinone oxidoreductase, the cytochrome bd-I, and the cytochrome bo3 terminal oxidases, and the FoF1 ATP-synthase. The fusions were integrated in the chromosome to prevent artifacts caused by protein overproduction. Biochemical analysis revealed that all modified complexes were fully assembled, active, and stable. The distribution of the OXPHOS complexes in living cells was determined using total internal reflection fluorescence microscopy. The dynamics within the membrane were detected by fluorescence recovery after photobleaching. All aerobic OXPHOS complexes showed an uneven distribution in large mobile patches within the E. coli cytoplasmic membrane. It is discussed whether the individual OXPHOS complexes are organized as clustered individual complexes, here called "segrazones."

  7. A novel molecular dynamics approach to evaluate the effect of phosphorylation on multimeric protein interface: the αB-Crystallin case study. (United States)

    Chiappori, Federica; Mattiazzi, Luca; Milanesi, Luciano; Merelli, Ivan


    Phosphorylation is one of the most important post-translational modifications (PTM) employed by cells to regulate several cellular processes. Studying the effects of phosphorylations on protein structures allows to investigate the modulation mechanisms of several proteins including chaperones, like the small HSPs, which display different multimeric structures according to the phosphorylation of a few serine residues. In this context, the proposed study is aimed at finding a method to correlate different PTM patterns (in particular phosphorylations at the monomers interface of multimeric complexes) with the dynamic behaviour of the complex, using physicochemical parameters derived from molecular dynamics simulations in the timescale of nanoseconds. We have developed a methodology relying on computing nine physicochemical parameters, derived from the analysis of short MD simulations, and combined with N identifiers that characterize the PTMs of the analysed protein. The nine general parameters were validated on three proteins, with known post-translational modified conformation and unmodified conformation. Then, we applied this approach to the case study of αB-Crystallin, a chaperone which multimeric state (up to 40 units) is supposed to be controlled by phosphorylation of Ser45 and Ser59. Phosphorylation of serines at the dimer interface induces the release of hexamers, the active state of αB-Crystallin. 30 ns of MD simulation were obtained for each possible combination of dimer phosphorylation state and average values of structural, dynamic, energetic and functional features were calculated on the equilibrated portion of the trajectories. Principal Component Analysis was applied to the parameters and the first five Principal Components, which summed up to 84 % of the total variance, were finally considered. The validation of this approach on multimeric proteins, which structures were known both modified and unmodified, allowed us to propose a new approach that

  8. Severe protein aggregate myopathy in a knockout mouse model points to an essential role of cofilin2 in sarcomeric actin exchange and muscle maintenance. (United States)

    Gurniak, Christine B; Chevessier, Frédéric; Jokwitz, Melanie; Jönsson, Friederike; Perlas, Emerald; Richter, Hendrik; Matern, Gabi; Boyl, Pietro Pilo; Chaponnier, Christine; Fürst, Dieter; Schröder, Rolf; Witke, Walter


    Mutations in the human actin depolymerizing factor cofilin2 result in an autosomal dominant form of nemaline myopathy. Here, we report on the targeted ablation of murine cofilin2, which leads to a severe skeletal muscle specific phenotype within the first two weeks after birth. Apart from skeletal muscle, cofilin2 is also expressed in heart and CNS, however the pathology was restricted to skeletal muscle. The two close family members of cofilin2 - ADF and cofilin1 - were co-expressed in muscle, but unable to compensate for the loss of cofilin2. While primary myofibril assembly and muscle development were unaffected in cofilin2 mutant mice, progressive muscle degeneration was observed between postnatal days 3 and 7. Muscle pathology was characterized by sarcoplasmic protein aggregates, fiber size disproportion, mitochondrial abnormalities and internal nuclei. The observed muscle pathology differed from nemaline myopathy, but showed combined features of actin-associated myopathy and myofibrillar myopathy. In cofilin2 mutant mice, the postnatal expression pattern and turnover of sarcomeric α-actin isoforms were altered. Levels of smooth muscle α-actin were increased and remained high in developing muscles, suggesting that cofilin2 plays a crucial role during the exchange of α-actin isoforms during the early postnatal remodeling of the sarcomere.

  9. Cofilin Regulates Nuclear Architecture through a Myosin-II Dependent Mechanotransduction Module (United States)

    Wiggan, O’Neil; Schroder, Bryce; Krapf, Diego; Bamburg, James R.; DeLuca, Jennifer G.


    Structural features of the nucleus including shape, size and deformability impact its function affecting normal cellular processes such as cell differentiation and pathological conditions such as tumor cell migration. Despite the fact that abnormal nuclear morphology has long been a defining characteristic for diseases such as cancer relatively little is known about the mechanisms that control normal nuclear architecture. Mounting evidence suggests close coupling between F-actin cytoskeletal organization and nuclear morphology however, mechanisms regulating this coupling are lacking. Here we identify that Cofilin/ADF-family F-actin remodeling proteins are essential for normal nuclear structure in different cell types. siRNA mediated silencing of Cofilin/ADF provokes striking nuclear defects including aberrant shapes, nuclear lamina disruption and reductions to peripheral heterochromatin. We provide evidence that these anomalies are primarily due to Rho kinase (ROCK) controlled excessive contractile myosin-II activity and not to elevated F-actin polymerization. Furthermore, we demonstrate a requirement for nuclear envelope LINC (linker of nucleoskeleton and cytoskeleton) complex proteins together with lamin A/C for nuclear aberrations induced by Cofilin/ADF loss. Our study elucidates a pivotal regulatory mechanism responsible for normal nuclear structure and which is expected to fundamentally influence nuclear function. PMID:28102353

  10. A dynamic view to the modulation of phosphorylation and O-GlcNAcylation by inhibition of O-GlcNAcase. (United States)

    Tang, Cuyue; Welty, Devin F


    Protein phosphorylation and O-GlcNAcylation are reciprocally regulated. As hyperphosphorylation is implicated in tau pathology, approaches have been exploited to reduce the magnitude of tau phosphorylation by increasing the level of tau O-GlcNAcylation. With mathematic models constructed to describe different kinetic scenarios, we analyzed the temporal change of an O-GlcNAcylated protein in contrast to that of the phosphorylated form upon inhibition of O-GlcNAcase (OGA). The analyses indicate that when degradation of the modified protein is negligible relative to the naked one, the magnitude of O-GlcNAcylated protein increase is proportional to the level of inhibition, while the extent of phosphorylated protein decline varies due to other factors. Furthermore, the increase of O-GlcNAcylated protein parallels with the decrease of phosphorylated form upon acute or short-term inhibition of OGA, as observed in many in vitro and short term in vivo studies. However, phosphorylated protein is predicted to return to its initial level while O-GlcNAcylated protein to achieve a higher steady level under sustained inhibition. This simulated result is in line with a recent report on long-term inhibition of OGA in transgenic mice. Noticeably, inhibition withdrawal is anticipated to cause a transient rise of phosphorylated protein. If degradation of modified proteins proceeds in addition to the naked one, the characteristic temporal profiles of each form in response to OGA inhibition would depend on the relative importance of individual degradation pathways. The models described herein may serve as a useful investigational tool that will provide insight into pharmacological intervention for tauopathies in particular and for reciprocally modulated reactions in general. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

    DEFF Research Database (Denmark)

    Mann, Matthias; Ong, Shao En; Grønborg, Mads


    In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling...

  12. The effects of threonine phosphorylation on the stability and dynamics of the central molecular switch region of 18.5-kDa myelin basic protein.

    Directory of Open Access Journals (Sweden)

    Kenrick A Vassall

    Full Text Available The classic isoforms of myelin basic protein (MBP are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence -T92-P93-R94-T95-P96-P97-P98-S99- containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90 upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72-S107 with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012. Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the

  13. The effects of threonine phosphorylation on the stability and dynamics of the central molecular switch region of 18.5-kDa myelin basic protein. (United States)

    Vassall, Kenrick A; Bessonov, Kyrylo; De Avila, Miguel; Polverini, Eugenia; Harauz, George


    The classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence -T92-P93-R94-T95-P96-P97-P98-S99-) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72-S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the global structure

  14. Monocular deprivation delays the dynamic changes of phosphorylated synapsin Ia/b at site-1 in contralateral visual cortex of juvenile mice. (United States)

    Fu, Tao; Su, Qing; Xi, Ping; Han, Song; Li, Junfa


    Synapsins as a family of presynaptic terminal phosphoprotein participates in neuronal development, but their role in the synaptic plasticity of visual cortex is unclear. In this study, the impact of monocular deprivation (MD) on dynamic changes of isoform-specific protein expression and site 1 phosphorylation of synapsins in visual cortex of the postnatal mice were observed by using the technique of Western blot analysis. The results showed that the total (T-) protein levels of synapsins including the isoform of Ia/b, IIa/b and IIIa were about 21-26% of adult level in visual cortex of mice at postnatal 7 days (P7), and then the T-synapsin Ia/b and IIb could quickly reach adult level at P35. However, the T-synapsin IIa and IIIa increased more slowly (71-74% at P35), and then kept increasing in the visual cortex of mice at P60. Unlike to the changes of T-synapsins, the level of phosphorylated (P-) synapsin Ia/b (not IIa/b and IIIa) at site 1 increased with development to the highest level at P21, and then decreased rapidly to a low level in visual cortex of mice at P35-60. In addition, we found that the levels of P-synapsin Ia/b increased significantly in left visual cortex of P28 and P35 (not P21 and P42) mice with 1-week MD of right eye; and no significant changes of T-synapsins were observed in both left and right sides of visual cortex in P21-42 mice with MD treatment. These results suggested that the isoform-specific protein expression and site-1 phosphorylation of synapsins might play a different role in the synaptic plasticity of visual cortex, and MD delays the dynamic changes of phosphorylated synapsin Ia/b at site-1 in contralateral visual cortex of juvenile mice.

  15. Directional and quantitative phosphorylation networks

    DEFF Research Database (Denmark)

    Jørgensen, Claus; Linding, Rune


    for unravelling phosphorylation-mediated cellular interaction networks. In particular, we will discuss how the combination of new quantitative mass-spectrometric technologies and computational algorithms together are enhancing mapping of these largely uncharted dynamic networks. By combining quantitative...

  16. The effect of Asp54 phosphorylation on the energetics and dynamics in the response regulator protein Spo0F studied by molecular dynamics

    DEFF Research Database (Denmark)

    Peters, Günther H.J.


    and recognition regions exhibit lower mobility relative to the apo-conformation. Phosphorylation of Asp54 (P-Asp54), in which the apostructure coordinates to the magnesium ion, results in extension of the sidechain, and depending on which carboxylate oxygen is phosphorylated, distinct interactions between P-Asp54...... and magnesium ion as well as residues in its proximity are established. However, phosphorylation does not affect the coordination number of the magnesium ion yielding, within the statistical uncertainties, the same free energy change....

  17. The NAV2 homolog Sickie regulates F-actin-mediated axonal growth in Drosophila mushroom body neurons via the non-canonical Rac-Cofilin pathway. (United States)

    Abe, Takashi; Yamazaki, Daisuke; Murakami, Satoshi; Hiroi, Makoto; Nitta, Yohei; Maeyama, Yuko; Tabata, Tetsuya


    The Rac-Cofilin pathway is essential for cytoskeletal remodeling to control axonal development. Rac signals through the canonical Rac-Pak-LIMK pathway to suppress Cofilin-dependent axonal growth and through a Pak-independent non-canonical pathway to promote outgrowth. Whether this non-canonical pathway converges to promote Cofilin-dependent F-actin reorganization in axonal growth remains elusive. We demonstrate that Sickie, a homolog of the human microtubule-associated protein neuron navigator 2, cell-autonomously regulates axonal growth of Drosophila mushroom body (MB) neurons via the non-canonical pathway. Sickie was prominently expressed in the newborn F-actin-rich axons of MB neurons. A sickie mutant exhibited axonal growth defects, and its phenotypes were rescued by exogenous expression of Sickie. We observed phenotypic similarities and genetic interactions among sickie and Rac-Cofilin signaling components. Using the MARCM technique, distinct F-actin and phospho-Cofilin patterns were detected in developing axons mutant for sickie and Rac-Cofilin signaling regulators. The upregulation of Cofilin function alleviated the axonal defect of the sickie mutant. Epistasis analyses revealed that Sickie suppresses the LIMK overexpression phenotype and is required for Pak-independent Rac1 and Slingshot phosphatase to counteract LIMK. We propose that Sickie regulates F-actin-mediated axonal growth via the non-canonical Rac-Cofilin pathway in a Slingshot-dependent manner.

  18. Human CAP1 is a key factor in the recycling of cofilin and actin for rapid actin turnover. (United States)

    Moriyama, Kenji; Yahara, Ichiro


    Cofilin-ADF (actin-depolymerizing factor) is an essential driver of actin-based motility. We discovered two proteins, p65 and p55, that are components of the actin-cofilin complex in a human HEK293 cell extract and identified p55 as CAP1/ASP56, a human homologue of yeast CAP/SRV2 (cyclase-associated protein). CAP is a bifunctional protein with an N-terminal domain that binds to Ras-responsive adenylyl cyclase and a C-terminal domain that inhibits actin polymerization. Surprisingly, we found that the N-terminal domain of CAP1, but not the C-terminal domain, is responsible for the interaction with the actin-cofilin complex. The N-terminal domain of CAP1 was also found to accelerate the depolymerization of F-actin at the pointed end, which was further enhanced in the presence of cofilin and/or the C-terminal domain of CAP1. Moreover, CAP1 and its C-terminal domain were observed to facilitate filament elongation at the barbed end and to stimulate ADP-ATP exchange on G-actin, a process that regenerates easily polymerizable G-actin. Although cofilin inhibited the nucleotide exchange on G-actin even in the presence of the C-terminal domain of CAP1, its N-terminal domain relieved this inhibition. Thus, CAP1 plays a key role in speeding up the turnover of actin filaments by effectively recycling cofilin and actin and through its effect on both ends of actin filament.

  19. Dynamics of yeast histone H2A and H2B phosphorylation in response to a double-strand break. (United States)

    Lee, Cheng-Sheng; Lee, Kihoon; Legube, Gaëlle; Haber, James E


    In budding yeast, a single double-strand break (DSB) triggers extensive Tel1 (ATM)- and Mec1 (ATR)-dependent phosphorylation of histone H2A around the DSB, to form γ-H2AX. We describe Mec1- and Tel1-dependent phosphorylation of histone H2B at T129. γ-H2B formation is impaired by γ-H2AX and its binding partner Rad9. High-density microarray analyses show similar γ-H2AX and γ-H2B distributions, but γ-H2B is absent near telomeres. Both γ-H2AX and γ-H2B are strongly diminished over highly transcribed regions. When transcription of GAL7, GAL10 and GAL1 genes is turned off, γ-H2AX is restored within 5 min, in a Mec1-dependent manner; after reinduction of these genes, γ-H2AX is rapidly lost. Moreover, when a DSB is induced near CEN2, γ-H2AX spreads to all other pericentromeric regions, again depending on Mec1. Our data provide new insights in the function and establishment of phosphorylation events occurring on chromatin after DSB induction.

  20. Dynamic fluorescence spectroscopy on single tryptophan mutants of EII(mtl) in detergent micelles. Effects of substrate binding and phosphorylation on the fluorescence and anisotropy decay. (United States)

    Dijkstra, D S; Broos, J; Visser, A J; van Hoek, A; Robillard, G T


    The effects of substrate and substrate analogue binding and phosphorylation on the conformational dynamics of the mannitol permease of Escherichia coli were investigated, using time-resolved fluorescence spectroscopy on mutants containing five single tryptophans situated in the membrane-embedded C domain of the enzyme [Swaving Dijkstra et al. (1996) Biochemistry 35, 6628-6634]. Since no fluorescent impurities are present in these mutants, the changes in fluorescence and anisotropy could be related with changes in the tryptophan microenvironment. Tryptophans at positions 30 and 42 showed changes in fluorescence intensity decay upon binding mannitol, which were reflected in the changes in lifetime distribution patterns. The disappearance of the shortest-lived decay component in these mutants, as well as in the mutant with a single tryptophan at position 109, indicates a change in the local environment such that quenching via neighboring side chains or solvent is reduced. Phosphorylation at histidine 554 and cysteine 384, located in the cytoplasmatic A and B domains of EII(mtl), respectively, induced an increase in the average fluorescence lifetimes of all of the tryptophans. The effect was most pronounced for tryptophans 30 and 109 which show large increases in the average fluorescence lifetime mainly due to loss of short-lived decay components. A correlation time distribution of the individual tryptophans deduced from an analysis of the anisotropy decay showed that they differed in their rotational mobility with tryptophan 30 showing the least local flexibility. Phosphorylation resulted in immobilization of W109 which, together with changes in the average fluorescence lifetime, is evidence for a conformational coupling between the phosphorylated B domain and the C domain. The influence of mannitol binding on the rotational behavior of the tryptophans is limited; it induces more internal flexibility at all tryptophan positions. A rotational correlation time of 30 ns

  1. High-resolution structure of the phosphorylated form of the histidine-containing phosphocarrier protein HPR from Escherichia coli determined by restrained molecular dynamics from NMR-NOE data

    NARCIS (Netherlands)

    van Nuland, Nicolaas; Boelens, R; Scheek, R.M.; Robillard, G.T.


    The solution structure of the phosphorylated form of the histidine-containing phosphocarrier protein, HPr, from Escherichia coli has been determined by NMR in combination with restrained molecular dynamics simulations. The structure of phospho-HPr (P-HPr) results from a molecular dynamics simulation

  2. Phosphorylation statuses at different residues of lamin B2, B1, and A/C dynamically and independently change throughout the cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Kuga, Takahisa, E-mail: [Laboratory of Proteome Research, National Institute of Biomedical Innovation, Ibaraki, Osaka 567-0085 (Japan); Department of Molecular Diagnosis (F8), Graduate School of Medicine, Chiba University, Chuo-ku, Chiba 260-8670 (Japan); Nozaki, Naohito [Department of Biochemistry and Molecular Biology, Kanagawa Dental College, Yokosuka, Kanagawa 238-8580 (Japan); Matsushita, Kazuyuki; Nomura, Fumio [Department of Molecular Diagnosis (F8), Graduate School of Medicine, Chiba University, Chuo-ku, Chiba 260-8670 (Japan); Tomonaga, Takeshi, E-mail: [Laboratory of Proteome Research, National Institute of Biomedical Innovation, Ibaraki, Osaka 567-0085 (Japan); Department of Molecular Diagnosis (F8), Graduate School of Medicine, Chiba University, Chuo-ku, Chiba 260-8670 (Japan)


    Lamins, major components of the nuclear lamina, undergo phosphorylation at multiple residues during cell cycle progression, but their detailed phosphorylation kinetics remain largely undetermined. Here, we examined changes in the phosphorylation of major phosphorylation residues (Thr14, Ser17, Ser385, Ser387, and Ser401) of lamin B2 and the homologous residues of lamin B1, A/C during the cell cycle using novel antibodies to the site-specific phosphorylation. The phosphorylation levels of these residues independently changed during the cell cycle. Thr14 and Ser17 were phosphorylated during G{sub 2}/M phase to anaphase/telophase. Ser385 was persistently phosphorylated during mitosis to G{sub 1} phase, whereas Ser387 was phosphorylated discontinuously in prophase and G{sub 1} phase. Ser401 phosphorylation was enhanced in the G{sub 1}/S boundary. Immunoprecipitation using the phospho-antibodies suggested that metaphase-phosphorylation at Thr14, Ser17, and Ser385 of lamins occurred simultaneously, whereas G{sub 1}-phase phosphorylation at Ser385 and Ser387 occurred in distinct pools or with different timings. Additionally, we showed that lamin B2 phosphorylated at Ser17, but not Ser385, Ser387 and Ser401, was exclusively non-ionic detergent soluble, depolymerized forms in growing cells, implicating specific involvement of Ser17 phosphorylation in lamin depolymerization and nuclear envelope breakdown. These results suggest that the phosphorylations at different residues of lamins might play specific roles throughout the cell cycle.

  3. Entamoeba invadens: identification of ADF/cofilin and their expression analysis in relation to encystation and excystation. (United States)

    Makioka, Asao; Kumagai, Masahiro; Hiranuka, Kazushi; Kobayashi, Seiki; Takeuchi, Tsutomu


    The differentiation processes of excystation and encystation of Entamoeba are essential for infection and completion of their life-cycle, and the processes need cell motility and its control by actin cytoskeletal reorganization. This study investigated actin depolymerizing factor (ADF)/cofilin (Cfl) family proteins, which are important molecules in actin cytoskeletal reorganization, in Entamoeba invadens in relation to the encystation and excystation. Axenic culture systems were used to induce encystation and excystation. A homology search of the E. invadens genome database and molecular cloning identified three ADF/Cfl family proteins of the parasite (named for short as EiCfl-1, EiCfl-2, and EiCfl-3). This is different from other Entamoeba species, i.e. Entamoeba histolytica and Entamoeba dispar, each of which has only one ADF/Cfl family protein. These ADF/Cfl of E. invadens do not have Ser3 (serine locates third from first methionine), similar to E. histolytica, E. dispar, Saccharomyces cerevisiae and Schizosaccharomyces pombe, although the activity of ADF/Cfl is negatively regulated by phosphorylation of the Ser3 in metazoans. Phylogenetic analysis revealed that Entamoeba Cfl formed a distinctive clade that is separate from other organisms, and the branches of the tree were separated in two consistent with the presence and absence of Ser3. Rabbit anti-EiCfl-2 serum reacted with all recombinant EiCfls and EiCfl in lysates of cysts, trophozoites and metacystic amoebae. Immunofluorescence staining with this antiserum showed co-localization of EiCfl with actin beneath the cell membrane through the life stages. Both proteins proved to be rich in pseudopodia of trophozoites and metacystic amoebae. Real-time RT-PCR showed that mRNAs of EiCfl-2 and actins were highly expressed, but there were few mRNA of EiCfl-1 and EiCfl-3. Remarkably decreased mRNA levels were observed in EiCfl-2 and actins during encystation. All three EiCfls and actins became transcribed after the

  4. Nucleolus disassembly in mitosis and apoptosis: dynamic redistribution of phosphorylated-c-Myc, fibrillarin and Ki-67

    Directory of Open Access Journals (Sweden)

    C Soldani


    Full Text Available The nucleolus may undergo disassembly either reversibly during mitosis, or irreversibly in apoptosis, thus allowing the redistribution of the nucleolar proteins.We investigated here by immunocytochemistry the fate of three representative proteins, namely phosphorylated c-Myc, fibrillarin and Ki-67, and found that they behave independently in both processes: they relocate in distinct compartments during mitosis, whereas during apoptosis they may either be cleaved (Ki-67 or be extruded into the cytoplasm with a different kinetics and following an ordered, non chaotic program. The separation of these nucleolar proteins which occurs in early apoptotic nuclei continues also in the cytoplasm, and culminates in the final formation of apoptotic blebs containing different nucleolar proteins: this evidence confirms that the apoptotic bodies may be variable in size, content and surface reactivity, and include heterogeneous aggregates of nuclear proteins and/or nucleic acids.

  5. Sialidase NEU3 dynamically associates to different membrane domains specifically modifying their ganglioside pattern and triggering Akt phosphorylation.

    Directory of Open Access Journals (Sweden)

    Dario Bonardi

    Full Text Available Lipid rafts are known to regulate several membrane functions such as signaling, trafficking and cellular adhesion. The local enrichment in sphingolipids and cholesterol together with the low protein content allows their separation by density gradient flotation after extraction with non-ionic detergent at low temperature. These structures are also referred to as detergent resistant membranes (DRM. Among sphingolipids, gangliosides play important roles in different biological events, including signal transduction and tumorigenesis. Sialidase NEU3 shows high enzymatic specificity toward gangliosides. Moreover, the enzyme is present both at the cell surface and in endosomal structures and cofractionates with caveolin. Although changes in the expression level of NEU3 have been correlated to different tumors, little is known about the precise distribution of the protein and its ability in modifying the ganglioside composition of DRM and non-DRM, thus regulating intracellular events. By means of inducible expression cell system we found that i newly synthesized NEU3 is initially associated to non-DRM; ii at steady state the protein is equally distributed between the two membrane subcompartments, i.e., DRM and non-DRM; iii NEU3 is degraded via the proteasomal pathway; iv the enzyme specifically modifies the ganglioside composition of the membrane areas where it resides; and v NEU3 triggers phosphorylation of Akt, even in absence of exogenously administered EGF. Taken together our data demonstrate that NEU3 regulates the DRM ganglioside content and it can be considered as a modulator of Akt phosphorylation, further supporting the role of this enzyme in cancer and tumorigenesis.

  6. Cofilin and Vangl2 cooperate in the initiation of planar cell polarity in the mouse embryo. (United States)

    Mahaffey, James P; Grego-Bessa, Joaquim; Liem, Karel F; Anderson, Kathryn V


    The planar cell polarity (PCP; non-canonical Wnt) pathway is required to orient the cells within the plane of an epithelium. Here, we show that cofilin 1 (Cfl1), an actin-severing protein, and Vangl2, a core PCP protein, cooperate to control PCP in the early mouse embryo. Two aspects of planar polarity can be analyzed quantitatively at cellular resolution in the mouse embryo: convergent extension of the axial midline; and posterior positioning of cilia on cells of the node. Analysis of the spatial distribution of brachyury(+) midline cells shows that the Cfl1 mutant midline is normal, whereas Vangl2 mutants have a slightly wider midline. By contrast, midline convergent extension fails completely in Vangl2 Cfl1 double mutants. Planar polarity is required for the posterior positioning of cilia on cells in the mouse node, which is essential for the initiation of left-right asymmetry. Node cilia are correctly positioned in Cfl1 and Vangl2 single mutants, but cilia remain in the center of the cell in Vangl2 Cfl1 double mutants, leading to randomization of left-right asymmetry. In both the midline and node, the defect in planar polarity in the double mutants arises because PCP protein complexes fail to traffic to the apical cell membrane, although other aspects of apical-basal polarity are unaffected. Genetic and pharmacological experiments demonstrate that F-actin remodeling is essential for the initiation, but not maintenance, of PCP. We propose that Vangl2 and cofilin cooperate to target Rab11(+) vesicles containing PCP proteins to the apical membrane during the initiation of planar cell polarity.

  7. Role of cyclic nucleotide-dependent actin cytoskeletal dynamics:Ca(2+](i and force suppression in forskolin-pretreated porcine coronary arteries.

    Directory of Open Access Journals (Sweden)

    Kyle M Hocking

    Full Text Available Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca(2+]i and phosphorylation of myosin light chains (MLC. However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca(2+]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.

  8. Aerobic exercise regulates Rho/cofilin pathways to rescue synaptic loss in aged rats (United States)

    Li, Yan; Zhao, Li; Gu, Boya; Cai, Jiajia; Lv, Yuanyuan; Yu, Laikang


    Purpose The role of exercise to prevent or reverse aging-induced cognitive decline has been widely reported. This neuroprotection is associated with changes in the synaptic structure plasticity. However, the mechanisms of exercise-induced synaptic plasticity in the aging brain are still unclear. Thus, the aim of the present study is to investigate the aging-related alterations of Rho-GTPase and the modulatory influences of exercise training. Methods Young and old rats were used in this study. Old rats were subjected to different schedules of aerobic exercise (12 m/min, 60 min/d, 3d/w or 5d/w) or kept sedentary for 12 w. After 12 w of aerobic exercise, the synapse density in the cortex and hippocampus was detected with immunofluorescent staining using synaptophysin as a marker. The total protein levels of RhoA, Rac1, Cdc42 and cofilin in the cortex and hippocampus were detected with Western Blot. The activities of RhoA, Rac1 and Cdc42 were determined using a pull down assay. Results We found that synapse loss occurred in aging rats. However, the change of expression and activity of RhoA, Rac1 and Cdc42 was different in the cortex and hippocampus. In the cortex, the expression and activity of Rac1 and Cdc42 was greatly increased with aging, whereas there were no changes in the expression and activity of RhoA. In the hippocampus, the expression and activity of Rac1 and Cdc42 was greatly decreased and there were no changes in the expression and activity of RhoA. As a major downstream substrate of the Rho GTPase family, the increased expression of cofilin was only observed in the cortex. High frequency exercise ameliorated all aging-related changes in the cortex and hippocampus. Conclusions These data suggest that aerobic exercise reverses synapse loss in the cortex and hippocampus in aging rats, which might be related to the regulation of Rho GTPases. PMID:28152068

  9. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay, E-mail:


    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

  10. Proteomic Approaches Identify Members of Cofilin Pathway Involved in Oral Tumorigenesis (United States)

    Polachini, Giovana M.; Sobral, Lays M.; Mercante, Ana M. C.; Paes-Leme, Adriana F.; Xavier, Flávia C. A.; Henrique, Tiago; Guimarães, Douglas M.; Vidotto, Alessandra; Fukuyama, Erica E.; Góis-Filho, José F.; Cury, Patricia M.; Curioni, Otávio A.; Michaluart Jr, Pedro; Silva, Adriana M. A.; Wünsch-Filho, Victor; Nunes, Fabio D.; Leopoldino, Andréia M.; Tajara, Eloiza H.


    The prediction of tumor behavior for patients with oral carcinomas remains a challenge for clinicians. The presence of lymph node metastasis is the most important prognostic factor but it is limited in predicting local relapse or survival. This highlights the need for identifying biomarkers that may effectively contribute to prediction of recurrence and tumor spread. In this study, we used one- and two-dimensional gel electrophoresis, mass spectrometry and immunodetection methods to analyze protein expression in oral squamous cell carcinomas. Using a refinement for classifying oral carcinomas in regard to prognosis, we analyzed small but lymph node metastasis-positive versus large, lymph node metastasis-negative tumors in order to contribute to the molecular characterization of subgroups with risk of dissemination. Specific protein patterns favoring metastasis were observed in the “more-aggressive” group defined by the present study. This group displayed upregulation of proteins involved in migration, adhesion, angiogenesis, cell cycle regulation, anti-apoptosis and epithelial to mesenchymal transition, whereas the “less-aggressive” group was engaged in keratinocyte differentiation, epidermis development, inflammation and immune response. Besides the identification of several proteins not yet described as deregulated in oral carcinomas, the present study demonstrated for the first time the role of cofilin-1 in modulating cell invasion in oral carcinomas. PMID:23227181

  11. Protein kinase A (PKA) phosphorylation of Na+/K+-ATPase opens intracellular C-terminal water pathway leading to third Na+-binding site in molecular dynamics simulations

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Nissen, Poul; Mouritsen, Ole G.;


    Phosphorylation is one of the major mechanisms for posttranscriptional modification of proteins. The addition of a compact, negatively charged moiety to a protein can significantly change its function and localization by affecting its structure and interaction network. We have used all-atom Molec...... the effects of S936 phosphorylation. The results establish a structural association of S936 with the C-terminus of NKA and indicate that phosphorylation of S936 can modulate pumping activity by changing the accessibility to the ion-binding site....

  12. Linear motif atlas for phosphorylation-dependent signaling

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Jensen, LJ; Diella, F;


    Systematic and quantitative analysis of protein phosphorylation is revealing dynamic regulatory networks underlying cellular responses to environmental cues. However, matching these sites to the kinases that phosphorylate them and the phosphorylation-dependent binding domains that may subsequently...... sequence models of linear motifs. The atlas is available as a community resource (

  13. Reconstitution and dissection of the 600-kDa Srv2/CAP complex: roles for oligomerization and cofilin-actin binding in driving actin turnover. (United States)

    Quintero-Monzon, Omar; Jonasson, Erin M; Bertling, Enni; Talarico, Lou; Chaudhry, Faisal; Sihvo, Maarit; Lappalainen, Pekka; Goode, Bruce L


    Srv2/cyclase-associated protein is expressed in virtually all plant, animal, and fungal organisms and has a conserved role in promoting actin depolymerizing factor/cofilin-mediated actin turnover. This is achieved by the abilities of Srv2 to recycle cofilin from ADP-actin monomers and to promote nucleotide exchange (ATP for ADP) on actin monomers. Despite this important and universal role in facilitating actin turnover, the mechanism underlying Srv2 function has remained elusive. Previous studies have demonstrated a critical functional role for the G-actin-binding C-terminal half of Srv2. Here we describe an equally important role in vivo for the N-terminal half of Srv2 in driving actin turnover. We pinpoint this activity to a conserved patch of surface residues on the N-terminal dimeric helical folded domain of Srv2, and we show that this functional site interacts with cofilin-actin complexes. Furthermore, we show that this site is essential for Srv2 acceleration of cofilin-mediated actin turnover in vitro. A cognate Srv2-binding site is identified on a conserved surface of cofilin, suggesting that this function likely extends to other organisms. In addition, our analyses reveal that higher order oligomerization of Srv2 depends on its N-terminal predicted coiled coil domain and that oligomerization optimizes Srv2 function in vitro and in vivo. Based on these data, we present a revised model for the mechanism by which Srv2 promotes actin turnover, in which coordinated activities of its N- and C-terminal halves catalyze sequential steps in recycling cofilin and actin monomers.

  14. Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin

    DEFF Research Database (Denmark)

    Larsen, Anna K; Lametsch, René; Elce, John S


    blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild-type phenotype in knockout cells. Proteomic comparison of wild-type and knockout cells identified decreased levels of RhoGDI-1 (Rho GDP-dissociation inhibitor) and cofilin 1, and increased levels...

  15. Reseach progress on cofilin and pulmonary disease%丝切蛋白与肺部疾病的研究进展

    Institute of Scientific and Technical Information of China (English)

    包志瑶; 周新; 韩黎


    Cofilin is an actin-binding protein,which is ubiquitous in eukaryotic cells.It prevents F actin assembly by both depolymerizing F-actin and inhibiting the polymerization of monomeric G-actin,and results in changes of cytoskeletal.Thus cofilin is involved in many different cellular processes that essential for cell proliferation,motility,adhesion and apoptosis.In recent years,there are more and more researches on cofilin and tumor metastasis,reproductive system diseases,nervous system disorders,however,fewer researches on pulmonary diseases.In this article,we focus on the progress on cofilin and pulmonary disease.%丝切蛋白(cofilin)是普遍存在于真核细胞中的一种肌动蛋白结合蛋白,它可促进细胞骨架主要成分肌动蛋白由丝状肌动蛋白解离为单体肌动蛋白,并抑制单体肌动蛋白的聚合,最终引起细胞骨架发生改变,从而影响细胞增殖、迁移、黏附及凋亡等重要生理功能.近年来cofilin作为靶蛋白已逐渐成为研究肿瘤转移、生殖系统疾病、神经系统疾病等的热点,但在呼吸系统疾病方面的研究还相对较少.文章将系统的对cofilin与呼吸系统疾病的相关性作一综述.

  16. Dynamic quantification of intracellular calcium and protein tyrosine phosphorylation in cryopreserved boar spermatozoa during short-time incubation with oviductal fluid. (United States)

    Kumaresan, A; González, R; Johannisson, A; Berqvist, A-S


    Freshly ejaculated boar spermatozoa require several hours of exposure to capacitating conditions to undergo capacitation. We hypothesized that cryopreserved boar spermatozoa might elicit a capacitation response after a relatively shorter time of exposure to capacitating conditions. Washed, frozen-thawed boar spermatozoa were incubated separately with pre-ovulatory isthmic oviductal fluid (EODF), post-ovulatory ODF (MODF), capacitation medium (CM), and noncapacitating medium (NCM) for 60 minutes. Aliquots of spermatozoa were taken at 0, 5, 15, 30, and 60 minutes during incubation and sperm kinematics, intracellular calcium [Ca2(+)]i content, and protein tyrosine phosphorylation (PTP) were studied. The proportion of motile spermatozoa increased significantly after 5 minutes of incubation with EODF. A similar increase was not observed in the other groups. During the initial 5 minutes of incubation, the proportion of spermatozoa with high [Ca(2+)]i decreased significantly in all four groups. The proportion of tyrosine phosphorylated spermatozoa increased from 6.49 ± 1.93% to 15.42 ± 3.58% and 18.41 ± 1.57% in EODF and MODF groups, respectively, at 5 minutes of incubation. Neither CM nor NCM elicited any immediate effect on PTP in spermatozoa. There was a positive and significant correlation between [Ca(2+)]i and sperm motility (P = 0.009). It may be concluded that frozen-thawed boar spermatozoa undergo capacitation-associated changes after a relatively short exposure to EODF, and there are some subpopulations of spermatozoa that undergo PTP despite possessing low [Ca(2+)]i.

  17. Compartment-Specific Phosphorylation of Squid Neurofilaments. (United States)

    Grant, Philip; Pant, Harish C


    Studies of the giant axon and synapse of third-order neurons in the squid stellate ganglion have provided a vast literature on neuronal physiology and axon transport. Large neuronal size also lends itself to comparative biochemical studies of cell body versus axon. These have focused on the regulation of synthesis, assembly, posttranslational modification and function of neuronal cytoskeletal proteins (microtubules (MTs) and neurofilaments (NFs)), the predominant proteins in axoplasm. These contribute to axonal organization, stability, transport, and impulse transmission responsible for rapid contractions of mantle muscles underlying jet propulsion. Studies of vertebrate NFs have established an extensive literature on NF structure, organization, and function; studies of squid NFs, however, have made it possible to compare compartment-specific regulation of NF synthesis, assembly, and function in soma versus axoplasm. Since NFs contain over 100 eligible sites for phosphorylation by protein kinases, the compartment-specific patterns of phosphorylation have been a primary focus of biochemical studies. We have learned that NF phosphorylation is tightly compartmentalized; extensive phosphorylation occurs only in the axonal compartment in squid and in vertebrate neurons. This extensive phosphorylation plays a key role in organizing NFs, in association with microtubules (MTs), into a stable, dynamic functional lattice that supports axon growth, diameter, impulse transmission, and synaptic activity. To understand how cytoskeletal phosphorylation is topographically regulated, the kinases and phosphatases, bound to NFs isolated from cell bodies and axoplasm, have also been studied.

  18. Structure and mechanism of mouse cyclase-associated protein (CAP1) in regulating actin dynamics. (United States)

    Jansen, Silvia; Collins, Agnieszka; Golden, Leslie; Sokolova, Olga; Goode, Bruce L


    Srv2/CAP is a conserved actin-binding protein with important roles in driving cellular actin dynamics in diverse animal, fungal, and plant species. However, there have been conflicting reports about whether the activities of Srv2/CAP are conserved, particularly between yeast and mammalian homologs. Yeast Srv2 has two distinct functions in actin turnover: its hexameric N-terminal-half enhances cofilin-mediated severing of filaments, while its C-terminal-half catalyzes dissociation of cofilin from ADP-actin monomers and stimulates nucleotide exchange. Here, we dissected the structure and function of mouse CAP1 to better understand its mechanistic relationship to yeast Srv2. Although CAP1 has a shorter N-terminal oligomerization sequence compared with Srv2, we find that the N-terminal-half of CAP1 (N-CAP1) forms hexameric structures with six protrusions, similar to N-Srv2. Further, N-CAP1 autonomously binds to F-actin and decorates the sides and ends of filaments, altering F-actin structure and enhancing cofilin-mediated severing. These activities depend on conserved surface residues on the helical-folded domain. Moreover, N-CAP1 enhances yeast cofilin-mediated severing, and conversely, yeast N-Srv2 enhances human cofilin-mediated severing, highlighting the mechanistic conservation between yeast and mammals. Further, we demonstrate that the C-terminal actin-binding β-sheet domain of CAP1 is sufficient to catalyze nucleotide-exchange of ADP-actin monomers, while in the presence of cofilin this activity additionally requires the WH2 domain. Thus, the structures, activities, and mechanisms of mouse and yeast Srv2/CAP homologs are remarkably well conserved, suggesting that the same activities and mechanisms underlie many of the diverse actin-based functions ascribed to Srv2/CAP homologs in different organisms.

  19. Conformational choreography of a molecular switch region in myelin basic protein--molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments. (United States)

    Polverini, Eugenia; Coll, Eoin P; Tieleman, D Peter; Harauz, George


    The 18.5 kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5 kDa myelin basic protein (residues Glu80-Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80-Val91 and extended poly-proline type II from Thr92-Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the

  20. Protein phosphorylation and photorespiration. (United States)

    Hodges, M; Jossier, M; Boex-Fontvieille, E; Tcherkez, G


    Photorespiration allows the recycling of carbon atoms of 2-phosphoglycolate produced by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenase activity, as well as the removal of potentially toxic metabolites. The photorespiratory pathway takes place in the light, encompasses four cellular compartments and interacts with several other metabolic pathways and functions. Therefore, the regulation of this cycle is probably of paramount importance to plant metabolism, however, our current knowledge is poor. To rapidly respond to changing conditions, proteins undergo a number of different post-translational modifications that include acetylation, methylation and ubiquitylation, but protein phosphorylation is probably the most common. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function, such as activity, subcellular localisation, capacity to interact with other proteins and stability. Recent data indicate that many photorespiratory enzymes can be phosphorylated, and thus it seems that the photorespiratory cycle is, in part, regulated by protein phosphorylation. In this review, the known phosphorylation sites of each Arabidopsis thaliana photorespiratory enzyme and several photorespiratory-associated proteins are described and discussed. A brief account of phosphoproteomic protocols is also given since the published data compiled in this review are the fruit of this approach.

  1. Dynamic changes in dopamine neuron function after DNSP-11 treatment: effects in vivo and increased ERK 1/2 phosphorylation in vitro. (United States)

    Fuqua, Joshua L; Littrell, Ofelia M; Lundblad, Martin; Turchan-Cholewo, Jadwiga; Abdelmoti, Lina G; Galperin, Emilia; Bradley, Luke H; Cass, Wayne A; Gash, Don M; Gerhardt, Greg A


    Glial cell-line derived neurotrophic factor (GDNF) has demonstrated robust effects on dopamine (DA) neuron function and survival. A post-translational processing model of the human GDNF proprotein theorizes the formation of smaller, amidated peptide(s) from the proregion that exhibit neurobiological function, including an 11-amino-acid peptide named dopamine neuron stimulating peptide-11 (DNSP-11). A single treatment of DNSP-11 was delivered to the substantia nigra in the rat to investigate effects on DA-neuron function. Four weeks after treatment, potassium (K+) and D-amphetamine evoked DA release were studied in the striatum using microdialysis. There were no significant changes in DA-release after DNSP-11 treatment determined by microdialysis. Dopamine release was further examined in discrete regions of the striatum using high-speed chronoamperometry at 1-, 2-, and 4-weeks after DNSP-11 treatment. Two weeks after DNSP-11 treatment, potassium-evoked DA release was increased in specific subregions of the striatum. However, spontaneous locomotor activity was unchanged by DNSP-11 treatment. In addition, we show that a single treatment of DNSP-11 in the MN9D dopaminergic neuronal cell line results in phosphorylation of ERK1/2, which suggests a novel cellular mechanism responsible for increases in DA function. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Roles of “junk phosphorylation” in modulating biomolecular association of phosphorylated proteins?

    DEFF Research Database (Denmark)

    Tan, Chris Soon Heng; Jørgensen, Claus; Linding, Rune


    Protein phosphorylation dynamically regulates cellular activities in response to environmental cues. Sequence conservation analysis of recent proteome-wide phosphorylation data revealed that many previously unidentified phosphorylation sites are not well-conserved leading to the proposal that man...... evolutionary approaches to interpret physiological important sites....

  3. Neurofilament Phosphorylation during Development and Disease: Which Came First, the Phosphorylation or the Accumulation?

    Directory of Open Access Journals (Sweden)

    Jeffrey M. Dale


    Full Text Available Posttranslational modification of proteins is a ubiquitous cellular mechanism for regulating protein function. Some of the most heavily modified neuronal proteins are cytoskeletal proteins of long myelinated axons referred to as neurofilaments (NFs. NFs are type IV intermediate filaments (IFs that can be composed of four subunits, neurofilament heavy (NF-H, neurofilament medium (NF-M, neurofilament light (NF-L, and α-internexin. Within wild type axons, NFs are responsible for mediating radial growth, a process that determines axonal diameter. NFs are phosphorylated on highly conserved lysine-serine-proline (KSP repeats located along the C-termini of both NF-M and NF-H within myelinated axonal regions. Phosphorylation is thought to regulate aspects of NF transport and function. However, a key pathological hallmark of several neurodegenerative diseases is ectopic accumulation and phosphorylation of NFs. The goal of this review is to provide an overview of the posttranslational modifications that occur in both normal and diseased axons. We review evidence that challenges the role of KSP phosphorylation as essential for radial growth and suggests an alternative role for NF phosphorylation in myelinated axons. Furthermore, we demonstrate that regulation of NF phosphorylation dynamics may be essential to avoiding NF accumulations.

  4. Phosphorylated LIM kinases colocalize with gamma-tubulin in centrosomes during early stages of mitosis. (United States)

    Chakrabarti, Ratna; Jones, Jennifer L; Oelschlager, Denise K; Tapia, Tenekua; Tousson, Albert; Grizzle, William E


    LIM kinases (LIMK1 and LIMK2) are LIM domain containing serine/threonine kinases that modulate reorganization of actin cytoskeleton through inactivating phosphorylation of cofilin. The Rho family of small GTPases regulates the catalytic activity of LIMK1 and LIMK2 through activating phosphorylation by ROCK or by p21 kinase. Recent studies have suggested that LIMK1 could play a role in modulation of cellular growth by alteration of the cell cycle in breast and prostate tumor cells; however, the direct mitogenic effects of LIMK1 in these tumor cells is yet to be elucidated. Via immunofluorescence, in this study, we show that phosphorylated LIM kinases (pLIMK1/2) are colocalized with gamma-tubulin in the centrosomes during the early mitotic phases of human breast and prostate cancer cells (MDA-MB-231 and DU145); apparent colocalization begins in the centrosomes in prophase. As shown by both bright field (MDA-MB-231) and fluorescent immunohistochemistry (MDA-MB-231 and DU145), pLIMK1/2 does not localize to centrosomes during interphase. By bright field immunohistochemistry, the largest area of the centrosome that is stained with pLIMK1/2 occurs at anaphase. In early telophase, reduced staining of pLIMK1/2 at the spindle poles and concomitant accumulation of pLIMK1/2 at the cleavage furrow begins to occur. In late telophase, loss of staining of pLIMK1/2 and of colocalization with gamma-tubulin occurs at the poles and pLIMK1/2 became further concentrated at the junction between the two daughter cells. Co-immunoprecipitation studies indicated that gamma-tubulin associates with phosphorylated LIMK1 and LIMK2 but not with dephosphorylated LIMK1 or LIMK2. The results suggest that activated LIMK1/2 may associate with gamma-tubulin and play a role in mitotic spindle assembly.

  5. Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode. (United States)

    Prihandoko, Rudi; Bradley, Sophie J; Tobin, Andrew B; Butcher, Adrian J


    G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype.

  6. Protein tyrosine phosphorylation in streptomycetes. (United States)

    Waters, B; Vujaklija, D; Gold, M R; Davies, J


    Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.

  7. Actin Tyrosine-53-Phosphorylation in Neuronal Maturation and Synaptic Plasticity. (United States)

    Bertling, Enni; Englund, Jonas; Minkeviciene, Rimante; Koskinen, Mikko; Segerstråle, Mikael; Castrén, Eero; Taira, Tomi; Hotulainen, Pirta


    Rapid reorganization and stabilization of the actin cytoskeleton in dendritic spines enables cellular processes underlying learning, such as long-term potentiation (LTP). Dendritic spines are enriched in exceptionally short and dynamic actin filaments, but the studies so far have not revealed the molecular mechanisms underlying the high actin dynamics in dendritic spines. Here, we show that actin in dendritic spines is dynamically phosphorylated at tyrosine-53 (Y53) in rat hippocampal and cortical neurons. Our findings show that actin phosphorylation increases the turnover rate of actin filaments and promotes the short-term dynamics of dendritic spines. During neuronal maturation, actin phosphorylation peaks at the first weeks of morphogenesis, when dendritic spines form, and the amount of Y53-phosphorylated actin decreases when spines mature and stabilize. Induction of LTP transiently increases the amount of phosphorylated actin and LTP induction is deficient in neurons expressing mutant actin that mimics phosphorylation. Actin phosphorylation provides a molecular mechanism to maintain the high actin dynamics in dendritic spines during neuronal development and to induce fast reorganization of the actin cytoskeleton in synaptic plasticity. In turn, dephosphorylation of actin is required for the stabilization of actin filaments that is necessary for proper dendritic spine maturation and LTP maintenance. Dendritic spines are small protrusions from neuronal dendrites where the postsynaptic components of most excitatory synapses reside. Precise control of dendritic spine morphology and density is critical for normal brain function. Accordingly, aberrant spine morphology is linked to many neurological diseases. The actin cytoskeleton is a structural element underlying the proper morphology of dendritic spines. Therefore, defects in the regulation of the actin cytoskeleton in neurons have been implicated in neurological diseases. Here, we revealed a novel mechanism for

  8. Skeletal muscle microRNA and messenger RNA profiling in cofilin-2 deficient mice reveals cell cycle dysregulation hindering muscle regeneration.

    Directory of Open Access Journals (Sweden)

    Sarah U Morton

    Full Text Available Congenital myopathies are rare skeletal muscle diseases presenting in early age with hypotonia and weakness often linked to a genetic defect. Mutations in the gene for cofilin-2 (CFL2 have been identified in several families as a cause of congenital myopathy with nemaline bodies and cores. Here we explore the global messenger and microRNA expression patterns in quadriceps muscle samples from cofillin-2-null mice and compare them with sibling-matched wild-type mice to determine the molecular pathways and mechanisms involved. Cell cycle processes are markedly dysregulated, with altered expression of genes involved in mitotic spindle formation, and evidence of loss of cell cycle checkpoint regulation. Importantly, alterations in cell cycle, apoptosis and proliferation pathways are present in both mRNA and miRNA expression patterns. Specifically, p21 transcript levels were increased, and the expression of p21 targets, such as cyclin D and cyclin E, was decreased. We therefore hypothesize that deficiency of cofilin-2 is associated with interruption of the cell cycle at several checkpoints, hindering muscle regeneration. Identification of these pathways is an important step towards developing appropriate therapies against various congenital myopathies.

  9. Oxidative phosphorylation in cancer cells. (United States)

    Solaini, Giancarlo; Sgarbi, Gianluca; Baracca, Alessandra


    Evidence suggests that mitochondrial metabolism may play a key role in controlling cancer cells life and proliferation. Recent evidence also indicates how the altered contribution of these organelles to metabolism and the resistance of cancer mitochondria against apoptosis-associated permeabilization are closely related. The hallmarks of cancer growth, increased glycolysis and lactate production in tumours, have raised attention due to recent observations suggesting a wide spectrum of oxidative phosphorylation deficit and decreased availability of ATP associated with malignancies and tumour cell expansion. More specifically, alteration in signal transduction pathways directly affects mitochondrial proteins playing critical roles in controlling the membrane potential as UCP2 and components of both MPTP and oxphos complexes, or in controlling cells life and death as the Bcl-2 proteins family. Moreover, since mitochondrial bioenergetics and dynamics, are also involved in processes of cells life and death, proper regulation of these mitochondrial functions is crucial for tumours to grow. Therefore a better understanding of the key pathophysiological differences between mitochondria in cancer cells and in their non-cancer surrounding tissue is crucial to the finding of tools interfering with these peculiar tumour mitochondrial functions and will disclose novel approaches for the prevention and treatment of malignant diseases. Here, we review the peculiarity of tumour mitochondrial bioenergetics and the mode it is linked to the cell metabolism, providing a short overview of the evidence accumulated so far, but highlighting the more recent advances.

  10. Phosphorylation of chicken growth hormone

    Energy Technology Data Exchange (ETDEWEB)

    Aramburo, C.; Montiel, J.L. (Universidad Nacional Autonoma de Mexico (Mexico)); Donoghue, D.; Scanes, C.G. (Rutgers Univ., New Brunswick, NJ (USA)); Berghman, L.R. (Laboratory for Neuroendocrinology and Immunological Biotechnology, Louvain (Belgium))


    The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and {gamma}-{sup 32}P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of {sup 32}P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of {sup 32}P-phosphate labeled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer.

  11. Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins. (United States)

    Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi


    During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

  12. Amyloid-β and proinflammatory cytokines utilize a prion protein-dependent pathway to activate NADPH oxidase and induce cofilin-actin rods in hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Keifer P Walsh

    Full Text Available Neurites of neurons under acute or chronic stress form bundles of filaments (rods containing 1∶1 cofilin∶actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (5-30 min in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors. In contrast, slow rod formation (50% of maximum response in ∼6 h occurs in a subpopulation (∼20% of hippocampal neurons upon exposure to soluble human amyloid-β dimer/trimer (Aβd/t at subnanomolar concentrations. Here we show that proinflammatory cytokines (TNFα, IL-1β, IL-6 also induce rods at the same rate and within the same neuronal population as Aβd/t. Neurons from prion (PrP(C-null mice form rods in response to glutamate or antimycin A, but not in response to proinflammatory cytokines or Aβd/t. Two pathways inducing rod formation were confirmed by demonstrating that NADPH-oxidase (NOX activity is required for prion-dependent rod formation, but not for rods induced by glutamate or energy depletion. Surprisingly, overexpression of PrP(C is by itself sufficient to induce rods in over 40% of hippocampal neurons through the NOX-dependent pathway. Persistence of PrP(C-dependent rods requires the continuous activity of NOX. Removing inducers or inhibiting NOX activity in cells containing PrP(C-dependent rods causes rod disappearance with a half-life of about 36 min. Cofilin-actin rods provide a mechanism for synapse loss bridging the amyloid and cytokine hypotheses for Alzheimer disease, and may explain how functionally diverse Aβ-binding membrane proteins induce synaptic dysfunction.

  13. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation. (United States)

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia


    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Chemistry of Phosphorylated Formaldehyde Derivatives. Part I

    Directory of Open Access Journals (Sweden)

    Vasily P. Morgalyuk


    Full Text Available The underinvestigated derivatives of unstable phosphorylated formaldehyde acetals and some of the structurally related compounds, such as thioacetals, aminonitriles, aminomethylphosphinoyl compounds, are considered. Separately considered are halogen aminals of phosphorylated formaldehyde, acetals of phosphorylated formaldehyde of H-phosphinate-type and a phosphorylated gem-diol of formaldehyde. Synthetic methods, chemical properties and examples of practical applications are given.

  15. Physical exercise improves brain cortex and cerebellum mitochondrial bioenergetics and alters apoptotic, dynamic and auto(mito)phagy markers. (United States)

    Marques-Aleixo, I; Santos-Alves, E; Balça, M M; Rizo-Roca, D; Moreira, P I; Oliveira, P J; Magalhães, J; Ascensão, A


    We here investigate the effects of two exercise modalities (endurance treadmill training-TM and voluntary free-wheel activity-FW) on the brain cortex and cerebellum mitochondrial bioenergetics, permeability transition pore (mPTP), oxidative stress, as well as on proteins involved in mitochondrial biogenesis, apoptosis, and quality control. Eighteen male rats were assigned to sedentary-SED, TM and FW groups. Behavioral alterations and ex vivo brain mitochondrial function endpoints were assessed. Proteins involved in oxidative phosphorylation (OXPHOS, including the adenine nucleotide translocator), oxidative stress markers and regulatory proteins (SIRT3, p66shc, UCP2, carbonyls, MDA, -SH, aconitase, Mn-SOD), as well as proteins involved in mitochondrial biogenesis (PGC1α, TFAM) were evaluated. Apoptotic signaling was measured through quantifying caspase 3, 8 and 9-like activities, Bax, Bcl2, CypD, and cofilin expression. Mitochondrial dynamics (Mfn1/2, OPA1 and DRP1) and auto(mito)phagy (LC3II, Beclin1, Pink1, Parkin, p62)-related proteins were also measured by Western blotting. Only the TM exercise group showed increased spontaneous alternation and exploratory activity. Both exercise regimens improved mitochondrial respiratory activity, increased OXPHOS complexes I, III and V subunits in both brain subareas and decreased oxidative stress markers. Increased resistance to mPTP and decreased apoptotic signaling were observed in the brain cortex from TM and in the cerebellum from TM and FW groups. Also, exercise increased the expression of proteins involved in mitochondrial biogenesis, autophagy and fusion, simultaneous with decreased expression of mitochondrial fission-related protein DRP1. In conclusion, physical exercise improves brain cortex and cerebellum mitochondrial function, decreasing oxidative stress and apoptotic related markers. It is also possible that favorable alterations in mitochondrial biogenesis, dynamics and autophagy signaling induced by exercise

  16. Adjusting ammonium uptake via phosphorylation. (United States)

    Lanquar, Viviane; Frommer, Wolf B


    In plants, AMT/MEP/Rh superfamily mediates high affinity ammonium uptake. AMT/MEP transporters form a trimeric complex, which requires a productive interaction between subunits in order to be functional. The AMT/MEP C-terminal domain is highly conserved in more than 700 AMT homologs from cyanobacteria to higher plants with no cases found to be lacking this domain. AMT1;1 exists in active and inactive states, probably controlled by the spatial positioning of the C-terminus. Ammonium triggers the phosphorylation of a conserved threonine residue (T460) in the C-terminus of AMT1;1 in a time- and concentration-dependent manner. The T460 phosphorylation level correlates with a decrease of root ammonium uptake. We propose that ammonium-induced phosphorylation modulates ammonium uptake as a general mechanism to protect against ammonium toxicity.

  17. Phosphorylation Variation during the Cell Cycle Scales with Structural Propensities of Proteins

    DEFF Research Database (Denmark)

    Tyanova, S.; Frishman, D.; Cox, J.;


    of the cell division cycle we investigate how the variation of the amount of phosphorylation correlates with the protein structure in the vicinity of the modified site. We find two distinct phosphorylation site groups: intrinsically disordered regions tend to contain sites with dynamically varying levels...

  18. Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

    DEFF Research Database (Denmark)

    Satpathy, Shankha; Wagner, Sebastian A; Beli, Petra;


    ) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system. BCR activation leads to rapid tyrosine phosphorylation and ubiquitylation...

  19. Propofol directly increases tau phosphorylation.

    Directory of Open Access Journals (Sweden)

    Robert A Whittington

    Full Text Available In Alzheimer's disease (AD and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of

  20. Nucleoside phosphorylation in amide solutions (United States)

    Schoffstall, A. M.; Kokko, B.


    The paper deals with phosphorylation in possible prebiotic nonaqueous solvents. To this end, phosphorylation of nucleosides using inorganic phosphates in amide solutions is studied at room and elevated temperatures. Reaction proceeds most readily in formamide and N-methylformamide. Products obtained at elevated temperature are nucleotides, nucleoside 2',3'-cyclic phosphates, and when the phosphate concentration is high, nucleoside diphosphates. At room temperature, adenosine afforded a mixture of nucleotides, but none of the cyclic nucleotide. Conditions leading to the highest relative percentage of cyclic nucleotide involve the use of low concentrations of phosphate and an excess of nucleoside.

  1. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr;


    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichin......Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat...

  2. Tyrosine phosphorylation in human lymphomas

    NARCIS (Netherlands)

    Haralambieva, E; Jones, M.; Roncador, GM; Cerroni, L; Lamant, L; Ott, G; Rosenwald, A; Sherman, C; Thorner, P; Kusec, R; Wood, KM; Campo, E; Falini, B; Ramsay, A; Marafioti, T; Stein, H; Kluin, PM; Pulford, K; Mason, DY


    In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated


    Energy Technology Data Exchange (ETDEWEB)



    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  4. Thiamine phosphorylated derivatives and bioelectrogenesis. (United States)

    Schoffeniels, E


    Kinetic as well as thermodynamic considerations favour the idea that the change in sodium conductance explaining the action potential, must result from a bimolecular reaction system. The fact that thiamine phosphorylated derivatives are associated with the specific protein forming the sodium channel could well mean that these thiamine derivatives and more specifically thiamine triphosphate are directly involved in the conductance change.

  5. Biocatalytic asymmetric phosphorylation of mevalonate

    NARCIS (Netherlands)

    Matsumi, R.; Hellriegel, C.; Schoenenberger, B.; Milesi, T.; Oost, van der J.; Wohlgemuth, R.


    The excellent selectivity of the mevalonate kinase-catalyzed phosphorylation of mevalonate simplifies lengthy multi-step routes to (R)-mevalonate-5-phosphate to a one-step biocatalytic reaction, because the phosphate group can be transferred directly and without any additional reaction steps

  6. Phosphorylation regulates coilin activity and RNA association

    Directory of Open Access Journals (Sweden)

    Hanna J. Broome


    The Cajal body (CB is a domain of concentrated components found within the nucleus of cells in an array of species that is functionally important for the biogenesis of telomerase and small nuclear ribonucleoproteins. The CB is a dynamic structure whose number and size change during the cell cycle and is associated with other nuclear structures and gene loci. Coilin, also known as the marker protein for the CB, is a phosphoprotein widely accepted for its role in maintaining CB integrity. Recent studies have been done to further elucidate functional activities of coilin apart from its structural role in the CB in an attempt to explore the rationale for coilin expression in cells that have few CBs or lack them altogether. Here we show that the RNA association profile of coilin changes in mitosis with respect to that during interphase. We provide evidence of transcriptional and/or processing dysregulation of several CB-related RNA transcripts as a result of ectopic expression of both wild-type and phosphomutant coilin proteins. We also show apparent changes in transcription and/or processing of these transcripts upon coilin knockdown in both transformed and primary cell lines. Additionally, we provide evidence of specific coilin RNase activity regulation, on both U2 and hTR transcripts, by phosphorylation of a single residue, serine 489. Collectively, these results point to additional functions for coilin that are regulated by phosphorylation.

  7. Affinity chromatography of phosphorylated proteins. (United States)

    Tchaga, Grigoriy S


    This chapter covers the use of immobilized metal ion affinity chromatography (IMAC) for enrichment of phosphorylated proteins. Some requirements for successful enrichment of these types of proteins are discussed. An experimental protocol and a set of application data are included to enable the scientist to obtain high-yield results in a very short time with pre-packed phospho-specific metal ion affinity resin (PMAC).

  8. Circadian KaiC phosphorylation: a multi-layer network.

    Directory of Open Access Journals (Sweden)

    Congxin Li


    Full Text Available Circadian KaiC phosphorylation in cyanobacteria reconstituted in vitro recently initiates a series of studies experimentally and theoretically to explore its mechanism. In this paper, we report a dynamic diversity in hexameric KaiC phosphoforms using a multi-layer reaction network based on the nonequivalence of the dual phosphorylation sites (S431 and T432 in each KaiC subunit. These diverse oscillatory profiles can generate a kaleidoscopic phase modulation pattern probably responsible for the genome-wide transcription rhythms directly and/or indirectly in cyanobacteria. Particularly, our model reveals that a single KaiC hexamer is an energy-based, phosphorylation-dependent and self-regulated circadian oscillator modulated by KaiA and KaiB. We suggest that T432 is the main regulator for the oscillation amplitude, while S431 is the major phase regulator. S431 and T432 coordinately control the phosphorylation period. Robustness of the Kai network was examined by mixing samples in different phases, and varying protein concentrations and temperature. Similar results were obtained regardless of the deterministic or stochastic method employed. Therefore, the dynamic diversities and robustness of Kai oscillator make it a qualified core pacemaker that controls the cellular processes in cyanobacteria pervasively and accurately.

  9. InlB-mediated Listeria monocytogenes internalization requires a balanced phospholipase D activity maintained through phospho-cofilin

    NARCIS (Netherlands)

    Han, Xuelin; Yu, Rentao; Ji, Lei; Zhen, Dongyu; Tao, Sha; Li, Shuai; Sun, Yansong; Huang, Liuyu; Feng, Zhe; Li, Xianping; Han, Gaige; Schmidt, Martina; Han, Li

    Internalization of Listeria monocytogenes into non-phagocytic cells is tightly controlled by host cell actin dynamics and cell membrane alterations. However, knowledge about the impact of phosphatidylcholine cleavage driven by host cell phospholipase D (PLD) on Listeria internalization into

  10. Proteomic investigation of phosphorylation sites in poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase. (United States)

    Gagné, Jean-Philippe; Moreel, Xavier; Gagné, Pierre; Labelle, Yves; Droit, Arnaud; Chevalier-Paré, Mélissa; Bourassa, Sylvie; McDonald, Darin; Hendzel, Michael J; Prigent, Claude; Poirier, Guy G


    Phosphorylation is a very common post-translational modification event known to modulate a wide range of biological responses. Beyond the regulation of protein activity, the interrelation of phosphorylation with other post-translational mechanisms is responsible for the control of diverse signaling pathways. Several observations suggest that phosphorylation of poly(ADP-ribose) polymerase-1 (PARP-1) regulates its activity. There is also accumulating evidence to suggest the establishment of phosphorylation-dependent assembly of PARP-1-associated multiprotein complexes. Although it is relatively straightforward to demonstrate phosphorylation of a defined target, identification of the actual amino acids involved still represents a technical challenge for many laboratories. With the use of a combination of bioinformatics-based predictions tools for generic and kinase-specific phosphorylation sites, in vitro phosphorylation assays and mass spectrometry analysis, we investigated the phosphorylation profile of PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), two major enzymes responsible for poly(ADP-ribose) turnover. Mass spectrometry analysis revealed the phosphorylation of several serine/threonine residues within important regulatory domains and motifs of both enzymes. With the use of in vivo microirradiation-induced DNA damage, we show that altered phosphorylation at specific sites can modify the dynamics of assembly and disassembly of PARP-1 at sites of DNA damage. By documenting and annotating a collection of known and newly identified phosphorylation sites, this targeted proteomics study significantly advances our understanding of the roles of phosphorylation in the regulation of PARP-1 and PARG.

  11. InlB-mediated Listeria monocytogenes internalization requires a balanced phospholipase D activity maintained through phospho-cofilin

    NARCIS (Netherlands)

    Han, Xuelin; Yu, Rentao; Ji, Lei; Zhen, Dongyu; Tao, Sha; Li, Shuai; Sun, Yansong; Huang, Liuyu; Feng, Zhe; Li, Xianping; Han, Gaige; Schmidt, Martina; Han, Li


    Internalization of Listeria monocytogenes into non-phagocytic cells is tightly controlled by host cell actin dynamics and cell membrane alterations. However, knowledge about the impact of phosphatidylcholine cleavage driven by host cell phospholipase D (PLD) on Listeria internalization into epitheli

  12. Phosphorylation of brain proteins in generalized convulsions

    Energy Technology Data Exchange (ETDEWEB)

    Horan, M.P.


    Phosphorylation of neuronal proteins is being proposed as a modulating influence on several aspects of neuronal function. By labeling proteins with radioactive phosphorus (/sup 32/P) and then separating these proteins by polyacrylamide gel electrophoresis, the author can determine what factors change the phosphorylation of these proteins. They have used such a system to analyze the effects of generalized convulsions on protein phosphorylation. Electroshock (ES) and pentylenetetrazol (PTZ) were utilized to produce generalized convulsions. Brain membranes, taken from rats immediately after a convulsion, exhibited an increase in protein phosphorylation in vitro. The most noticeable change took place in proteins in the 18,000-20,000 MW range. They have designated these proteins as the low molecular weight (LMW) proteins. The change in phosphorylation was basically the same after one convulsions as after six daily convulsions. Twenty-four hours after a single convulsion no change in phosphorylation was observed. When rat membranes are exposed to PTZ in vitro, phosphorylation is increased at 20 sec but has returned to control level at 90 sec of incubation. This effect is produced without a convulsion. In general, as the concentration of magnesium is increased from 5 mM to 10 mM phosphorylation is increased. Increasing the incubation time from 20 sec to 90 sec and increasing the calcium concentration to 10 mM both decrease phosphorylation of the LMW proteins. Human temporal cortex samples present with phosphorylated proteins having patterns very similar to those in rat membranes.

  13. Plk phosphorylation regulates the microtubule-stabilizing protein TCTP. (United States)

    Yarm, Frederic R


    The mitotic polo-like kinases have been implicated in the formation and function of bipolar spindles on the basis of their respective localizations and mutant phenotypes. To date, this putative regulation has been limited to a kinesin-like motor protein, a centrosomal structural protein, and two microtubule-associated proteins (MAPs). In this study, another spindle-regulating protein, the mammalian non-MAP microtubule-binding and -stabilizing protein, the translationally controlled tumor protein (TCTP), was identified as a putative Plk-interacting clone by a two-hybrid screen. Plk phosphorylates TCTP on two serine residues in vitro and cofractionates with the majority of kinase activity toward TCTP in mitotic cell lysates. In addition, these sites were demonstrated to be phosphorylated in vivo. Overexpression of a Plk phosphorylation site-deficient mutant of TCTP induced a dramatic increase in the number of multinucleate cells, rounded cells with condensed ball-like nuclei, and cells undergoing cell death, similar to both the reported anti-Plk antibody microinjection and the low-concentration taxol treatment phenotypes. These results suggest that phosphorylation decreases the microtubule-stabilizing activity of TCTP and promotes the increase in microtubule dynamics that occurs after metaphase.

  14. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation (United States)

    Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.


    Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4–fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

  15. OGlcNAcylation and phosphorylation have opposing structural effects in tau: phosphothreonine induces particular conformational order. (United States)

    Brister, Michael A; Pandey, Anil K; Bielska, Agata A; Zondlo, Neal J


    Phosphorylation and OGlcNAcylation are dynamic intracellular protein post-translational modifications that frequently are alternatively observed on the same serine and threonine residues. Phosphorylation and OGlcNAcylation commonly occur in natively disordered regions of proteins, and often have opposing functional effects. In the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of Alzheimer's disease, whereas OGlcNAcylation stabilizes the soluble form of tau. A series of peptides derived from the proline-rich domain (residues 174-251) of tau was synthesized, with free Ser/Thr hydroxyls, phosphorylated Ser/Thr (pSer/pThr), OGlcNAcylated Ser/Thr, and diethylphosphorylated Ser/Thr. Phosphorylation and OGlcNAcylation were found by CD and NMR to have opposing structural effects on polyproline helix (PPII) formation, with phosphorylation favoring PPII, OGlcNAcylation opposing PPII, and the free hydroxyls intermediate in structure, and with phosphorylation structural effects greater than OGlcNAcylation. For tau196-209, phosphorylation and OGlcNAcylation had similar structural effects, opposing a nascent α-helix. Phosphomimic Glu exhibited PPII-favoring structural effects. Structural changes due to Thr phosphorylation were greater than those of Ser phosphorylation or Glu, with particular conformational restriction as the dianion, with mean (3)JαN = 3.5 Hz (pThr) versus 5.4 Hz (pSer), compared to 7.2, 6.8, and 6.2 Hz for Thr, Ser, and Glu, respectively, values that correlate with the backbone torsion angle ϕ. Dianionic phosphothreonine induced strong phosphothreonine amide protection and downfield amide chemical shifts (δmean = 9.63 ppm), consistent with formation of a stable phosphate-amide hydrogen bond. These data suggest potentially greater structural importance of threonine phosphorylation than serine phosphorylation due to larger induced structural effects.

  16. Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Feng; Camp, David G.; Gritsenko, Marina A.; Luo, Quanzhou; Kelly, Ryan T.; Clauss, Therese RW; Brinkley, William R.; Smith, Richard D.; Stenoien, David L.


    The chromosomal passenger complex (CPC) is a critical regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation specific antibody that labels the CPC using liquid chromatography coupled to mass spectrometry. A mitotic phosphorylation motif (PX{G/T/S}{L/M}[pS]P or WGL[pS]P) was identified in 11 proteins including Fzr/Cdh1 and RIC-8, two proteins with potential links to the CPC. Phosphoprotein complexes contained known CPC components INCENP, Aurora-B and TD-60, as well as SMAD2, 14-3-3 proteins, PP2A, and Cdk1, a likely kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins including SMAD2, Plk3 and INCENP. Mitotic SMAD2 and Plk3 phosphorylation was confirmed using phosphorylation specific antibodies, and in the case of Plk3, phosphorylation correlates with its localization to the mitotic apparatus. A mutagenesis approach was used to show INCENP phosphorylation is required for midbody localization. These results provide evidence for a shared phosphorylation event that regulates localization of critical proteins during mitosis.

  17. Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus. (United States)

    Yang, Feng; Camp, David G; Gritsenko, Marina A; Luo, Quanzhou; Kelly, Ryan T; Clauss, Therese R W; Brinkley, William R; Smith, Richard D; Stenoien, David L


    The chromosomal passenger complex (CPC) is a crucial regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, using liquid chromatography coupled to mass spectrometry (LC-MS), we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation-specific antibody that labels the CPC. A mitotic phosphorylation motif {PX[G/T/S][L/M]S(P) P or WGLS(P) P} was identified by MS in 11 proteins, including FZR1 (Cdh1) and RIC8A-two proteins with potential links to the CPC. Phosphoprotein complexes contained the known CPC components INCENP, Aurora-B (Aurkb) and TD-60 (Rcc2, RCC1-like), as well as SMAD2, 14-3-3 proteins, PP2A and Cdk1 (Cdc2a), a probable kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins, including SMAD2, PLK3 and INCENP. Mitotic SMAD2 and PLK3 phosphorylation was confirmed using phosphorylation-specific antibodies, and, in the case of Plk3, phosphorylation correlated with its localization to the mitotic apparatus and the midbody. A mutagenesis approach was used to show that INCENP phosphorylation is required for its localization to the midbody. These results provide evidence for a shared phosphorylation event that regulates localization of crucial proteins during mitosis.

  18. Methods for the Analysis of Protein Phosphorylation-Mediated Cellular Signaling Networks (United States)

    White, Forest M.; Wolf-Yadlin, Alejandro


    Protein phosphorylation-mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks.

  19. Analysis of mitotic phosphorylation of Borealin

    Directory of Open Access Journals (Sweden)

    Date Dipali A


    Full Text Available Abstract Background The main role of the chromosomal passenger complex is to ensure that Aurora B kinase is properly localized and activated before and during mitosis. Borealin, a member of the chromosomal passenger complex, shows increased expression during G2/M phases and is involved in targeting the complex to the centromere and the spindle midzone, where it ensures proper chromosome segregation and cytokinesis. Borealin has a consensus CDK1 phosphorylation site, threonine 106 and can be phosphorylated by Aurora B Kinase at serine 165 in vitro. Results Here, we show that Borealin is phosphorylated during mitosis in human cells. Dephosphorylation of Borealin occurs as cells exit mitosis. The phosphorylated form of Borealin is found in an INCENP-containing complex in mitosis. INCENP-containing complexes from cells in S phase are enriched in the phosphorylated form suggesting that phosphorylation may encourage entry of Borealin into the chromosomal passenger complex. Although Aurora B Kinase is found in complexes that contain Borealin, it is not required for the mitotic phosphorylation of Borealin. Mutation of T106 or S165 of Borealin to alanine does not alter the electrophoretic mobility shift of Borealin. Experiments with cyclohexamide and the phosphatase inhibitor sodium fluoride suggest that Borealin is phosphorylated by a protein kinase that can be active in interphase and mitosis and that the phosphorylation may be regulated by a short-lived phosphatase that is active in interphase but not mitosis. Conclusion Borealin is phosphorylated during mitosis. Neither residue S165, T106 nor phosphorylation of Borealin by Aurora B Kinase is required to generate the mitotic, shifted form of Borealin. Suppression of phosphorylation during interphase is ensured by a labile protein, possibly a cell cycle regulated phosphatase.

  20. Dynamic interplay between phosphorylation and O-glycosylation of neurofilaments in neurodegenerative diseases%神经丝蛋白质糖基化与磷酸化的相互调节和神经退行性疾病

    Institute of Scientific and Technical Information of China (English)

    崔冉亮; 胡海燕; 吕朴; 戎凯; 陈宁; 邓艳秋


    Neurofilaments (NFs), assembled from three subunits of different molecular masses, namely NFL (neurofilament light, 68 kDa), NFM (neurofilament medium, 160 kDa), and NFH (neurofilament heavy, 200 kDa), are one of major cytoskeletal elements in neurons and play a very significant role in stabilizing morphology and structure of cells and maintaining axon transportation. Two important posttranslational modifications exist in NFs protein are phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc), which modify the same or proximal hydroxyl groups of serine or threonine, therefore, there may exist competitive inter-regulation between phosphorylation and O-glycosylation, which may play an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Amyotrophic lateral sclerosis (ALS) and Spinal muscular atrophy (SMA).%神经丝(neurofnament,NF)蛋白质是神经元细胞骨架的主要成分,由低(68 kDa)、中(160 kDa)和高(200 kDa)分子量的三种哑基聚合而成,在维持细胞骨架、稳定细胞形态和轴突转运方面均有十分重要意义.NF蛋白质存在两个非常重要的翻译后修饰--磷酸化和O位N-乙酰葡萄糖胺(O-linked N-acetylgltlcosamine.O-GlcNAc)糖基化.由于它们修饰同一蛋白质的相同或邻近丝氨酸和苏氨酸羟基,因此磷酸化和糖基化修饰可能存在着竞争性调节,在神经退行性变性疾病如阿尔茨海默病、肌萎缩性脊髓侧索硬化症和进行性肌肉萎缩症等发病机制中可能起十分重要的作用.

  1. Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation



    We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC inc...

  2. Site-specific phosphorylation of Tau protein is associated with deacetylation of microtubules in mouse spermatogenic cells during meiosis. (United States)

    Inoue, Hiroki; Hiradate, Yuuki; Shirakata, Yoshiki; Kanai, Kenta; Kosaka, Keita; Gotoh, Aina; Fukuda, Yasuhiro; Nakai, Yutaka; Uchida, Takafumi; Sato, Eimei; Tanemura, Kentaro


    Tau is one of the microtubule-associated proteins and a major component of paired helical filaments, a hallmark of Alzheimer's disease. Its expression has also been indicated in the testis. However, its function and modification in the testis have not been established. Here, we analyzed the dynamics of phosphorylation patterns during spermatogenesis. The expression of Tau protein and its phosphorylation were shown in the mouse testis. Immunohistochemistry revealed that the phosphorylation was strongly detected during meiosis. Correspondingly, the expression of acetylated tubulin was inversely weakened during meiosis. These results suggest that phosphorylation of Tau protein contributes to spermatogenesis, especially in meiosis.

  3. Proteomic analysis of tyrosine phosphorylation during human liver transplantation

    Directory of Open Access Journals (Sweden)

    Boutros Tarek


    Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

  4. SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J


    spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

  5. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

    NARCIS (Netherlands)

    Yamaguchi, Tomoya; Goto, Hidemasa; Yokoyama, Tomoya; Silljé, Herman; Hanisch, Anja; Uldschmid, Andreas; Takai, Yasushi; Oguri, Takashi; Nigg, Erich A; Inagaki, Masaki


    Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its si

  6. Characterizing the Microenvironment Surrounding Phosphorylated Protein Sites

    Institute of Scientific and Technical Information of China (English)

    Shi-Cai Fan; Xue-Gong Zhang


    Protein phosphorylation plays an important role in various cellular processes. Due to its high complexity, the mechanism needs to be further studied. In the last few years, many methods have been contributed to this field, but almost all of them investigated the mechanism based on protein sequences around protein sites. In this study, we implement an exploration by characterizing the microenvironment surrounding phosphorylated protein sites with a modified shell model, and obtain some significant properties by the rank-sum test, such as the lack of some classes of residues, atoms, and secondary structures. Furthermore, we find that the depletion of some properties affects protein phosphorylation remarkably. Our results suggest that it is a meaningful direction to explore the mechanism of protein phosphorylation from microenvironment and we expect further findings along with the increasing size of phosphorylation and protein structure data.

  7. Identification of phosphorylated proteins in erythrocytes infected by the human malaria parasite Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Xia Dong


    Full Text Available Abstract Background Previous comparative proteomic analysis on Plasmodium falciparum isolates of different adhesion properties suggested that protein phosphorylation varies between isolates with different cytoadherence properties. But the extent and dynamic changes in phosphorylation have not been systematically studied. As a baseline for these future studies, this paper examined changes in the phosphoproteome of parasitized red blood cells (pRBC. Methods Metabolic labelling with [35S] methionine on pRBC and 2D gel electrophoresis (2-DE has previously been used to show the expression of parasite proteins and changes in protein iso-electric point (PI. 2-DE of different parasite strains was combined with immunoblotting using monoclonal antibodies specifically to phosphorylated serine/threonine and tyrosine, to obtain the phosphorylation profiles throughout the erythrocytic lifecycle. Affinity chromatography was used to purify/enrich phosphorylated proteins and these proteins from mature trophozoite stages which were identified using high-accuracy mass spectrometry and MASCOT search. Results 2D-immunoblots showed that P. falciparum infection greatly increased phosphorylation of a set of proteins in pRBC, the dominant size classes for phosphorylated tyrosine proteins were 95, 60, 50 and 30 kDa and for phosphorylated serine/threonine were 120, 95, 60, 50, 43, 40 and 30 kDa. The most abundant molecules from 2D-gel mapping of phosphorylated proteins in ItG infected RBCs were identified by MALDI-TOF. A proteomic overview of phosphorylated proteins in pRBC was achieved by using complementary phosphorylated protein enrichment techniques combined with nano-flow LC/MS/MS analysis and MASCOT MS/MS ions search with phosphorylation as variable modifications. The definite phosphoproteins of pRBC are reported and discussed. Conclusion Protein phosphorylation is a major process in P. falciparum-parasitized erythrocytes. Preliminary screens identified 170 P

  8. Muscle-specific splicing factors ASD-2 and SUP-12 cooperatively switch alternative pre-mRNA processing patterns of the ADF/cofilin gene in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Genta Ohno

    Full Text Available Pre-mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre-mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre-mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre-mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre-mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA-binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT-PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators

  9. Phosphorylation of EB2 by Aurora B and CDK1 ensures mitotic progression and genome stability. (United States)

    Iimori, Makoto; Watanabe, Sugiko; Kiyonari, Shinichi; Matsuoka, Kazuaki; Sakasai, Ryo; Saeki, Hiroshi; Oki, Eiji; Kitao, Hiroyuki; Maehara, Yoshihiko


    Temporal regulation of microtubule dynamics is essential for proper progression of mitosis and control of microtubule plus-end tracking proteins by phosphorylation is an essential component of this regulation. Here we show that Aurora B and CDK1 phosphorylate microtubule end-binding protein 2 (EB2) at multiple sites within the amino terminus and a cluster of serine/threonine residues in the linker connecting the calponin homology and end-binding homology domains. EB2 phosphorylation, which is strictly associated with mitotic entry and progression, reduces the binding affinity of EB2 for microtubules. Expression of non-phosphorylatable EB2 induces stable kinetochore microtubule dynamics and delays formation of bipolar metaphase plates in a microtubule binding-dependent manner, and leads to aneuploidy even in unperturbed mitosis. We propose that Aurora B and CDK1 temporally regulate the binding affinity of EB2 for microtubules, thereby ensuring kinetochore microtubule dynamics, proper mitotic progression and genome stability.

  10. Phosphorylation of human Sgo1 by NEK2A is essential for chromosome congression in mitosis

    Institute of Scientific and Technical Information of China (English)

    Guosheng Fu; Xia Ding; Kai Yuan; Felix Aikhionbare; Jianhui Yao; Xin Cai; Kai Jiang; Xuebiao Yao


    Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules. Our recent study shows that NEK2A interacts with MAD1 at the kinetochore and possibly functions as a novel integrator of spindle checkpoint signaling. However, it is unclear how NEK2 A regulates kinetochore-microtubule attachment in mitosis. Here we show that NEK2A phosphorylates human Sgol and such phosphorylation is essential for faithful chromosome congression in mitosis. NEK2A binds directly to HsSgol in vitro and co-distributes with HsSgol to the kinetochore of mitotic cells. Our in vitro phosphorylation experiment demonstrated that HsSgol is a substrate of NEK2A and the phosphorylation sites were mapped to Ser14 and Ser507 as judged by the incorporation of 32P. Although such phosphorylation is not required for assembly of HsSgol to the kinetochore, expression of non-phosphorylatable mutant HsSgol perturbed chromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic and monotelic attachments. These findings reveal a key role for the NEK2A-mediated phosphorylation of HsSgol in orchestrating dynamic kinetochore-microtubule interaction. We propose that NEK2A-mediated phosphorylation of human Sgol provides a link between centromeric cohesion and spindle microtubule attachment at the kinetochores.

  11. In vivo analysis of Yorkie phosphorylation sites. (United States)

    Oh, H; Irvine, K D


    The co-activator Yorkie (Yki) mediates transcriptional regulation effected by the Drosophila Fat-Warts (Wts)-Hippo (Hpo) pathways. Yki is inhibited by Wts-mediated phosphorylation, and a Wts phosphorylation site at Ser168 has been identified. Here we identify two additional Wts phosphorylation sites on Yki, and examine the respective contribution of all three sites to Yki nuclear localization and activity. Our results show that although Ser168 is the most critical site, all three phosphorylation sites influence Yki localization and activity in vivo, and can be sites of regulation by Wts. Thus, investigations of the role of Yki and its mammalian homolog Yes-associated protein (YAP) in development and oncogenesis should include evaluations of additional sites. The WW domains of Yki are not required for its phosphorylation, but instead are positively required for its activity. We also identify two potential sites of phosphorylation by an unknown kinase, which could influence phosphorylation of Ser168 by Wts, suggesting that there are additional mechanisms for regulating Yki/YAP activity.

  12. Challenges and strategies for targeted phosphorylation site identification and quantification using mass spectrometry analysis. (United States)

    Blackburn, Kevin; Goshe, Michael B


    Despite its importance, the 'ultimate' method to identify and quantify site-specific protein phosphorylation using mass spectrometry (MS) has yet to be established. This is as much a function of the dynamic range of instrumentation as it is the complexities surrounding the isolation and behavior of phosphopeptides. Phosphorylation site analysis using MS can be quite challenging when analyzing just one protein and quickly becomes a daunting task when attempting to perform proteome-wide measurements. Data-dependent tandem MS-based methods which are useful for the discovery and characterization of novel phosphorylation sites often lack the dynamic range and quantitative aspect required for studying the temporal phases of phosphorylation. While targeted methods such as multiple reaction monitoring do provide a highly specific and quantitative methodology for studying phosphorylation changes over time, they are not suited for initial discovery of previously unreported sites of phosphorylation. Data-independent acquisition represents a relatively new approach for simultaneous qualitative and quantitative sample analysis which holds promise for filling this technological gap.

  13. Tyrosine-1 and threonine-4 phosphorylation marks complete the RNA polymerase II CTD phospho-code. (United States)

    Heidemann, Martin; Eick, Dirk


    Eukaryotic RNA polymerase II (RNAP II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation of Ser2, Ser5 and Ser7 residues orchestrates the binding of transcription and RNA processing factors to the transcription machinery. Recent studies show that the two remaining potential phosphorylation sites, tyrosine-1 and threonine-4, are phosphorylated as well and contribute to the previously proposed "CTD code". With the impairment of binding of CTD interacting factors, these novel phosphorylation marks add an accessory layer of regulation to the RNAP II transcription cycle.

  14. Different mechanisms of homologous and heterologous μ-opioid receptor phosphorylation. (United States)

    Mann, Anika; Illing, Susann; Miess, Elke; Schulz, Stefan


    The efficiency of μ-opioid receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite-specific μ-opioid receptor antibodies, which have proved to be excellent tools for monitoring the spatial and temporal dynamics of receptor phosphorylation and dephosphorylation. Agonist-induced phosphorylation of μ-opioid receptors occurs at a conserved 10 residue sequence (370) TREHPSTANT(379) in the receptor's carboxyl-terminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at S375, present in the middle of this sequence, but only high-efficacy opioids have the ability to drive higher order phosphorylation on flanking residues (T370, T376 and T379). S375 is the initiating residue in a hierarchical phosphorylation cascade. In contrast, agonist-independent heterologous μ-opioid receptor phosphorylation occurs primarily at T370. The combination of phosphosite-specific antibodies and siRNA knockdown screening also facilitated the identification of relevant kinases and phosphatases. In fact, morphine induces a selective S375 phosphorylation that is predominantly catalysed by GPCR kinase 5 (GRK5), whereas multisite phosphorylation induced by high-efficacy opioids specifically requires GRK2/3. By contrast, T370 phosphorylation stimulated by phorbol esters or heterologous activation of Gq -coupled receptors is mediated by PKCα. Rapid μ-opioid receptor dephosphorylation occurs at or near the plasma membrane and is catalysed by protein phosphatase 1γ (PP1γ). These findings suggest that there are distinct phosphorylation motifs for homologous and heterologous regulation of μ-opioid receptor phosphorylation. However, it remains to be seen to what extent different μ-opioid receptor phosphorylation patterns contribute to the development of tolerance and dependence in vivo. This article

  15. Phosphorylation of Intrinsically Disordered Regions in Remorin Proteins

    Directory of Open Access Journals (Sweden)

    Macarena eMarín


    Full Text Available Plant-specific remorin proteins reside in subdomains of plasma membranes, originally termed membrane rafts. They probably facilitate cellular signal transduction by direct interaction with signalling proteins such as receptor-like kinases (RLKs and may dynamically modulate their lateral segregation within plasma membranes. Recent evidence suggests such functions of remorins during plant-microbe interactions and innate immune responses, where differential phosphorylation of some of these proteins has been described to be dependent on the perception of the microbe-associated molecular pattern (MAMP flg22 and the presence of the NBS-LRR resistance protein RPM1. A number of specifically phosphorylated residues in their highly variable and intrinsically disordered N-terminal regions have been identified. Sequence diversity of these evolutionary distinct domains suggests that remorins may serve a wide range of biological functions. Here, we describe patterns and features of intrinsic disorder in remorin protein and discuss possible functional implications of phosphorylation within these rapidly evolving domains.

  16. HSP20 phosphorylation and airway smooth muscle relaxation

    Directory of Open Access Journals (Sweden)

    Mariam Ba


    Full Text Available Mariam Ba1, Cherie A Singer1, Manoj Tyagi2, Colleen Brophy3, Josh E Baker4, Christine Cremo4, Andrew Halayko5, William T Gerthoffer21Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; 2Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; 3Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; 5Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, CanadaAbstract: HSP20 (HSPB6 is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1, and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa to smaller complexes (<60 kDa. The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway

  17. Tyrosyl phosphorylation toggles a Runx1 switch


    Benjamin G. Neel; Speck, Nancy A.


    The Runx1 transcription factor is post-translationally modified by seryl/threonyl phosphorylation, acetylation, and methylation that control its interactions with transcription factor partners and epigenetic coregulators. In this Perspective, the study by Huang et al. (in this issue), which describes how the regulation of Runx1 tyrosyl phosphorylation by Src family kinases and the Shp2 phosphatase toggle Runx1's interactions between different coregulatory molecules, is discussed.

  18. Fibronectin phosphorylation by ecto-protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru (Meiji Institute of Health Science, Odawara (Japan))


    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  19. Protein phosphorylation: Localization in regenerating optic axons

    Energy Technology Data Exchange (ETDEWEB)

    Larrivee, D. (Cornell Univ. Medical College, New York, NY (USA))


    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  20. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seon Sook; Lee, Eun Hye [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Lee, Kooyeon [Department of Bio-Health Technology, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Jo, Su-Hyun, E-mail: [Department of Physiology, BK21 Plus Graduate Program, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Seo, Su Ryeon, E-mail: [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)


    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  1. Phosphorylation of Cdc5 regulates its accumulation

    Directory of Open Access Journals (Sweden)

    Simpson-Lavy Kobi J


    Full Text Available Abstract Background Cdc5 (polo kinase/Plk1 is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28 phosphorylation site present. Findings Cdc5T23A introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts in cis and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/CCdh1 independent and requires new protein synthesis. Cdc5T23E is hyperactive, and reduces the levels of Cdc5 (in trans and drastically reduces Clb2 levels. Conclusions Phosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1 caps Cdc5 and other CLB2 cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/CCdh1 inactivation.

  2. Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation. (United States)

    Karim, Christine B; Espinoza-Fonseca, L Michel; James, Zachary M; Hanse, Eric A; Gaynes, Jeffrey S; Thomas, David D; Kelekar, Ameeta


    We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa's BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

  3. Dynamics

    CERN Document Server

    Goodman, Lawrence E


    Beginning text presents complete theoretical treatment of mechanical model systems and deals with technological applications. Topics include introduction to calculus of vectors, particle motion, dynamics of particle systems and plane rigid bodies, technical applications in plane motions, theory of mechanical vibrations, and more. Exercises and answers appear in each chapter.

  4. Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation (United States)

    Rigatti, Marc; Le, Andrew V.; Gerber, Claire; Moraru, Ion I.; Dodge-Kafka, Kimberly L.


    Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca2+ cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca2+ re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100–200nM) to regulate the phosphorylation of large quantities of PLB (50µM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100–200 fold lower concentrations. PMID:26027516

  5. Phasic phosphorylation of caldesmon and ERK 1/2 during contractions in human myometrium. (United States)

    Paul, Jonathan; Maiti, Kaushik; Read, Mark; Hure, Alexis; Smith, Julia; Chan, Eng-Cheng; Smith, Roger


    Human myometrium develops phasic contractions during labor. Phosphorylation of caldesmon (h-CaD) and extracellular signal-regulated kinase 1/2 (ERK 1/2) has been implicated in development of these contractions, however the phospho-regulation of these proteins is yet to be examined during periods of both contraction and relaxation. We hypothesized that protein phosphorylation events are implicated in the phasic nature of myometrial contractions, and aimed to examine h-CaD and ERK 1/2 phosphorylation in myometrium snap frozen at specific stages, including; (1) prior to onset of contractions, (2) at peak contraction and (3) during relaxation. We aimed to compare h-CaD and ERK 1/2 phosphorylation in vitro against results from in vivo studies that compared not-in-labor (NIL) and laboring (L) myometrium. Comparison of NIL (n = 8) and L (n = 8) myometrium revealed a 2-fold increase in h-CaD phosphorylation (ser-789; P = 0.012) during onset of labor in vivo, and was associated with significantly up-regulated ERK2 expression (P = 0.022), however no change in ERK2 phosphorylation was observed (P = 0.475). During in vitro studies (n = 5), transition from non-contracting tissue to tissue at peak contraction was associated with increased phosphorylation of both h-CaD and ERK 1/2. Furthermore, tissue preserved at relaxation phase exhibited diminished levels of h-CaD and ERK 1/2 phosphorylation compared to tissue preserved at peak contraction, thereby producing a phasic phosphorylation profile for h-CaD and ERK 1/2. h-CaD and ERK 1/2 are phosphorylated during myometrial contractions, however their phospho-regulation is dynamic, in that h-CaD and ERK 1/2 are phosphorylated and dephosphorylated in phase with contraction and relaxation respectively. Comparisons of NIL and L tissue are at risk of failing to detect these changes, as L samples are not necessarily preserved in the midst of an active contraction.

  6. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.


    phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...

  7. Mapping of p140Cap phosphorylation sites

    DEFF Research Database (Denmark)

    Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta


    Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes phosp...

  8. Phosphorylation sites within Ebola virus nucleoprotein

    Institute of Scientific and Technical Information of China (English)

    Sora; Yasri; Viroj; Wiwanitkit


    To understand the infection process, the viral multiplication and entry to the cell is widely studied. The Ebola virus nucleoprotein is the important problem for the pathological process. Focusing on the specific biological process, the post translational modification is needed. Here, the authors used the bioinformatics study to find the phosphorylation sites within the Ebola virus nucleoprotein and could identify many new sites.

  9. Ion channels, phosphorylation and mammalian sperm capacitation. (United States)

    Visconti, Pablo E; Krapf, Dario; de la Vega-Beltrán, José Luis; Acevedo, Juan José; Darszon, Alberto


    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.

  10. Ion channels, phosphorylation and mammalian sperm capacitation

    Institute of Scientific and Technical Information of China (English)

    Pablo E Visconti; Dario Krapf; José Luis de la Vega-Beltrán; Juan José Acevedo; Alberto Darszon


    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.

  11. Phosphorylated α-synuclein in Parkinson's disease

    DEFF Research Database (Denmark)

    Stewart, Tessandra; Sossi, Vesna; Aasly, Jan O;


    INTRODUCTION: α-Synuclein (α-syn) is a key protein in Parkinson's disease (PD), and one of its phosphorylated forms, pS129, is higher in PD patients than healthy controls. However, few studies have examined its levels in longitudinally collected cerebrospinal fluid (CSF) or in preclinical cases. ...

  12. Transferases for alkylation, glycosylation and phosphorylation

    NARCIS (Netherlands)

    Auriol, D.; ter Halle, R.; Lefèvre, F.; Visser, D.F.; Gordon, G.E.R.; Bode, M.L.; Mathiba, K.; Brady, D.; De Winter, K.; Desmet, T.; Cerdobbel, A.; Soetaert, W.; van Herk, T.; Hartog, A.F.; Wever, R.; Brzezińska-rodak, M.; Klimek-Ochab, M.; Żymańczyk-Duda, E.; Mukherjee, J.; Gupta, M.N.; Yin, W.B.; Li, S.M.; Gruber-Khadjawi, M.; Whittall, J.; Sutton, P.W.


    This chapter contains sections titled: Industrial Production of Caffeic Acid-α-D-O-Glucoside Enzymatic Synthesis of 5-Methyluridine by Transglycosylation of Guanosine and Thymine Preparation and Use of Sucrose Phosphorylase as Cross-Linked Enzyme Aggregate (CLEA) Enzymatic Synthesis of Phosphorylate

  13. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    Energy Technology Data Exchange (ETDEWEB)

    Karen S. Browning


    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  14. Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

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    Jang, Deok-Jin; Wang, Daojing


    Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

  15. Phosphorylation and proteome dynamics in pathogen-resistant tomato plants

    NARCIS (Netherlands)

    Stulemeijer, I.J.E.


    Microbial plant pathogens impose a continuous threat on global food production. Similar to disease resistance in mammals, an innate immune system allows plants to recognise pathogens and swiftly activate defence. For the work described in this thesis, the interaction between tomato and the extracell

  16. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.


    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during

  17. Phosphorylation and proteome dynamics in pathogen-resistant tomato plants

    NARCIS (Netherlands)

    Stulemeijer, I.J.E.


    Microbial plant pathogens impose a continuous threat on global food production. Similar to disease resistance in mammals, an innate immune system allows plants to recognise pathogens and swiftly activate defence. For the work described in this thesis, the interaction between tomato and the

  18. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.


    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during different

  19. Phosphorylation and proteome dynamics in pathogen-resistant tomato plants

    NARCIS (Netherlands)

    Stulemeijer, I.J.E.


    Microbial plant pathogens impose a continuous threat on global food production. Similar to disease resistance in mammals, an innate immune system allows plants to recognise pathogens and swiftly activate defence. For the work described in this thesis, the interaction between tomato and the extracell

  20. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Van Hoof, Dennis; Muñoz, Javier; Braam, Stefan R


    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during...

  1. Reconstruction and analysis of nutrient-induced phosphorylation networks in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Guangyou eDuan


    Full Text Available Elucidating the dynamics of molecular processes in living organisms in response to external perturbations is a central goal in modern systems biology. We investigated the dynamics of protein phosphorylation events in Arabidopsis thaliana exposed to changing nutrient conditions. Phosphopeptide expression levels were detected at five consecutive time points over a time interval of 30 minutes after nutrient resupply following prior starvation. The three tested inorganic, ionic nutrients NH4+, NO3-, PO43- elicited similar phosphosignaling responses that were distinguishable from those invoked by the sugars mannitol, sucrose. When embedded in the protein-protein interaction network of Arabidopsis thaliana, phosphoproteins were found to exhibit a higher degree compared to average proteins. Based on the time-series data, we reconstructed a network of regulatory interactions mediated by phosphorylation. The performance of different network inference methods was evaluated by the observed likelihood of physical interactions within and across different subcellular compartments and based on gene ontology semantic similarity. The dynamic phosphorylation network was then reconstructed using a Pearson correlation method with added directionality based on partial variance differences. The topology of the inferred integrated network corresponds to an information dissemination architecture, in which the phosphorylation signal is passed on to an increasing number of phosphoproteins stratified into an initiation, processing, and effector layer. Specific phosphorylation peptide motifs associated with the distinct layers were identified indicating the action of layer-specific kinases. Despite the limited temporal resolution, combined with information on subcellular location, the available time-series data proved useful for reconstructing the dynamics of the molecular signaling cascade in response to nutrient stress conditions in the plant Arabidopsis thaliana.

  2. Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database

    DEFF Research Database (Denmark)

    Nühse, Thomas S; Stensballe, Allan; Jensen, Ole N;


    Functional genomic technologies are generating vast amounts of data describing the presence of transcripts or proteins in plant cells. Together with classical genetics, these approaches broaden our understanding of the gene products required for specific responses. Looking to the future, the focus...... of research must shift to the dynamic aspects of biology: molecular mechanisms of function and regulation. Phosphorylation is a key regulatory factor in all aspects of plant biology; but it is difficult, if not impossible, for most researchers to identify in vivo phosphorylation sites within their proteins...... will be a valuable resource for many fields of plant biology and overcome a major impediment to the elucidation of signal transduction pathways. We present an analysis of the characteristics of phosphorylation sites, their conservation among orthologs and paralogs, and the existence of putative motifs surrounding...

  3. Insights into the Phosphoryl Transfer Mechanism of Human Ubiquitous Mitochondrial Creatine Kinase. (United States)

    Li, Quanjie; Fan, Shuai; Li, Xiaoyu; Jin, Yuanyuan; He, Weiqing; Zhou, Jinming; Cen, Shan; Yang, ZhaoYong


    Human ubiquitous mitochondrial creatine kinase (uMtCK) is responsible for the regulation of cellular energy metabolism. To investigate the phosphoryl-transfer mechanism catalyzed by human uMtCK, in this work, molecular dynamic simulations of uMtCK∙ATP-Mg(2+)∙creatine complex and quantum mechanism calculations were performed to make clear the puzzle. The theoretical studies hereof revealed that human uMtCK utilizes a two-step dissociative mechanism, in which the E227 residue of uMtCK acts as the catalytic base to accept the creatine guanidinium proton. This catalytic role of E227 was further confirmed by our assay on the phosphatase activity. Moreover, the roles of active site residues in phosphoryl transfer reaction were also identified by site directed mutagenesis. This study reveals the structural basis of biochemical activity of uMtCK and gets insights into its phosphoryl transfer mechanism.

  4. ERK Activation Globally Downregulates miRNAs through Phosphorylating Exportin-5. (United States)

    Sun, Hui-Lung; Cui, Ri; Zhou, JianKang; Teng, Kun-Yu; Hsiao, Yung-Hsuan; Nakanishi, Kotaro; Fassan, Matteo; Luo, Zhenghua; Shi, Guqin; Tili, Esmerina; Kutay, Huban; Lovat, Francesca; Vicentini, Caterina; Huang, Han-Li; Wang, Shih-Wei; Kim, Taewan; Zanesi, Nicola; Jeon, Young-Jun; Lee, Tae Jin; Guh, Jih-Hwa; Hung, Mien-Chie; Ghoshal, Kalpana; Teng, Che-Ming; Peng, Yong; Croce, Carlo M


    MicroRNAs (miRNA) are mostly downregulated in cancer. However, the mechanism underlying this phenomenon and the precise consequence in tumorigenesis remain obscure. Here we show that ERK suppresses pre-miRNA export from the nucleus through phosphorylation of exportin-5 (XPO5) at T345/S416/S497. After phosphorylation by ERK, conformation of XPO5 is altered by prolyl isomerase Pin1, resulting in reduction of pre-miRNA loading. In liver cancer, the ERK-mediated XPO5 suppression reduces miR-122, increases microtubule dynamics, and results in tumor development and drug resistance. Analysis of clinical specimens further showed that XPO5 phosphorylation is associated with poor prognosis for liver cancer patients. Our study reveals a function of ERK in miRNA biogenesis and suggests that modulation of miRNA export has potential clinical implications.

  5. Cell stress promotes the association of phosphorylated HspB1 with F-actin.

    Directory of Open Access Journals (Sweden)

    Joseph P Clarke

    Full Text Available Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

  6. Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity. (United States)

    Ahmad, Shabbir; Ytterberg, A Jimmy; Thulasingam, Madhuranayaki; Tholander, Fredrik; Bergman, Tomas; Zubarev, Roman; Wetterholm, Anders; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z


    Leukotriene C4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C4 (LTC4). LTC4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser(36) as the major p70S6k phosphorylation site, along with a low frequency site at Thr(40), using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 μmol/min/mg) of the activity of WT enzyme (95 μmol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser(36) is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser(36) upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg(104) in the adjacent subunit. Because Arg(104) is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser(36) inhibits the catalytic function of LTC4S by interference with the catalytic machinery. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Development of a STAT5 phosphorylation assay as a rapid bioassay to assess interleukin-7 potency. (United States)

    Zumpe, C; Engel, K; Wiedemann, N; Metzger, A U; Pischetsrieder, M; Bachmann, C L


    Interleukin (IL)-7 is a cytokine inducing the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway. As a consequence of IL-7 activating this pathway, STAT5 is phosphorylated. In pharmaceutical quality control, the potency of biopharmaceuticals is commonly assessed by proliferation assays. This is also possible for IL-7 conjugates. However, the disadvantage of these classical "endpoint-assays" is that they require very long incubation times, up to several days, since they measure the downstream events of a cellular response. As an alternative to this, we developed a rapid intracellular phosphorylation assay, measuring IL-7 induced STAT5 phosphorylation in Kit 225 cells. The Kit 225 human T cell line expresses the IL-7 receptor and is responsive to IL-7, therefore making it a good candidate cell line for assay development. Like the Kinase receptor activation (KIRA) assay, developed by Sadick et al. [1], the STAT5 phosphorylation assay was performed using two separate microtiter plates: the first one for cell stimulation and lysis, the second one for enzyme-linked immuno sorbent assay (ELISA). The assay showed a high accuracy and precision with a mean recovery of 102% and a mean coefficient of variation of 9%. In comparison to the classical proliferation assay, the phosphorylation assay is much faster. Thus, the assay procedure time can at least be reduced from six to three days by using STAT5 phosphorylation instead of proliferation as an endpoint due to the shorter incubation time with IL-7. Moreover, the phosphorylation assay shows a wider dynamic range and higher signal to noise ratios and is thus more robust than the proliferation assay.mAs a consequence, this assay could serve as reliable, accurate, precise and fast alternative to the classical proliferation assay for IL-7. This study also serves as an example for the typical steps during development and qualification / validation of a potency assay for quality control testing.

  8. A Cell-Signaling Network Temporally Resolves Specific versus Promiscuous Phosphorylation

    Directory of Open Access Journals (Sweden)

    Evgeny Kanshin


    Full Text Available If specific and functional kinase- or phosphatase-substrate interactions are optimized for binding compared to promiscuous interactions, then changes in phosphorylation should occur faster on functional versus promiscuous substrates. To test this hypothesis, we designed a high temporal resolution global phosphoproteomics protocol to study the high-osmolarity glycerol (HOG response in the budding yeast Saccharomyces cerevisiae. The method provides accurate, stimulus-specific measurement of phosphoproteome changes, quantitative analysis of phosphodynamics at sub-minute temporal resolution, and detection of more phosphosites. Rates of evolution of dynamic phosphosites were comparable to those of known functional phosphosites and significantly lower than static or longer-time-frame dynamic phosphosites. Kinetic profile analyses indicated that putatively functional kinase- or phosphatase-substrate interactions occur more rapidly, within 60 s, than promiscuous interactions. Finally, we report many changes in phosphorylation of proteins implicated in cytoskeletal and mitotic spindle dynamics that may underlie regulation of cell cycle and morphogenesis.

  9. Characterization of in vivo phosphorylation modification of differentially accumulated proteins in cotton fiber-initiation process. (United States)

    Liu, Wenying; Zhang, Bing; He, Wenying; Wang, Zi; Li, Guanqiao; Liu, Jinyuan


    Initiation of cotton fiber from ovule epidermal cells determines the ultimate number of fibers per cotton ovule, making it one of the restriction factors of cotton fiber yield. Previous comparative proteomics studies have collectively revealed 162 important differentially accumulated proteins (DAPs) in cotton fiber-initiation process, however, whether and how post-translational modifications, especially phosphorylation modification, regulate the expression and function of the DAPs are still unclear. Here we reported the successful identification of 17 phosphopeptides from 16 phosphoproteins out of the 162 DAPs using the integrated bioinformatics analyses of peptide mass fingerprinting data and targeted MS/MS identification method. In-depth analyses indicated that 15 of the 17 phosphorylation sites were novel phosphorylation sites first identified in plants, whereas 6 of the 16 phosphoproteins were found to be the phosphorylated isoforms of 6 proteins. The phosphorylation-regulated dynamic protein network derived from this study not only expanded our understanding of the cotton fiber-initiation process, but also provided a valuable resource for future functional studies of the phosphoproteins. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail:

  10. The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation. (United States)

    Magron, Audrey; Elowe, Sabine; Carreau, Madeleine


    The Fanconi anemia (FA) proteins are involved in a signaling network that assures the safeguard of chromosomes. To understand the function of FA proteins in cellular division events, we investigated the interaction between Stathmin-1 (STMN1) and the FA group C (FANCC) protein. STMN1 is a ubiquitous cytosolic protein that regulates microtubule dynamics. STMN1 activities are regulated through phosphorylation-dephosphorylation mechanisms that control assembly of the mitotic spindle, and dysregulation of STMN1 phosphorylation is associated with mitotic aberrancies leading to chromosome instability and cancer progression. Using different biochemical approaches, we showed that FANCC interacts and co-localizes with STMN1 at centrosomes during mitosis. We also showed that FANCC is required for STMN1 phosphorylation, as mutations in FANCC reduced serine 16- and 38-phosphorylated forms of STMN1. Phosphorylation of STMN1 at serine 16 is likely an event dependent on a functional FA pathway, as it is reduced in FANCA- and FANCD2-mutant cells. Furthermore, FA-mutant cells exhibited mitotic spindle anomalies such as supernumerary centrosomes and shorter mitotic spindles. These results suggest that FA proteins participate in the regulation of cellular division via the microtubule-associated protein STMN1.

  11. The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation.

    Directory of Open Access Journals (Sweden)

    Audrey Magron

    Full Text Available The Fanconi anemia (FA proteins are involved in a signaling network that assures the safeguard of chromosomes. To understand the function of FA proteins in cellular division events, we investigated the interaction between Stathmin-1 (STMN1 and the FA group C (FANCC protein. STMN1 is a ubiquitous cytosolic protein that regulates microtubule dynamics. STMN1 activities are regulated through phosphorylation-dephosphorylation mechanisms that control assembly of the mitotic spindle, and dysregulation of STMN1 phosphorylation is associated with mitotic aberrancies leading to chromosome instability and cancer progression. Using different biochemical approaches, we showed that FANCC interacts and co-localizes with STMN1 at centrosomes during mitosis. We also showed that FANCC is required for STMN1 phosphorylation, as mutations in FANCC reduced serine 16- and 38-phosphorylated forms of STMN1. Phosphorylation of STMN1 at serine 16 is likely an event dependent on a functional FA pathway, as it is reduced in FANCA- and FANCD2-mutant cells. Furthermore, FA-mutant cells exhibited mitotic spindle anomalies such as supernumerary centrosomes and shorter mitotic spindles. These results suggest that FA proteins participate in the regulation of cellular division via the microtubule-associated protein STMN1.

  12. Enhancement of tunability of MAPK cascade due to coexistence of processive and distributive phosphorylation mechanisms. (United States)

    Sun, Jianqiang; Yi, Ming; Yang, Lijian; Wei, Wenbin; Ding, Yiming; Jia, Ya


    The processive phosphorylation mechanism becomes important when there is macromolecular crowding in the cytoplasm. Integrating the processive phosphorylation mechanism with the traditional distributive one, we propose a mixed dual-site phosphorylation (MDP) mechanism in a single-layer phosphorylation cycle. Further, we build a degree model by applying the MDP mechanism to a three-layer mitogen-activated protein kinase (MAPK) cascade. By bifurcation analysis, our study suggests that the crowded-environment-induced pseudoprocessive mechanism can qualitatively change the response of this biological network. By adjusting the degree of processivity in our model, we find that the MAPK cascade is able to switch between the ultrasensitivity, bistability, and oscillatory dynamical states. Sensitivity analysis shows that the theoretical results remain unchanged within a reasonably chosen variation of parameter perturbation. By scaling the reaction rates and also introducing new connections into the kinetic scheme, we further construct a proportion model of the MAPK cascade to validate our findings. Finally, it is illustrated that the spatial propagation of the activated MAPK signal can be improved (or attenuated) by increasing the degree of processivity of kinase (or phosphatase). Our research implies that the MDP mechanism makes the MAPK cascade become a flexible signal module, and the coexistence of processive and distributive phosphorylation mechanisms enhances the tunability of the MAPK cascade.

  13. The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

    Directory of Open Access Journals (Sweden)

    Herrmann Harald


    Full Text Available Abstract Background Colocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested in vitro the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed. Results The results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-α-helical amino-terminal domain of vimentin in vitro. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated in vivo. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association. Conclusion We hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.

  14. Dynamin Reduces Pyk2 Y402 Phosphorylation and Src Binding in Osteoclasts ▿ † (United States)

    Bruzzaniti, Angela; Neff, Lynn; Sandoval, Amanda; Du, Liping; Horne, William C.; Baron, Roland


    Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin's GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2. PMID:19380485

  15. Dynamin reduces Pyk2 Y402 phosphorylation and SRC binding in osteoclasts. (United States)

    Bruzzaniti, Angela; Neff, Lynn; Sandoval, Amanda; Du, Liping; Horne, William C; Baron, Roland


    Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin's GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2.

  16. The role of cyclase-associated protein in regulating actin filament dynamics - more than a monomer-sequestration factor. (United States)

    Ono, Shoichiro


    Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cell biological events. A variety of actin-regulatory proteins modulate polymerization and depolymerization of actin and contribute to actin cytoskeletal reorganization. Cyclase-associated protein (CAP) is a conserved actin-monomer-binding protein that has been studied for over 20 years. Early studies have shown that CAP sequesters actin monomers; recent studies, however, have revealed more active roles of CAP in actin filament dynamics. CAP enhances the recharging of actin monomers with ATP antagonistically to ADF/cofilin, and also promotes the severing of actin filaments in cooperation with ADF/cofilin. Self-oligomerization and binding to other proteins regulate activities and localization of CAP. CAP has crucial roles in cell signaling, development, vesicle trafficking, cell migration and muscle sarcomere assembly. This Commentary discusses the recent advances in our understanding of the functions of CAP and its implications as an important regulator of actin cytoskeletal dynamics, which are involved in various cellular activities.

  17. Lys169 of human glucokinase is a determinant for glucose phosphorylation: implication for the atomic mechanism of glucokinase catalysis.

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    Full Text Available Glucokinase (GK, a glucose sensor, maintains plasma glucose homeostasis via phosphorylation of glucose and is a potential therapeutic target for treating maturity-onset diabetes of the young (MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI. To characterize the catalytic mechanism of glucose phosphorylation by GK, we combined molecular modeling, molecular dynamics (MD simulations, quantum mechanics/molecular mechanics (QM/MM calculations, experimental mutagenesis and enzymatic kinetic analysis on both wild-type and mutated GK. Our three-dimensional (3D model of the GK-Mg(2+-ATP-glucose (GMAG complex, is in agreement with a large number of mutagenesis data, and elucidates atomic information of the catalytic site in GK for glucose phosphorylation. A 10-ns MD simulation of the GMAG complex revealed that Lys169 plays a dominant role in glucose phosphorylation. This prediction was verified by experimental mutagenesis of GK (K169A and enzymatic kinetic analyses of glucose phosphorylation. QM/MM calculations were further used to study the role of Lys169 in the catalytic mechanism of the glucose phosphorylation and we found that Lys169 enhances the binding of GK with both ATP and glucose by serving as a bridge between ATP and glucose. More importantly, Lys169 directly participates in the glucose phosphorylation as a general acid catalyst. Our findings provide mechanistic details of glucose phorphorylation catalyzed by GK, and are important for understanding the pathogenic mechanism of MODY.

  18. Functional analysis of phosphorylation of the mitotic centromere-associated kinesin by Aurora B kinase in human tumor cells. (United States)

    Ritter, Andreas; Sanhaji, Mourad; Friemel, Alexandra; Roth, Susanne; Rolle, Udo; Louwen, Frank; Yuan, Juping


    Mitotic centromere-associated kinesin (MCAK) is the best characterized member of the kinesin-13 family and plays important roles in microtubule dynamics during mitosis. Its activity and subcellular localization is tightly regulated by an orchestra of mitotic kinases, such as Aurora B. It is well known that serine 196 of MCAK is the major phosphorylation site of Aurora B in Xenopus leavis extracts and that this phosphorylation regulates its catalytic activity and subcellular localization. In the current study, we have addressed the conserved phosphorylation site serine 192 in human MCAK to characterize its function in more depth in human cancer cells. Our data confirm that S192 is the major phosphorylation site of Aurora B in human MCAK and that this phosphorylation has crucial roles in regulating its catalytic activity and localization at the kinetochore/centromere region in mitosis. Interfering with this phosphorylation leads to a delayed progression through prometa- and metaphase associated with mitotic defects in chromosome alignment and segregation. We show further that MCAK is involved in directional migration and invasion of tumor cells, and interestingly, interference with the S192 phosphorylation affects this capability of MCAK. These data provide the first molecular explanation for clinical observation, where an overexpression of MCAK was associated with lymphatic invasion and lymph node metastasis in gastric and colorectal cancer patients.

  19. BAD Phosphorylation: A Novel Link between Apoptosis and Cancer


    Polzien, Lisa


    BAD (Bcl-2 antagonist of cell death, Bcl-2 associated death promoter) is a pro-apoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD (mBAD), little data are available with respect to phosphorylation of human BAD (hBAD) protein. In this work, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating se...

  20. Flux control through protein phosphorylation in yeast

    DEFF Research Database (Denmark)

    Chen, Yu; Nielsen, Jens


    describe the development of phosphoproteomics in yeast as well as approaches to analysing the phosphoproteomics data. Finally, we focus on integrated analyses with other omics data sets and genome-scale metabolic models. Despite the advances, future studies improving both experimental technologies......Protein phosphorylation is one of the most important mechanisms regulating metabolism as it can directly modify metabolic enzymes by the addition of phosphate groups. Attributed to such a rapid and reversible mechanism, cells can adjust metabolism rapidly in response to temporal changes. The yeast...... as well as identify mechanisms underlying human metabolic diseases. Here we collect functional phosphorylation events of 41 enzymes involved in yeast metabolism and demonstrate functional mechanisms and the application of this information in metabolic engineering. From a systems biology perspective, we...

  1. Phosphorylation sites within Ebola virus nucleoprotein

    Directory of Open Access Journals (Sweden)

    Sora Yasri


    Full Text Available To understand the infection process, the viral multiplication and entry to the cell is widely studied. The Ebola virus nucleoprotein is the important problem for the pathological process. Focusing on the specific biological process, the post translational modification is needed. Here, the authors used the bioinformatics study to find the phosphorylation sites within the Ebola virus nucleoprotein and could identify many new sites.

  2. Protein phosphorylation in bcterial signaling and regulation

    KAUST Repository

    Mijakovic, Ivan


    In 2003, it was demonstrated for the first time that bacteria possess protein-tyrosine kinases (BY-kinases), capable of phosphorylating other cellular proteins and regulating their activity. It soon became apparent that these kinases phosphorylate a number of protein substrates, involved in different cellular processes. More recently, we found out that BY-kinases can be activated by several distinct protein interactants, and are capable of engaging in cross-phosphorylation with other kinases. Evolutionary studies based on genome comparison indicate that BY-kinases exist only in bacteria. They are non-essential (present in about 40% bacterial genomes), and their knockouts lead to pleiotropic phenotypes, since they phosphorylate many substrates. Surprisingly, BY-kinase genes accumulate mutations at an increased rate (non-synonymous substitution rate significantly higher than other bacterial genes). One direct consequence of this phenomenon is no detectable co-evolution between kinases and their substrates. Their promiscuity towards substrates thus seems to be “hard-wired”, but why would bacteria maintain such promiscuous regulatory devices? One explanation is the maintenance of BY-kinases as rapidly evolving regulators, which can readily adopt new substrates when environmental changes impose selective pressure for quick evolution of new regulatory modules. Their role is clearly not to act as master regulators, dedicated to triggering a single response, but they might rather be employed to contribute to fine-tuning and improving robustness of various cellular responses. This unique feature makes BY-kinases a potentially useful tool in synthetic biology. While other bacterial kinases are very specific and their signaling pathways insulated, BY-kinase can relatively easily be engineered to adopt new substrates and control new biosynthetic processes. Since they are absent in humans, and regulate some key functions in pathogenic bacteria, they are also very promising

  3. Solid polymer electrolyte from phosphorylated chitosan

    Energy Technology Data Exchange (ETDEWEB)

    Fauzi, Iqbal, E-mail:; Arcana, I Made, E-mail: [Inorganic and Physical Chemistry Research Groups, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132 (Indonesia)


    Recently, the need of secondary battery application continues to increase. The secondary battery which using a liquid electrolyte was indicated had some weakness. A solid polymer electrolyte is an alternative electrolytes membrane which developed in order to replace the liquid electrolyte type. In the present study, the effect of phosphorylation on to polymer electrolyte membrane which synthesized from chitosan and lithium perchlorate salts was investigated. The effect of the component’s composition respectively on the properties of polymer electrolyte, was carried out by analyzed of it’s characterization such as functional groups, ion conductivity, and thermal properties. The mechanical properties i.e tensile resistance and the morphology structure of membrane surface were determined. The phosphorylation processing of polymer electrolyte membrane of chitosan and lithium perchlorate was conducted by immersing with phosphoric acid for 2 hours, and then irradiated on a microwave for 60 seconds. The degree of deacetylation of chitosan derived from shrimp shells was obtained around 75.4%. Relative molecular mass of chitosan was obtained by viscometry method is 796,792 g/mol. The ionic conductivity of chitosan membrane was increase from 6.33 × 10{sup −6} S/cm up to 6.01 × 10{sup −4} S/cm after adding by 15 % solution of lithium perchlorate. After phosphorylation, the ionic conductivity of phosphorylated lithium chitosan membrane was observed 1.37 × 10{sup −3} S/cm, while the tensile resistance of 40.2 MPa with a better thermal resistance. On the strength of electrolyte membrane properties, this polymer electrolyte membrane was suggested had one potential used for polymer electrolyte in field of lithium battery applications.

  4. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Brunak, Søren; Olsen, JV


    ) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells....

  5. Phosphorylation of erythrocyte membrane liberates calcium

    Energy Technology Data Exchange (ETDEWEB)

    Chauhan, V.P.S.; Brockerhoff, H.


    Phosphorylation of permeabilized erythrocyte ghost membranes with ATP results in an increase free calcium level as measured with the help of Ca/sup 2 +/ electrode and /sup 45/Ca. This effect could not be observed in the presence of p/sup -/ chloromercuric benzoate, an inhibitor of kinases. The rise in the free calcium due to phosphorylation of the membrane was accompanied by a decrease in the level of phosphatidylinositol (PI) and an increase in phosphatidylinositolmonophosphate (PIP) and phosphatidylinositolbisphosphate (PIP/sub 2/). These results support the proposal that an inositol shuttle, PI in equilibrium PIP in equilibrium PIP/sub 2/, operates to maintain the intracellular calcium concentration. The cation is believed to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with the participation of both PO and fatty acid ester CO groups. When the inositol group of such a cage is phosphorylated, inter-headgroup hydrogen bonding between the lipids is broken. As a result the cage opens and calcium is released.

  6. Phosphorylation of Astrin Regulates Its Kinetochore Function. (United States)

    Chung, Hee Jin; Park, Ji Eun; Lee, Nam Soo; Kim, Hongtae; Jang, Chang-Young


    The error-free segregation of chromosomes, which requires the precisely timed search and capture of chromosomes by spindles during early mitotic and meiotic cell division, is responsible for genomic stability and is achieved by the spindle assembly checkpoint in the metaphase-anaphase transition. Mitotic kinases orchestrate M phase events, such as the reorganization of cell architecture and kinetochore (KT) composition with the exquisite phosphorylation of mitotic regulators, to ensure timely and temporal progression. However, the molecular mechanisms underlying the changes of KT composition for stable spindle attachment during mitosis are poorly understood. Here, we show that the sequential action of the kinase Cdk1 and the phosphatase Cdc14A control spindle attachment to KTs. During prophase, the mitotic spindle protein Spag5/Astrin is transported into centrosomes by Kinastrin and phosphorylated at Ser-135 and Ser-249 by Cdk1, which, in prometaphase, is loaded onto the spindle and targeted to KTs. We also demonstrate that Cdc14A dephosphorylates Astrin, and therefore the overexpression of Cdc14A sequesters Astrin in the centrosome and results in aberrant chromosome alignment. Mechanistically, Plk1 acts as an upstream kinase for Astrin phosphorylation by Cdk1 and targeting phospho-Astrin to KTs, leading to the recruitment of outer KT components, such as Cenp-E, and the stable attachment of spindles to KTs. These comprehensive findings reveal a regulatory circuit for protein targeting to KTs that controls the KT composition change of stable spindle attachment and chromosome integrity.

  7. Control mechanisms in mitochondrial oxidative phosphorylation

    Institute of Scientific and Technical Information of China (English)

    Jana Hroudová; Zdeněk Fi(s)ar


    Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5'- triphosphate production is regulated by many control mechanism–firstly by oxygen, substrate level, adenosine-5'-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by "second control mechanisms," such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5'-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.

  8. Flagellin FliC Phosphorylation Affects Type 2 Protease Secretion and Biofilm Dispersal in Pseudomonas aeruginosa PAO1 (United States)

    Suriyanarayanan, Tanujaa; Periasamy, Saravanan; Lin, Miao-Hsia; Ishihama, Yasushi; Swarup, Sanjay


    Protein phosphorylation has a major role in controlling the life-cycle and infection stages of bacteria. Proteome-wide occurrence of S/T/Y phosphorylation has been reported for many prokaryotic systems. Previously, we reported the phosphoproteome of Pseudomonas aeruginosa and Pseudomonas putida. In this study, we show the role of S/T phosphorylation of one motility protein, FliC, in regulating multiple surface-associated phenomena of P. aeruginosa PAO1. This is the first report of occurrence of phosphorylation in the flagellar protein, flagellin FliC in its highly conserved N-terminal NDO domain across several Gram negative bacteria. This phosphorylation is likely a well-regulated phenomenon as it is growth phase dependent in planktonic cells. The absence of phosphorylation in the conserved T27 and S28 residues of FliC, interestingly, did not affect swimming motility, but affected the secretome of type 2 secretion system (T2SS) and biofilm formation of PAO1. FliC phosphomutants had increased levels and activities of type 2 secretome proteins. The secretion efficiency of T2SS machinery is associated with flagellin phosphorylation. FliC phosphomutants also formed reduced biofilms at 24 h under static conditions and had delayed biofilm dispersal under dynamic flow conditions, respectively. The levels of type 2 secretome and biofilm formation under static conditions had an inverse correlation. Hence, increase in type 2 secretome levels was accompanied by reduced biofilm formation in the FliC phosphomutants. As T2SS is involved in nutrient acquisition and biofilm dispersal during survival and spread of P. aeruginosa, we propose that FliC phosphorylation has a role in ecological adaptation of this opportunistic environmental pathogen. Altogether, we found a system of phosphorylation that affects key surface related processes such as proteases secretion by T2SS, biofilm formation and dispersal. PMID:27701473

  9. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Rashna Bhandari; Roy Mathew; K Vijayachandra; Sandhya S Visweswariah


    Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.

  10. A phosphorylation cascade controls the degradation of active SREBP1. (United States)

    Bengoechea-Alonso, Maria T; Ericsson, Johan


    Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes.

  11. A strategy to quantitate global phosphorylation of bone matrix proteins. (United States)

    Sroga, Grażyna E; Vashishth, Deepak


    Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured "in bulk" are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.

  12. Multistep phosphorylation systems: tunable components of biological signaling circuits. (United States)

    Valk, Evin; Venta, Rainis; Ord, Mihkel; Faustova, Ilona; Kõivomägi, Mardo; Loog, Mart


    Multisite phosphorylation of proteins is a powerful signal processing mechanism that plays crucial roles in cell division and differentiation as well as in disease. We recently demonstrated a novel phenomenon in cell cycle regulation by showing that cyclin-dependent kinase-dependent multisite phosphorylation of a crucial substrate is performed sequentially in the N-to-C terminal direction along the disordered protein. The process is controlled by key parameters, including the distance between phosphorylation sites, the distribution of serines and threonines in sites, and the position of docking motifs. According to our model, linear patterns of phosphorylation along disordered protein segments determine the signal-response function of a multisite phosphorylation switch. Here we discuss the general advantages and engineering principles of multisite phosphorylation networks as processors of kinase signals. We also address the idea of using the mechanistic logic of linear multisite phosphorylation networks to design circuits for synthetic biology applications.

  13. Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria. (United States)

    Kavanagh, N I; Ainscow, E K; Brand, M D


    Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.

  14. Phosphorylated testis-specific serine/threonine kinase 4 may phosphorylate Crem at Ser-117. (United States)

    Fu, Guolong; Wei, Youheng; Wang, Xiaoli; Yu, Long


    We aimed to investigate the internal existence status of testis-specific serine/threonine kinase 4 (Tssk4) and the interaction of Tssk4 and Cre-responsive element modulator (Crem). The internal existence status of Tssk4 in testis of mice was detected using western blotting and dephosphorylation method. The interaction of Tssk4 and Crem was analyzed by western blotting, immunohistochemistry, immunofluorescence, in vitro co-immunoprecipitation assays, and in vitro kinase assay. The results revealed that Tssk4 existed in testis both in phosphorylation and unphosphorylation status by a temporal manner with the development of testis. Immunofluorescence results showed that Tssk4 had identical distribution pattern with Crem in testis, which was utterly different to the localization of Cre-responsive element binding (Creb). In conclusion, our study demonstrated that phosphorylated Tssk4 might participate in testis genes expressions by phosphorylating Crem at Ser-117.

  15. PhosTryp: a phosphorylation site predictor specific for parasitic protozoa of the family trypanosomatidae

    Directory of Open Access Journals (Sweden)

    Palmeri Antonio


    Full Text Available Abstract Background Protein phosphorylation modulates protein function in organisms at all levels of complexity. Parasites of the Leishmania genus undergo various developmental transitions in their life cycle triggered by changes in the environment. The molecular mechanisms that these organisms use to process and integrate these external cues are largely unknown. However Leishmania lacks transcription factors, therefore most regulatory processes may occur at a post-translational level and phosphorylation has recently been demonstrated to be an important player in this process. Experimental identification of phosphorylation sites is a time-consuming task. Moreover some sites could be missed due to the highly dynamic nature of this process or to difficulties in phospho-peptide enrichment. Results Here we present PhosTryp, a phosphorylation site predictor specific for trypansomatids. This method uses an SVM-based approach and has been trained with recent Leishmania phosphosproteomics data. PhosTryp achieved a 17% improvement in prediction performance compared with Netphos, a non organism-specific predictor. The analysis of the peptides correctly predicted by our method but missed by Netphos demonstrates that PhosTryp captures Leishmania-specific phosphorylation features. More specifically our results show that Leishmania kinases have sequence specificities which are different from their counterparts in higher eukaryotes. Consequently we were able to propose two possible Leishmania-specific phosphorylation motifs. We further demonstrate that this improvement in performance extends to the related trypanosomatids Trypanosoma brucei and Trypanosoma cruzi. Finally, in order to maximize the usefulness of PhosTryp, we trained a predictor combining all the peptides from L. infantum, T. brucei and T. cruzi. Conclusions Our work demonstrates that training on organism-specific data results in an improvement that extends to related species. PhosTryp is freely

  16. Activity-dependent Phosphorylation of Neuronal Kv2.1 Potassium Channels by CDK5*


    Cerda, Oscar; Trimmer, James S.


    Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible,...

  17. Genetic Manipulation of Neurofilament Protein Phosphorylation. (United States)

    Jones, Maria R; Villalón, Eric; Garcia, Michael L


    Neurofilament biology is important to understanding structural properties of axons, such as establishment of axonal diameter by radial growth. In order to study the function of neurofilaments, a series of genetically modified mice have been generated. Here, we describe a brief history of genetic modifications used to study neurofilaments, as well as an overview of the steps required to generate a gene-targeted mouse. In addition, we describe steps utilized to analyze neurofilament phosphorylation status using immunoblotting. Taken together, these provide comprehensive analysis of neurofilament function in vivo, which can be applied to many systems.

  18. A Simple Hydraulic Analog Model of Oxidative Phosphorylation. (United States)

    Willis, Wayne T; Jackman, Matthew R; Messer, Jeffrey I; Kuzmiak-Glancy, Sarah; Glancy, Brian


    Mitochondrial oxidative phosphorylation is the primary source of cellular energy transduction in mammals. This energy conversion involves dozens of enzymatic reactions, energetic intermediates, and the dynamic interactions among them. With the goal of providing greater insight into the complex thermodynamics and kinetics ("thermokinetics") of mitochondrial energy transduction, a simple hydraulic analog model of oxidative phosphorylation is presented. In the hydraulic model, water tanks represent the forward and back "pressures" exerted by thermodynamic driving forces: the matrix redox potential (ΔGredox), the electrochemical potential for protons across the mitochondrial inner membrane (ΔGH), and the free energy of adenosine 5'-triphosphate (ATP) (ΔGATP). Net water flow proceeds from tanks with higher water pressure to tanks with lower pressure through "enzyme pipes" whose diameters represent the conductances (effective activities) of the proteins that catalyze the energy transfer. These enzyme pipes include the reactions of dehydrogenase enzymes, the electron transport chain (ETC), and the combined action of ATP synthase plus the ATP-adenosine 5'-diphosphate exchanger that spans the inner membrane. In addition, reactive oxygen species production is included in the model as a leak that is driven out of the ETC pipe by high pressure (high ΔGredox) and a proton leak dependent on the ΔGH for both its driving force and the conductance of the leak pathway. Model water pressures and flows are shown to simulate thermodynamic forces and metabolic fluxes that have been experimentally observed in mammalian skeletal muscle in response to acute exercise, chronic endurance training, and reduced substrate availability, as well as account for the thermokinetic behavior of mitochondria from fast- and slow-twitch skeletal muscle and the metabolic capacitance of the creatine kinase reaction.

  19. Ultrasensitivity in phosphorylation-dephosphorylation cycles with little substrate.

    Directory of Open Access Journals (Sweden)

    Bruno M C Martins

    Full Text Available Cellular decision-making is driven by dynamic behaviours, such as the preparations for sunrise enabled by circadian rhythms and the choice of cell fates enabled by positive feedback. Such behaviours are often built upon ultrasensitive responses where a linear change in input generates a sigmoidal change in output. Phosphorylation-dephosphorylation cycles are one means to generate ultrasensitivity. Using bioinformatics, we show that in vivo levels of kinases and phosphatases frequently exceed the levels of their corresponding substrates in budding yeast. This result is in contrast to the conditions often required by zero-order ultrasensitivity, perhaps the most well known means for how such cycles become ultrasensitive. We therefore introduce a mechanism to generate ultrasensitivity when numbers of enzymes are higher than numbers of substrates. Our model combines distributive and non-distributive actions of the enzymes with two-stage binding and concerted allosteric transitions of the substrate. We use analytical and numerical methods to calculate the Hill number of the response. For a substrate with [Formula: see text] phosphosites, we find an upper bound of the Hill number of [Formula: see text], and so even systems with a single phosphosite can be ultrasensitive. Two-stage binding, where an enzyme must first bind to a binding site on the substrate before it can access the substrate's phosphosites, allows the enzymes to sequester the substrate. Such sequestration combined with competition for each phosphosite provides an intuitive explanation for the sigmoidal shifts in levels of phosphorylated substrate. Additionally, we find cases for which the response is not monotonic, but shows instead a peak at intermediate levels of input. Given its generality, we expect the mechanism described by our model to often underlay decision-making circuits in eukaryotic cells.

  20. Phosphorylation of hepatitis C virus RNA polymerases ser29 and ser42 by protein kinase C-related kinase 2 regulates viral RNA replication. (United States)

    Han, Song-Hee; Kim, Seong-Jun; Kim, Eun-Jung; Kim, Tae-Eun; Moon, Jae-Su; Kim, Geon-Woo; Lee, Seung-Hoon; Cho, Kun; Yoo, Jong Shin; Son, Woo Sung; Rhee, Jin-Kyu; Han, Seung Hyun; Oh, Jong-Won


    NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation in the Δ1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and their impacts on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  1. Phenobarbital Meets Phosphorylation of Nuclear Receptors. (United States)

    Negishi, Masahiko


    Phenobarbital was the first therapeutic drug to be characterized for its induction of hepatic drug metabolism. Essentially at the same time, cytochrome P450, an enzyme that metabolizes drugs, was discovered. After nearly 50 years of investigation, the molecular target of phenobarbital induction has now been delineated to phosphorylation at threonine 38 of the constitutive androstane receptor (NR1I3), a member of the nuclear receptor superfamily. Determining this mechanism has provided us with the molecular basis to understand drug induction of drug metabolism and disposition. Threonine 38 is conserved as a phosphorylation motif in the majority of both mouse and human nuclear receptors, providing us with an opportunity to integrate diverse functions of nuclear receptors. Here, I review the works and accomplishments of my laboratory at the National Institutes of Health National Institute of Environmental Health Sciences and the future research directions of where our study of the constitutive androstane receptor might take us. U.S. Government work not protected by U.S. copyright.

  2. Modelling the Krebs cycle and oxidative phosphorylation. (United States)

    Korla, Kalyani; Mitra, Chanchal K


    The Krebs cycle and oxidative phosphorylation are the two most important sets of reactions in a eukaryotic cell that meet the major part of the total energy demands of a cell. In this paper, we present a computer simulation of the coupled reactions using open source tools for simulation. We also show that it is possible to model the Krebs cycle with a simple black box with a few inputs and outputs. However, the kinetics of the internal processes has been modelled using numerical tools. We also show that the Krebs cycle and oxidative phosphorylation together can be combined in a similar fashion - a black box with a few inputs and outputs. The Octave script is flexible and customisable for any chosen set-up for this model. In several cases, we had no explicit idea of the underlying reaction mechanism and the rate determining steps involved, and we have used the stoichiometric equations that can be easily changed as and when more detailed information is obtained. The script includes the feedback regulation of the various enzymes of the Krebs cycle. For the electron transport chain, the pH gradient across the membrane is an essential regulator of the kinetics and this has been modelled empirically but fully consistent with experimental results. The initial conditions can be very easily changed and the simulation is potentially very useful in a number of cases of clinical importance.

  3. Prebiotic Phosphorylation Reactions on the Early Earth

    Directory of Open Access Journals (Sweden)

    Maheen Gull


    Full Text Available Phosphorus (P is an essential element for life. It occurs in living beings in the form of phosphate, which is ubiquitous in biochemistry, chiefly in the form of C-O-P (carbon, oxygen and phosphorus, C-P, or P-O-P linkages to form life. Within prebiotic chemistry, several key questions concerning phosphorus chemistry have developed: what were the most likely sources of P on the early Earth? How did it become incorporated into the biological world to form the P compounds that life employs today? Can meteorites be responsible for the delivery of P? What were the most likely solvents on the early Earth and out of those which are favorable for phosphorylation? Or, alternatively, were P compounds most likely produced in relatively dry environments? What were the most suitable temperature conditions for phosphorylation? A route to efficient formation of biological P compounds is still a question that challenges astrobiologists. This article discusses these important issues related to the origin of biological P compounds.

  4. A proteomic approach for quantitation of phosphorylation using stable isotope labeling in cell culture. (United States)

    Ibarrola, Nieves; Kalume, Dario E; Gronborg, Mads; Iwahori, Akiko; Pandey, Akhilesh


    Posttranslational modifications are major mechanisms of regulating protein activity and function in vertebrate cells. It is essential to obtain qualitative information about posttranslational modification patterns of proteins to understand signal transduction mechanisms in greater detail. However, it is equally important to measure the dynamics of posttranslational modifications such as phosphorylation to approach signaling networks from a systems biology perspective. Despite a number of advances, methods to quantitate posttranslational modifications remain difficult to implement due to a number of factors including lack of a generic method, elaborate chemical steps, and requirement for large amounts of sample. We have previously shown that stable isotope-containing amino acids in cell culture (SILAC) can be used to differentially label growing cell populations for quantitation of protein levels. In this report, we extend the use of SILAC as a novel proteomic approach for the relative quantitation of posttranslational modifications such as phosphorylation. We have used SILAC to quantitate the extent of known phosphorylation sites as well as to identify and quantitate novel phosphorylation sites.

  5. Gβ promotes pheromone receptor polarization and yeast chemotropism by inhibiting receptor phosphorylation. (United States)

    Ismael, Amber; Tian, Wei; Waszczak, Nicholas; Wang, Xin; Cao, Youfang; Suchkov, Dmitry; Bar, Eli; Metodiev, Metodi V; Liang, Jie; Arkowitz, Robert A; Stone, David E


    Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin cable-dependent delivery of vesicles to the plasma membrane (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone receptor by interacting with Yck1/2 and locally inhibiting receptor phosphorylation. We also found that receptor phosphorylation is essential for chemotropism, independently of its role in inducing receptor internalization. A mathematical model supports the idea that the interaction between Gβγ and Yck1/2 results in differential phosphorylation and internalization of the pheromone receptor and accounts for its polarization before the initiation of directed secretion.

  6. New insights into roles of intermediate filament phosphorylation and progeria pathogenesis. (United States)

    Goto, Hidemasa; Inagaki, Masaki


    Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Because of their insoluble nature, cellular IFs had been considered to be stable for a long time. The discovery that a purified protein kinase phosphorylated a purified IF protein and in turn induced the disassembly of IF structure in vitro led to the novel concept of dynamic IF regulation. Since then, a variety of protein kinases have been identified to phosphorylate IF proteins such as vimentin in a spatiotemporal regulated manner. A series of studies using cultured cells have demonstrated that preventing IF phosphorylation during mitosis inhibits cytokinesis by the retention of an IF bridge-like structure (IF-bridge) connecting the two daughter cells. Knock-in mice expressing phosphodeficient vimentin variants developed binucleation/aneuploidy in lens epithelial cells, which promoted microophthalmia and lens cataract. Therefore, mitotic phosphorylation of vimentin is of great importance in the completion of cytokinesis, the impairment of which promotes chromosomal instability and premature aging. © 2014 IUBMB Life, 2014.

  7. Vimentin expression influences flow dependent VASP phosphorylation and regulates cell migration and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Lund, Natalie [University Hospital Luebeck, Medical Department II, Experimental Angiology, Luebeck (Germany); Henrion, Daniel [Universite d' Angers, INSERM U771, CNRS UMR6214, UFR Sciences Mediales, Angers (France); Tiede, Petra [University Hospital Hamburg Eppendorf, Department of Hepatobiliary and Transplant Surgery, Hamburg (Germany); Ziche, Marina [University of Siena, Department of Molecular Biology, Siena (Italy); Schunkert, Heribert [University Hospital Luebeck, Medical Department II, Experimental Angiology, Luebeck (Germany); Ito, Wulf D., E-mail: [University Hospital Luebeck, Medical Department II, Experimental Angiology, Luebeck (Germany); Cardiovascular Center Oberallgaeu, Academic Teaching Hospital, University of Ulm, Immenstadt (Germany)


    The cytoskeleton plays a central role for the integration of biochemical and biomechanical signals across the cell required for complex cellular functions. Recent studies indicate that the intermediate filament vimentin is necessary for endothelial cell morphogenesis e.g. in the context of leukocyte transmigration. Here, we present evidence, that the scaffold provided by vimentin is essential for VASP localization and PKG mediated VASP phosphorylation and thus controls endothelial cell migration and proliferation. Vimentin suppression using siRNA technique significantly decreased migration velocity by 50% (videomicroscopy), diminished transmigration activity by 42.5% (Boyden chamber) and reduced proliferation by 43% (BrdU-incorporation). In confocal microscopy Vimentin colocalized with VASP and PKG in endothelial cells. Vimentin suppression was accompanied with a translocation of VASP from focal contacts to the perinuclear region. VASP/Vimentin and PKG/Vimentin colocalization appeared to be essential for proper PKG mediated VASP phosphorylation because we detected a diminished expression of PKG and p{sup Ser239}-VASP in vimentin-suppressed cells, Furthermore, the induction of VASP phosphorylation in perfused arteries was markedly decreased in vimentin knockout mice compared to wildtypes. A link is proposed between vimentin, VASP phosphorylation and actin dynamics that delivers an explanation for the important role of vimentin in controlling endothelial cell morphogenesis.

  8. Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism. (United States)

    Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich


    Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

  9. Diverse roles for auxiliary subunits in phosphorylation-dependent regulation of mammalian brain voltage-gated potassium channels. (United States)

    Vacher, Helene; Trimmer, James S


    Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse partly due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here, we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels.

  10. Kinetic analyses of phosphorylated and non-phosphorylated eIFiso4E binding to mRNA cap analogues. (United States)

    Khan, Mateen A; Goss, Dixie J


    Phosphorylation of eukaryotic initiation factors was previously shown to interact with m(7)G cap and play an important role in the regulation of translation initiation of protein synthesis. To gain further insight into the phosphorylation process of plant protein synthesis, the kinetics of phosphorylated wheat eIFiso4E binding to m(7)G cap analogues were examined. Phosphorylation of wheat eIFiso4E showed similar kinetic effects to human eIF4E binding to m(7)-G cap. Phosphorylation of eIFiso4E decreased the kinetic rate (2-fold) and increased the dissociation rate (2-fold) as compared to non-phosphorylated eIFiso4E binding to both mono- and di-nucleotide analogues at 22°C. Phosphorylated and non-phosphorylated eIFiso4E-m(7)G cap binding rates were found to be independent of concentration, suggesting conformational changes were rate limiting. Rate constant for phosphorylated and non-phosphorylated eIFiso4E binding to m(7)-G cap increased with temperature. Phosphorylation of eIFiso4E decreased (2-fold) the activation energy for both m(7)-G cap analogues binding as compared to non-phosphorylated eIFiso4E. The reduced energy barrier for the formation of eIFiso4E-m(7)-G cap complex suggests a more stable platform for further initiation complex formation and possible means of adapting variety of environmental conditions. Furthermore, the formation of phosphorylated eIFiso4E-cap complex may contribute to modulation of the initiation of protein synthesis in plants. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides (United States)

    Bertran-Vicente, Jordi; Penkert, Martin; Nieto-Garcia, Olaia; Jeckelmann, Jean-Marc; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P. R.


    In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

  12. Phosphorylation modifies the molecular stability of β-amyloid deposits (United States)

    Rezaei-Ghaleh, Nasrollah; Amininasab, Mehriar; Kumar, Sathish; Walter, Jochen; Zweckstetter, Markus


    Protein aggregation plays a crucial role in neurodegenerative diseases. A key feature of protein aggregates is their ubiquitous modification by phosphorylation. Little is known, however, about the molecular consequences of phosphorylation of protein aggregates. Here we show that phosphorylation of β-amyloid at serine 8 increases the stability of its pathogenic aggregates against high-pressure and SDS-induced dissociation. We further demonstrate that phosphorylation results in an elevated number of hydrogen bonds at the N terminus of β-amyloid, the region that is critically regulated by a variety of post-translational modifications. Because of the increased lifetime of phosphorylated β-amyloid aggregates, phosphorylation can promote the spreading of β-amyloid in Alzheimer pathogenesis. Our study suggests that regulation of the molecular stability of protein aggregates by post-translational modifications is a crucial factor for disease progression in the brain.

  13. Control of Collagen Triple Helix Stability by Phosphorylation. (United States)

    Acevedo-Jake, Amanda M; Ngo, Daniel H; Hartgerink, Jeffrey D


    The phosphorylation of the collagen triple helix plays an important role in collagen synthesis, assembly, signaling, and immune response, although no reports detailing the effect this modification has on the structure and stability of the triple helix exist. Here we investigate the changes in stability and structure resulting from the phosphorylation of collagen. Additionally, the formation of pairwise interactions between phosphorylated residues and lysine is examined. In all tested cases, phosphorylation increases helix stability. When charged-pair interactions are possible, stabilization via phosphorylation can play a very large role, resulting inasmuch as a 13.0 °C increase in triple helix stability. Two-dimensional NMR and molecular modeling are used to study the local structure of the triple helix. Our results suggest a mechanism of action for phosphorylation in the regulation of collagen and also expand upon our understanding of pairwise amino acid stabilization of the collagen triple helix.

  14. Chemical Approaches to Studying Labile Amino Acid Phosphorylation. (United States)

    Marmelstein, Alan M; Moreno, Javier; Fiedler, Dorothea


    Phosphorylation of serine, threonine, and tyrosine residues is the archetypal posttranslational modification of proteins. While phosphorylation of these residues has become standard textbook knowledge, phosphorylation of other amino acid side chains is underappreciated and minimally characterized by comparison. This disparity is rooted in the relative instability of these chemically distinct amino acid side chain moieties, namely phosphoramidates, acyl phosphates, thiophosphates, and phosphoanhydrides. In the case of the O-phosphorylated amino acids, synthetic constructs were critical to assessing their stability and developing tools for their study. As the chemical biology community has become more aware of these alternative phosphorylation sites, methodology has been developed for the synthesis of well-characterized standards and close mimics of these phosphorylated amino acids as well. In this article, we review the synthetic chemistry that is a prerequisite to progress in this field.

  15. Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria. (United States)

    Gnad, Florian; Forner, Francesca; Zielinska, Dorota F; Birney, Ewan; Gunawardena, Jeremy; Mann, Matthias


    High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the

  16. Synthesis of O-Phosphorylated Oligopeptides Using Phosphoramidite

    Institute of Scientific and Technical Information of China (English)


    Reversible protein phosphorylation is of great importance in the regulation of many cellular processes. Structurally well-defined compounds are needed for the study of the roles of the phospho-proteins in biological processes. In this paper, O-phosphorylated oligopeptides were synthesized using bis-alkyloxy-N,N-dialkylphosphoramidite reacting with the oligopeptide followed by oxidation. Many hydroxyl groups in oligopeptides can be phosphorylated in one step.

  17. Constitutive phosphorylation of Shc proteins in human tumors

    DEFF Research Database (Denmark)

    Pelicci, G; Lanfrancone, L; Salcini, A E


    cells. In tumor cells with known TK gene alterations Shc proteins were constitutively phosphorylated and complexed with the activated TK. No constitutive Shc phosphorylation was found in primary cell cultures and normal tissues. In 14 of 27 tumor cell lines with no reported TK alterations, Shc proteins...... activated TKs and that the analysis of Shc phosphorylation allow the identification of tumors with constitutive TK activation....

  18. A New Intermolecular Phosphoryl Transfer between Serine and Histidine Residues

    Institute of Scientific and Technical Information of China (English)

    SU,Yu-Qian; NIU,Ming-Yu; CAO,Shu-Xia; ZHANG,Jian-Chen; QU,Ling-Bo; LIAO,Xin-Cheng; ZHAO,Yu-Fen


    @@ Phosphoryl transfer constitutes one of the most important reactions in functionalized molecules, bioorganic chemistry and biochemistry.[1] The transformations are involved in diverse processes, such as activated state change of phosphorus, DNA/RNA synthesis, energy metabolism and signal transduction. So, phosphoryl transfer reaction which can be performed by either intramolecular or intermolecular phosphorylation and dephosphorylation mechanism has been investigated by many scientists in wide fields.

  19. A Crystallographic Snapshot of Tyrosine Trans-phosphorylation in Action

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Xu, C; Ma, J; Eliseenkova, A; Li, W; Pollock, P; Pitteloud, N; Miller, W; Neubert, T; Mohammadi, M


    Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 {angstrom} away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

  20. The importance of intrinsically disordered segments of cardiac troponin in modulating function by phosphorylation and disease-causing mutations

    Directory of Open Access Journals (Sweden)

    Maria Papadaki


    Full Text Available Troponin plays a central role in regulation of muscle contraction. It is the Ca2+ switch of striated muscles including the heart and in the cardiac muscle is physiologically modulated by PKA-dependent phosphorylation at Ser22 and 23. Many cardiomyopathy-related mutations affect Ca2+ regulation and/or disrupt the relationship between Ca2+ binding and phosphorylation. Unlike the mechanism of heart activation, the modulation of Ca2+-sensitivity by phosphorylation of the cardiac specific N-terminal segment of TnI (1-30 is structurally subtle and has proven hard to investigate. The crystal structure of cardiac troponin describes only the relatively stable core of the molecule and the crucial mobile parts of the molecule are missing including TnI C terminal region, TnI (1-30, TnI (134-149 (‘inhibitory’ peptide and the C-terminal 28 amino acids of TnT that are intrinsically disordered.Recent studies over the years have been performed to answer this matter by building structural models of cardiac troponin in phosphorylated and dephosphorylated states based on peptide NMR studies. Now these have been updated by more recent concepts derived from molecular dynamic simulations treating troponin as a dynamic structure. The emerging model confirms the stable core structure of troponin and the mobile structure of the intrinsically disordered segments. We will discuss how we can describe these segments in terms of dynamic transitions between a small number of states with the probability distributions being altered by phosphorylation and by HCM or DCM-related mutations that can explain how Ca2+-sensitivity is modulated by phosphorylation and the effects of mutations.

  1. TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux (United States)

    Fu, Jingyan; Bian, Minglei; Xin, Guangwei; Deng, Zhaoxuan; Luo, Jia; Guo, Xiao; Chen, Hao; Wang, Yao; Jiang, Qing


    A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A–dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux. PMID:26240182

  2. Phosphorylation of a central clock transcription factor is required for thermal but not photic entrainment.

    Directory of Open Access Journals (Sweden)

    Euna Lee


    Full Text Available Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK, the master transcription factor driving cyclical gene expression and the rate-limiting component in the Drosophila circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a Drosophila tissue culture system, we identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A rescues the arrythmicity of Clk(out flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression.

  3. A grammar inference approach for predicting kinase specific phosphorylation sites. (United States)

    Datta, Sutapa; Mukhopadhyay, Subhasis


    Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.

  4. Phosphorylation Modulates Ameloblastin Self-assembly and Ca2+ Binding

    Directory of Open Access Journals (Sweden)

    Øystein Stakkestad


    Full Text Available Ameloblastin (AMBN, an important component of the self-assembled enamel extra cellular matrix, contains several in silico predicted phosphorylation sites. However, to what extent these sites actually are phosphorylated and the possible effects of such post-translational modifications are still largely unknown. Here we report on in vitro experiments aimed at investigating what sites in AMBN are phosphorylated by casein kinase 2 (CK2 and protein kinase A (PKA and the impact such phosphorylation has on self-assembly and calcium binding. All predicted sites in AMBN can be phosphorylated by CK2 and/or PKA. The experiments show that phosphorylation, especially in the exon 5 derived part of the molecule, is inversely correlated with AMBN self-assembly. These results support earlier findings suggesting that AMBN self-assembly is mostly dependent on the exon 5 encoded region of the AMBN gene. Phosphorylation was significantly more efficient when the AMBN molecules were in solution and not present as supramolecular assemblies, suggesting that post-translational modification of AMBN must take place before the enamel matrix molecules self-assemble inside the ameloblast cell. Moreover, phosphorylation of exon 5, and the consequent reduction in self-assembly, seem to reduce the calcium binding capacity of AMBN suggesting that post-translational modification of AMBN also can be involved in control of free Ca2+ during enamel extra cellular matrix biomineralization. Finally, it is speculated that phosphorylation can provide a functional crossroad for AMBN either to be phosphorylated and act as monomeric signal molecule during early odontogenesis and bone formation, or escape phosphorylation to be subsequently secreted as supramolecular assemblies that partake in enamel matrix structure and mineralization.

  5. Differential effects of an O-GlcNAcase inhibitor on tau phosphorylation.

    Directory of Open Access Journals (Sweden)

    Yang Yu

    Full Text Available Abnormal hyperphosphorylation of microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD. The aggregation of hyperphosphorylated tau into neurofibrillary tangles is also a hallmark brain lesion of AD. Tau phosphorylation is regulated by tau kinases, tau phosphatases, and O-GlcNAcylation, a posttranslational modification of proteins on the serine or threonine residues with β-N-acetylglucosamine (GlcNAc. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase, the enzyme catalyzing the transfer of GlcNAc to proteins, and N-acetylglucosaminidase (OGA, the enzyme catalyzing the removal of GlcNAc from proteins. Thiamet-G is a recently synthesized potent OGA inhibitor, and initial studies suggest it can influence O-GlcNAc levels in the brain, allowing OGA inhibition to be a potential route to altering disease progression in AD. In this study, we injected thiamet-G into the lateral ventricle of mice to increase O-GlcNAcylation of proteins and investigated the resulting effects on site-specific tau phosphorylation. We found that acute thiamet-G treatment led to a decrease in tau phosphorylation at Thr181, Thr212, Ser214, Ser262/Ser356, Ser404 and Ser409, and an increase in tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 in the mouse brain. Investigation of the major tau kinases showed that acute delivery of a high dose of thiamet-G into the brain also led to a marked activation of glycogen synthase kinase-3β (GSK-3β, possibly as a consequence of down-regulation of its upstream regulating kinase, AKT. However, the elevation of tau phosphorylation at the sites above was not observed and GSK-3β was not activated in cultured adult hippocampal progenitor cells or in PC12 cells after thiamet-G treatment. These results suggest that acute high-dose thiamet-G injection can not only directly antagonize tau phosphorylation, but also stimulate GSK-3β activity, with the downstream consequence

  6. Inhibition of CRMP2 phosphorylation repairs CNS by regulating neurotrophic and inhibitory responses. (United States)

    Nagai, Jun; Owada, Kazuki; Kitamura, Yoshiteru; Goshima, Yoshio; Ohshima, Toshio


    Central nervous system (CNS) regeneration is restricted by both the lack of neurotrophic responses and the presence of inhibitory factors. As of yet, a common mediator of these two pathways has not been identified. Microtubule dynamics is responsible for several key processes after CNS injuries: intracellular trafficking of receptors for neurotrophic factors, axonal retraction by inhibitory factors, and secondary tissue damages by inflammation and scarring. Kinases regulating microtubule organization, such as Cdk5 or GSK3β, may play pivotal roles during CNS recovery, but the molecular mechanisms remain to be elucidated. Collapsin response mediator protein 2 (CRMP2) stabilizes cytoskeletal polymerization, while CRMP2 phosphorylation by Cdk5 and GSK3β loses its affinity for cytoskeleton proteins, leading to the inhibition of axonal growth. Here, we characterized CRMP2 phosphorylation as the first crucial factor regulating neurotrophic and inhibitory responses after spinal cord injury (SCI). We found that pharmacological inhibition of GSK3β enhanced brain-derived neurotrophic factor (BDNF)-induced axonal growth response in cultured dorsal root ganglion (DRG) neurons. DRG neurons from CRMP2 knock-in (Crmp2KI/KI) mice, where CRMP2 phosphorylation was eliminated, showed elevated sensitivity to BDNF as well. Additionally, cultured Crmp2KI/KI neurons exhibited suppressed axonal growth inhibition by chondroitin sulfate proteoglycan (CSPG). These data suggest a couple of new molecular insights: the BDNF/GSK3β/CRMP2 and CSPG/GSK3β/CRMP2 pathways. Next, we tested the significance of CRMP2 phosphorylation after CNS injury in vivo. The phosphorylation level of CRMP2 was enhanced in the injured spinal cord. Crmp2KI/KI mice exhibited prominent recovery of locomotive and nociceptive functions after SCI, which correlated with the enhanced axonal growth of the motor and sensory neurons. Neuroprotective effects against SCI, such as microtubule stabilization, reduced inflammation

  7. Microtubule Destabilizer KIF2A Undergoes Distinct Site-Specific Phosphorylation Cascades that Differentially Affect Neuronal Morphogenesis

    Directory of Open Access Journals (Sweden)

    Tadayuki Ogawa


    Full Text Available Neurons exhibit dynamic structural changes in response to extracellular stimuli. Microtubules (MTs provide rapid and dramatic cytoskeletal changes within the structural framework. However, the molecular mechanisms and signaling networks underlying MT dynamics remain unknown. Here, we have applied a comprehensive and quantitative phospho-analysis of the MT destabilizer KIF2A to elucidate the regulatory mechanisms of MT dynamics within neurons in response to extracellular signals. Interestingly, we identified two different sets of KIF2A phosphorylation profiles that accelerate (A-type and brake (B-type the MT depolymerization activity of KIF2A. Brain-derived neurotrophic factor (BDNF stimulates PAK1 and CDK5 kinases, which decrease the MT depolymerizing activity of KIF2A through B-type phosphorylation, resulting in enhanced outgrowth of neural processes. In contrast, lysophosphatidic acid (LPA induces ROCK2 kinase, which suppresses neurite outgrowth from round cells via A-type phosphorylation. We propose that these two mutually exclusive forms of KIF2A phosphorylation differentially regulate neuronal morphogenesis during development.

  8. Convergence of Ubiquitylation and Phosphorylation Signaling in Rapamycin-Treated Yeast Cells

    DEFF Research Database (Denmark)

    Iesmantavicius, Vytautas; Weinert, Brian Tate; Choudhary, Chuna Ram


    The target of rapamycin (TOR) kinase senses the availability of nutrients and coordinates cellular growth and proliferation with nutrient abundance. Inhibition of TOR mimics nutrient starvation and leads to the reorganization of many cellular processes, including autophagy, protein translation......, phosphorylation, and proteome changes in rapamycin-treated yeast cells. Our data constitutes a detailed proteomic analysis of rapamycin-treated yeast with 3,590 proteins, 8,961 phosphorylation sites, and 2,498 di-Gly modified lysines (putative ubiquitylation sites) quantified. The phosphoproteome was extensively...... modulated by rapamycin treatment, with more than 900 up-regulated sites one hour after rapamycin treatment. Dynamically regulated phosphoproteins were involved in diverse cellular processes, prominently including transcription, membrane organization, vesicle-mediated transport, and autophagy. Several...

  9. Analysis of the Protein phosphotome of Entamoeba histolytica reveals an intricate phosphorylation network. (United States)

    Anwar, Tamanna; Gourinath, Samudrala


    Phosphorylation is the most common mechanism for the propagation of intracellular signals. Protein phosphatases and protein kinases play a dynamic antagonistic role in protein phosphorylation. Protein phosphatases make up a significant fraction of eukaryotic proteome. In this article, we report the identification and analysis of protein phosphatases in the intracellular parasite Entamoeba histolytica. Based on an in silico analysis, we classified 250 non-redundant protein phosphatases in E. histolytica. The phosphotome of E. histolytica is 3.1% of its proteome and 1.3 times of the human phosphotome. In this extensive study, we identified 42 new putative phosphatases (39 hypothetical proteins and 3 pseudophosphatases). The presence of pseudophosphatases may have an important role in virulence of E. histolytica. A comprehensive phosphotome analysis of E. histolytica shows spectacular low similarity to human phosphatases, making them potent candidates for drug target.

  10. Bax mitochondrial relocation is linked to its phosphorylation and its interaction with Bcl-xL. (United States)

    Garenne, David; Renault, Thibaud T; Manon, Stéphen


    The heterologous expression of Bax, and other Bcl-2 family members, in the yeast Saccharomyces cerevisiae, has proved to be a valuable reporter system to investigate the molecular mechanisms underlying their interaction with mitochondria. By combining the co-expression of Bax and Bcl-xL mutants with analyzes of their localization and interaction in mitochondria and post-mitochondrial supernatants, we showed that the ability of Bax and Bcl-xL to interact is dependent both on Bax phosphorylation - mimicked by a substitution S184D - and by Bax and Bcl-xL localization. This, and previous data, provide the molecular basis for a model of dynamic equilibrium for Bax localization and activation, regulated both by phosphorylation and Bcl-xL.

  11. Multisite phosphorylation provides an effective and flexible mechanism for switch-like protein degradation. (United States)

    Varedi K, S Marjan; Ventura, Alejandra C; Merajver, Sofia D; Lin, Xiaoxia Nina


    Phosphorylation-triggered degradation is a common strategy for elimination of regulatory proteins in many important cell signaling processes. Interesting examples include cyclin-dependent kinase inhibitors such as p27 in human and Sic1 in yeast, which play crucial roles during the G1/S transition in the cell cycle. In this work, we have modeled and analyzed the dynamics of multisite-phosphorylation-triggered protein degradation systematically. Inspired by experimental observations on the Sic1 protein and a previous intriguing theoretical conjecture, we develop a model to examine in detail the degradation dynamics of a protein featuring multiple phosphorylation sites and a threshold site number for elimination in response to a kinase signal. Our model explains the role of multiple phosphorylation sites, compared to a single site, in the regulation of protein degradation. A single-site protein cannot convert a graded input of kinase increase to much sharper output, whereas multisite phosphorylation is capable of generating a highly switch-like temporal profile of the substrate protein with two characteristics: a temporal threshold and rapid decrease beyond the threshold. We introduce a measure termed temporal response coefficient to quantify the extent to which a response in the time domain is switch-like and further investigate how this property is determined by various factors including the kinase input, the total number of sites, the threshold site number for elimination, the order of phosphorylation, the kinetic parameters, and site preference. Some interesting and experimentally verifiable predictions include that the non-degradable fraction of the substrate protein exhibits a more switch-like temporal profile; a sequential system is more switch-like, while a random system has the advantage of increased robustness; all the parameters, including the total number of sites, the threshold site number for elimination and the kinetic parameters synergistically

  12. The in vivo phosphorylation sites of rat brain dynamin I

    DEFF Research Database (Denmark)

    Graham, Mark E; Anggono, Victor; Bache, Nicolai


    tail splice variant dynIxa and was not hierarchical. Co-purified, (32)P-labeled dynIII was phosphorylated at Ser(759), Ser(763), and Ser(853). Ser(853) is homologous to Ser(851) in dynIxa. The results identify all major and several minor phosphorylation sites in dynI and provide the first measure...

  13. Phosphorylation of proteins during human myometrial contractions: A phosphoproteomic approach. (United States)

    Hudson, Claire A; López Bernal, Andrés


    Phasic myometrial contractility is a key component of human parturition and the contractions are driven by reversible phosphorylation of myosin light chains catalyzed by the calcium (Ca(2+))-dependent enzyme myosin light chain kinase (MYLK). Other yet unknown phosphorylation or de-phosphorylation events may contribute to myometrial contraction and relaxation. In this study we have performed a global phosphoproteomic analysis of human myometrial tissue using tandem mass tagging to detect changes in the phosphorylation status of individual myometrial proteins during spontaneous and oxytocin-driven phasic contractions. We were able to detect 22 individual phosphopeptides whose relative ratio changed (fold > 2 or contraction. The most significant changes in phosphorylation were to MYLK on serine 1760, a site associated with reductions in calmodulin binding and subsequent kinase activity. Phosphorylated MYLK (ser1760) increased significantly during spontaneous (9.83 ± 3.27 fold, P contractions and we were able to validate these data using immunoblotting. Pathway analysis suggested additional proteins involved in calcium signalling, cGMP-PRKG signalling, adrenergic signalling and oxytocin signalling were also phosphorylated during contractions. This study demonstrates that a global phosphoproteomic analysis of myometrial tissue is a sensitive approach to detect changes in the phosphorylation of proteins during myometrial contractions, and provides a platform for further validation of these changes and for identification of their functional significance.

  14. Intermolecular Phosphoryl Transfer Between Serine and Histidine Residues

    Institute of Scientific and Technical Information of China (English)

    Yu Qian SU; Ming Yu NIU; Shu Xia CAO; Jian Chen ZHANG; Yu Fen ZHAO


    A novel intermolecular phosphoryl transfer from O-trimethylsilyl-N-(O, O-diisopropyl) phosphoryl serine trimethylsilyl ester to N, N'-bis(trimethylsilyl) histidine trimethylsilyl ester was studied through electrospray ionization mass spectrometry (ESI-MS). It was proposed that the transfer reaction went through penta-coordinated phosphorus intermediate.

  15. Phosphorylation of the Epstein-Barr virus nuclear antigen 2

    DEFF Research Database (Denmark)

    Grässer, F A; Göttel, S; Haiss, P


    A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK...

  16. Phosphorylation of the Goodpasture antigen by type A protein kinases. (United States)

    Revert, F; Penadés, J R; Plana, M; Bernal, D; Johansson, C; Itarte, E; Cervera, J; Wieslander, J; Quinones, S; Saus, J


    Collagen IV is the major component of basement membranes. The human alpha 3 chain of collagen IV contains an antigenic domain called the Goodpasture antigen that is the target for the circulating immunopathogenic antibodies present in patients with Goodpasture syndrome. Characteristically, the gene region encoding the Goodpasture antigen generates multiple alternative products that retain the antigen amino-terminal region with a five-residue motif (KRGDS). The serine therein appears to be the major in vitro cAMP-dependent protein kinase phosphorylation site in the isolated antigen and can be phosphorylated in vitro by two protein kinases of approximately 50 and 41 kDa associated with human kidney plasma membrane, suggesting that it can also be phosphorylated in vivo. Consistent with this, the Goodpasture antigen is isolated from human kidney in phosphorylated and non-phosphorylated forms and only the non-phosphorylated form is susceptible to phosphorylation in vitro. Since this motif is exclusive to the human alpha 3(IV) chain and includes the RGD cell adhesion motif, its phosphorylation might play a role in pathogenesis and influence cell attachment to basement membrane.

  17. Regulation of endothelial permeability and transendothelial migration of cancer cells by tropomyosin-1 phosphorylation

    Directory of Open Access Journals (Sweden)

    Simoneau Bryan


    Full Text Available Abstract Background Loss of endothelial cell integrity and selective permeability barrier is an early event in the sequence of oxidant-mediated injury and may result in atherosclerosis, hypertension and facilitation of transendothelial migration of cancer cells during metastasis. We already reported that endothelial cell integrity is tightly regulated by the balanced co-activation of p38 and ERK pathways. In particular, we showed that phosphorylation of tropomyosin-1 (tropomyosin alpha-1 chain = Tm1 at Ser283 by DAP kinase, downstream of the ERK pathway might be a key event required to maintain the integrity and normal functions of the endothelium in response to oxidative stress. Methods Endothelial permeability was assayed by monitoring the passage of Dextran-FITC through a tight monolayer of HUVECs grown to confluence in Boyden chambers. Actin and Tm1 dynamics and distribution were evaluated by immunofluorescence. We modulated the expression of Tm1 by siRNA and lentiviral-mediated expression of wild type and mutated forms of Tm1 insensitive to the siRNA. Transendothelial migration of HT-29 colon cancer cells was monitored in Boyden chambers similarly as for permeability. Results We provide evidence indicating that Tm1 phosphorylation at Ser283 is essential to regulate endothelial permeability under oxidative stress by modulating actin dynamics. Moreover, the transendothelial migration of colon cancer cells is also regulated by the phosphorylation of Tm1 at Ser283. Conclusion Our finding strongly support the role for the phosphorylation of endothelial Tm1 at Ser283 to prevent endothelial barrier dysfunction associated with oxidative stress injury.

  18. Stathmin slows down guanosine diphosphate dissociation from tubulin in a phosphorylation-controlled fashion. (United States)

    Amayed, P; Carlier, M F; Pantaloni, D


    Stathmin is an important protein that interacts with tubulin and regulates microtubule dynamics in a phosphorylation-controlled fashion. Here we show that the dissociation of guanosine 5'-diphosphate (GDP) from beta-tubulin is slowed 20-fold in the (tubulin)(2)-stathmin ternary complex (T(2)S). The kinetics of GDP or guanosine 5'-triphosphate (GTP) dissociation from tubulin have been monitored by the change in tryptophan fluorescence of tubulin upon exchanging 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-diphosphate (S6-GDP) for tubulin-bound guanine nucleotide. At molar ratios of stathmin to tubulin lower than 0.5, biphasic kinetics were observed, indicating that the dynamics of the complex is extremely slow, consistent with its high stability. The method was used to characterize the effects of phosphorylation of stathmin on its interaction with tubulin. The serine-to-glutamate substitution of all four phosphorylatable serines of stathmin (4E-stathmin) weakens the stability of the T(2)S complex by about 2 orders of magnitude. The phosphorylation of serines 16 and 63 in stathmin has a more severe effect and weakens the stability of T(2)S 10(4)-fold. The rate of GDP dissociation is lowered only 7-fold and 4-fold in the complexes of tubulin with 4E-stathmin and diphosphostathmin, respectively. Sedimentation velocity studies support the conclusions of nucleotide exchange data and show that the T(2)S complexes formed between tubulin and 4E-stathmin or diphosphostathmin are less compact than the highly stable T(2)S complex. The correlation between the effect of phosphorylation of stathmin on the stability of T(2)S complex measured in vitro and on the function of stathmin in vivo is discussed.

  19. Exploring the diversity of protein modifications: special bacterial phosphorylation systems

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad


    that has been most thoroughly investigated. Unlike in eukarya, a large diversity of enzyme families has been shown to phosphorylate and dephosphorylate proteins on various amino acids with different chemical properties in bacteria. In this review, after a brief overview of the known bacterial...... phosphorylation systems, we focus on more recently discovered and less widely known kinases and phosphatases. Namely, we describe in detail tyrosine- and arginine-phosphorylation together with some examples of unusual serine-phosphorylation systems and discuss their potential role and function in bacterial...... physiology, and regulatory networks. Investigating these unusual bacterial kinase and phosphatases is not only important to understand their role in bacterial physiology but will help to generally understand the full potential and evolution of protein phosphorylation for signal transduction, protein...

  20. Systematic inference of functional phosphorylation events in yeast metabolism

    DEFF Research Database (Denmark)

    Chen, Yu; Wang, Yonghong; Nielsen, Jens


    Motivation: Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification...... of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics...... has received increased attention.Results: We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution...

  1. Phosphorylation of the Epstein-Barr virus nuclear antigen 2

    DEFF Research Database (Denmark)

    Grässer, F A; Göttel, S; Haiss, P


    A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK......-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic...

  2. Interaction between O-GlcNAc modification and tyrosine phosphorylation of prohibitin: implication for a novel binary switch.

    Directory of Open Access Journals (Sweden)

    Sudharsana R Ande

    Full Text Available Prohibitin (PHB or PHB1 is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked beta-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114 and tyrosine 259 (Tyr259 in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121 and threonine 258 (Thr258 respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s or known tyrosine phosphorylation site(s revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.

  3. Septin phosphorylation and coiled-coil domains function in cell and septin ring morphology in the filamentous fungus Ashbya gossypii. (United States)

    Meseroll, Rebecca A; Occhipinti, Patricia; Gladfelter, Amy S


    Septins are a class of GTP-binding proteins conserved throughout many eukaryotes. Individual septin subunits associate with one another and assemble into heteromeric complexes that form filaments and higher-order structures in vivo. The mechanisms underlying the assembly and maintenance of higher-order structures in cells remain poorly understood. Septins in several organisms have been shown to be phosphorylated, although precisely how septin phosphorylation may be contributing to the formation of high-order septin structures is unknown. Four of the five septins expressed in the filamentous fungus, Ashbya gossypii, are phosphorylated, and we demonstrate here the diverse roles of these phosphorylation sites in septin ring formation and septin dynamics, as well as cell morphology and viability. Intriguingly, the alteration of specific sites in Cdc3p and Cdc11p leads to a complete loss of higher-order septin structures, implicating septin phosphorylation as a regulator of septin structure formation. Introducing phosphomimetic point mutations to specific sites in Cdc12p and Shs1p causes cell lethality, highlighting the importance of normal septin modification in overall cell function and health. In addition to discovering roles for phosphorylation, we also present diverse functions for conserved septin domains in the formation of septin higher-order structure. We previously showed the requirement for the Shs1p coiled-coil domain in limiting septin ring size and reveal here that, in contrast to Shs1p, the coiled-coil domains of Cdc11p and Cdc12p are required for septin ring formation. Our results as a whole reveal novel roles for septin phosphorylation and coiled-coil domains in regulating septin structure and function.

  4. Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

    Directory of Open Access Journals (Sweden)

    Danming Tang


    GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.

  5. Phosphoryl functionalized mesoporous silica for uranium adsorption (United States)

    Xue, Guo; Yurun, Feng; Li, Ma; Dezhi, Gao; Jie, Jing; Jincheng, Yu; Haibin, Sun; Hongyu, Gong; Yujun, Zhang


    Phosphoryl functionalized mesoporous silica (TBP-SBA-15) was synthesized by modified mesoporous silica with γ-amino propyl triethoxy silane and tributyl phosphate. The obtained samples were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), small angle X-ray diffraction (SAXRD), thermo-gravimetric/differential thermalanalyzer (TG/DTA), N2 adsorption-desorption (BET) and Fourier transform infrared spectroscopy (FT-IR) techniques. Results showed that TBP-SBA-15 had large surface areas with ordered channel structure. Moreover, the effects of adsorption time, sorbent dose, solution pH, initial uranium concentration and temperature on the uranium adsorption behaviors were investigated. TBP-SBA-15 showed a high uranium adsorption capacity in a broad range of pH values. The U(VI) adsorption rate of TBP-SBA-15 was fast and nearly achieved completion in 10 min with the sorbent dose of 1 g/L. The U(VI) adsorption of TBP-SBA-15 followed the pseudo-second-order kinetic model and Freundlich isotherm model, indicating that the process was belonged to chemical adsorption. Furthermore, the thermodynamic parameters (ΔG0, ΔH0 and ΔS0) confirmed that the adsorption process was endothermic and spontaneous.

  6. A negative-feedback loop regulating ERK1/2 activation and mediated by RasGPR2 phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jinqi [Departments of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Cook, Aaron A.; Bergmeier, Wolfgang [Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Sondek, John, E-mail: [Departments of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States)


    The dynamic regulation of ERK1 and -2 (ERK1/2) is required for precise signal transduction controlling cell proliferation, differentiation, and survival. However, the underlying mechanisms regulating the activation of ERK1/2 are not completely understood. In this study, we show that phosphorylation of RasGRP2, a guanine nucleotide exchange factor (GEF), inhibits its ability to activate the small GTPase Rap1 that ultimately leads to decreased activation of ERK1/2 in cells. ERK2 phosphorylates RasGRP2 at Ser394 located in the linker region implicated in its autoinhibition. These studies identify RasGRP2 as a novel substrate of ERK1/2 and define a negative-feedback loop that regulates the BRaf–MEK–ERK signaling cascade. This negative-feedback loop determines the amplitude and duration of active ERK1/2. -- Highlights: •ERK2 phosphorylates the guanine nucleotide exchange factor RasGRP2 at Ser394. •Phosphorylated RasGRP2 has decreased capacity to active Rap1b in vitro and in cells. •Phosphorylation of RasGRP2 by ERK1/2 introduces a negative-feedback loop into the BRaf-MEK-ERK pathway.

  7. Differences in c-Jun N-terminal kinase recognition and phosphorylation of closely related stathmin-family members. (United States)

    Yip, Yan Y; Yeap, Yvonne Y C; Bogoyevitch, Marie A; Ng, Dominic C H


    The stathmin (STMN) family of tubulin-binding phosphoproteins are critical regulators of interphase microtubule dynamics and organization in a broad range of cellular processes. c-Jun N-terminal kinase (JNK) signalling to STMN family proteins has been implicated specifically in neuronal maturation, degeneration and cell stress responses more broadly. Previously, we characterized mechanisms underlying JNK phosphorylation of STMN at proline-flanked serine residues (Ser25 and Ser38) that are conserved across STMN-like proteins. In this study, we demonstrated using in vitro kinase assays and alanine replacement of serine residues that JNK phosphorylated the STMN-like domain (SLD) of SCG10 on Ser73, consistent with our previous finding that STMN Ser38 was the primary JNK target site. In addition, we confirmed that a JNK binding motif ((41)KKKDLSL(47)) that facilitates JNK targeting of STMN is conserved in SCG10. In contrast, SCLIP was phosphorylated by JNK primarily on Ser60 which corresponds to Ser25 on STMN. Moreover, although the JNK-binding motif identified in STMN and SCG10 was not conserved in SCLIP, JNK phosphorylation of SCLIP was inhibited by a substrate competitive peptide (TI-JIP) highlighting kinase-substrate interaction as required for JNK targeting. Thus, STMN and SCG10 are similarly targeted by JNK but there are clear differences in JNK recognition and phosphorylation of the closely related family member, SCLIP.

  8. Expression and phosphorylation of neurofilament protein in different neuronal tissues

    Institute of Scientific and Technical Information of China (English)


    The neurofilament proteins (NFPs) from different neuronal tissues including Alzheimer and Huntington disease gray matter, rat brain gray, white matter and spinal cord were separated biochemically into two major fractions. A systematic investigation on the distribution, expression and phosphorylation of NFPs in those fractions was undertaken in the present study. It was found that only non-phosphorylated NF-H and NF-M, but not NF-L subunit were detected in Alzheimer brain gray matter high speed supernatant, whereas all neurofilament subunits including non-phosphorylated and phosphorylated were measured in high speed pellet fraction of the same tissue. The hyperphosphorylation of NF-H and NF-M in Alzheimer brain was shown by phosphorylation dependent monoclonal antibodies SMI31 and SMI34. This hyperphosphorylation was confirmed by non-phosphorylation dependent antibody SMI32 with dephosphosphorylation of the samples. Furthermore, an increased amount of NF-H, NH-M and NF-L, detected by SMI33 and NR4 respectively, was also observed in Alzheimer samples, in which the elevation in NF-L was significant. A significantly different immunoblot patterns in distribution, expression and phosphorylation were determined in various position of the neural system and alternative fractions. To our best knowledge, this is the first data shown definite abnormality of NFPs in Alzheimer disease. The information obtained in the present study will be extremely valuable in further study of the proteins both in physiological and pathological conditions.

  9. Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

    DEFF Research Database (Denmark)

    Caspersen, M.B.; Qiu, J.-L.; Zhang, X.


    The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

  10. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy (United States)

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang


    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knockin mice expressing non-phosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knockin mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knockin mice compared to their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knockin mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knockin and wild type mice, indicating that mTORC1 was still activated in the knockin mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knockin mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth. PMID:25229342

  11. Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

    Directory of Open Access Journals (Sweden)

    Thomas H Sanderson

    Full Text Available Recent advancements in isolation techniques for cytochrome c (Cytc have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

  12. Acute exercise modifies titin phosphorylation and increases cardiac myofilament stiffness

    Directory of Open Access Journals (Sweden)

    Anna Eliane Müller


    Full Text Available Titin-based myofilament stiffness is largely modulated by phosphorylation of its elastic I-band regions N2-Bus (decreases passive stiffness, PT and PEVK (increases PT. Here, we tested the hypothesis that acute exercise changes titin phosphorylation and modifies myofilament stiffness. Adult rats were exercised on a treadmill for 15min, untrained animals served as controls. Titin phosphorylation was determined by Western blot analysis using phosphospecific antibodies to Ser4099 and Ser4010 in the N2-Bus region (PKG and PKA-dependent. respectively, and to Ser11878 and Ser 12022 in the PEVK region (PKCα and CaMKIIδ-dependent, respectively. Passive tension was determined by step-wise stretching of isolated skinned cardiomyocytes to sarcomere length ranging from 1.9-2.4µm and showed a significantly increased PT from exercised samples, compared to controls. In cardiac samples titin N2-Bus phosphorylation was significantly decreased by 40% at Ser4099, however, no significant changes were observed at Ser4010. PEVK phosphorylation at Ser11878 was significantly increased, which is probably mediated by the observed exercise-induced increase in PKCα activity. Interestingly, relative phosphorylation of Ser12022 was substantially decreased in the exercised samples. Surprisingly, in skeletal samples from acutely exercised animals we detected a significant decrease in PEVK phosphorylation at Ser11878 and an increase in Ser12022 phosphorylation; however, PKCα activity remained unchanged. In summary, our data show that a single exercise bout of 15 min affects titin domain phosphorylation and titin-based myocyte stiffness with obviously divergent effects in cardiac and skeletal muscle tissues. The observed changes in titin stiffness could play an important role in adapting the passive and active properties of the myocardium and the skeletal muscle to increased physical activity.

  13. PKC isoforms interact with and phosphorylate DNMT1

    Directory of Open Access Journals (Sweden)

    Pradhan Sriharsa


    Full Text Available Abstract Background DNA methyltransferase 1 (DNMT1 has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. Results Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity. Conclusions Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.

  14. Rosamines targeting the cancer oxidative phosphorylation pathway.

    Directory of Open Access Journals (Sweden)

    Siang Hui Lim

    Full Text Available Reprogramming of energy metabolism is pivotal to cancer, so mitochondria are potential targets for anticancer therapy. A prior study has demonstrated the anti-proliferative activity of a new class of mitochondria-targeting rosamines. This present study describes in vitro cytotoxicity of second-generation rosamine analogs, their mode of action, and their in vivo efficacies in a tumor allografted mouse model. Here, we showed that these compounds exhibited potent cytotoxicity (average IC50<0.5 µM, inhibited Complex II and ATP synthase activities of the mitochondrial oxidative phosphorylation pathway and induced loss of mitochondrial transmembrane potential. A NCI-60 cell lines screen further indicated that rosamine analogs 4 and 5 exhibited potent antiproliferative effects with Log10GI50 = -7 (GI50 = 0.1 µM and were more effective against a colorectal cancer sub-panel than other cell lines. Preliminary in vivo studies on 4T1 murine breast cancer-bearing female BALB/c mice indicated that treatment with analog 5 in a single dosing of 5 mg/kg or a schedule dosing of 3 mg/kg once every 2 days for 6 times (q2d×6 exhibited only minimal induction of tumor growth delay. Our results suggest that rosamine analogs may be further developed as mitochondrial targeting agents. Without a doubt proper strategies need to be devised to enhance tumor uptake of rosamines, i.e. by integration to carrier molecules for better therapeutic outcome.

  15. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Thingholm, Tine E; Jensen, Ole N


    Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation i...

  16. Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability. (United States)

    Fairfax, Benjamin P; Pitcher, Julie A; Scott, Mark G H; Calver, Andrew R; Pangalos, Menelas N; Moss, Stephen J; Couve, Andrés


    GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.

  17. Modulation of RNA polymerase II phosphorylation downstream of pathogen perception orchestrates plant immunity. (United States)

    Li, Fangjun; Cheng, Cheng; Cui, Fuhao; de Oliveira, Marcos V V; Yu, Xiao; Meng, Xiangzong; Intorne, Aline C; Babilonia, Kevin; Li, Maoying; Li, Bo; Chen, Sixue; Ma, Xianfeng; Xiao, Shunyuan; Zheng, Yi; Fei, Zhangjun; Metz, Richard P; Johnson, Charles D; Koiwa, Hisashi; Sun, Wenxian; Li, Zhaohu; de Souza Filho, Gonçalo A; Shan, Libo; He, Ping


    Perception of microbe-associated molecular patterns (MAMPs) elicits host transcriptional reprogramming as part of the immune response. Although pathogen perception is well studied, the signaling networks orchestrating immune gene expression remain less clear. In a genetic screen for components involved in the early immune gene transcription reprogramming, we identified Arabidopsis RNA polymerase II C-terminal domain (CTD) phosphatase-like 3 (CPL3) as a negative regulator of immune gene expression. MAMP perception induced rapid and transient cyclin-dependent kinase C (CDKC)-mediated phosphorylation of Arabidopsis CTD. The CDKCs, which are in turn phosphorylated and activated by a canonical MAP kinase (MAPK) cascade, represent a point of signaling convergence downstream of multiple immune receptors. CPL3 directly dephosphorylated CTD to counteract MAPK-mediated CDKC regulation. Thus, modulation of the phosphorylation dynamics of eukaryotic RNA polymerase II transcription machinery by MAPKs, CTD kinases, and phosphatases constitutes an essential mechanism for rapid orchestration of host immune gene expression and defense upon pathogen attacks.

  18. PCTAIRE1 phosphorylates p27 and regulates mitosis in cancer cells. (United States)

    Yanagi, Teruki; Krajewska, Maryla; Matsuzawa, Shu-ichi; Reed, John C


    PCTAIRE1 is distant relative of the cyclin-dependent kinase family that has been implicated in spermatogenesis and neuronal development, but it has not been studied in cancer. Here, we report that PCTAIRE1 is expressed in prostate, breast, and cervical cancer cells, where its RNAi-mediated silencing causes growth inhibition with aberrant mitosis due to defects in centrosome dynamics. PCTAIRE1 was not similarly involved in proliferation of nontransformed cells, including diploid human IMR-90 fibroblasts. Through yeast two-hybrid screening, we identified tumor suppressor p27 as a PCTAIRE1 interactor. In vitro kinase assays showed PCTAIRE1 phosphorylates p27 at Ser10. PCTAIRE1 silencing modulated Ser10 phosphorylation on p27 and led to its accumulation in cancer cells but not in nontransformed cells. In a mouse xenograft model of PPC1 prostate cancer, conditional silencing of PCTAIRE1 restored p27 protein expression and suppressed tumor growth. Mechanistic studies in HeLa cells showed that PCTAIRE1 phosphorylates p27 during the S and M phases of the cell cycle. Notably, p27 silencing was sufficient to rescue cells from mitotic arrest caused by PCTAIRE1 silencing. Clinically, PCTAIRE1 was highly expressed in primary breast and prostate tumors compared with adjacent normal epithelial tissues. Together our findings reveal an unexpected role for PCTAIRE1 in regulating p27 stability, mitosis, and tumor growth, suggesting PCTAIRE1 as a candidate cancer therapeutic target.

  19. Role of Ser7 phosphorylation of the CTD during transcription of snRNA genes (United States)

    Egloff, Sylvain


    The largest subunit of RNA polymerase (pol) II, Rpb1, contains an unusual carboxyl-terminal domain (CTD) composed of consecutive repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). During transcription, Ser2, Ser5 and Ser7 are subjected to dynamic phosphorylation and dephosphorylation by CTD kinases and phosphatases, creating a characteristic CTD phosphorylation pattern along genes. This CTD “code” allows the coupling of transcription with co-transcriptional RNA processing, through the timely recruitment of the appropriate factors at the right point of the transcription cycle. In mammals, phosphorylation of Ser7 (Ser7P) is detected on all pol II-transcribed genes, but is only essential for expression of a sub-class of genes encoding small nuclear (sn)RNAs. The molecular mechanisms by which Ser7P influences expression of these particular genes are becoming clearer. Here, I discuss our recent findings clarifying how Ser7P facilitates transcription of these genes and 3′end processing of the transcripts, through recruitment of the RPAP2 phosphatase and the snRNA gene-specific Integrator complex. PMID:22858677

  20. Specific threonine-4 phosphorylation and function of RNA polymerase II CTD during M phase progression. (United States)

    Hintermair, Corinna; Voß, Kirsten; Forné, Ignasi; Heidemann, Martin; Flatley, Andrew; Kremmer, Elisabeth; Imhof, Axel; Eick, Dirk


    Dynamic phosphorylation of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 heptad-repeats in the C-terminal domain (CTD) of the large subunit coordinates progression of RNA polymerase (Pol) II through the transcription cycle. Here, we describe an M phase-specific form of Pol II phosphorylated at Thr4, but not at Tyr1, Ser2, Ser5, and Ser7 residues. Thr4 phosphorylated Pol II binds to centrosomes and midbody and interacts with the Thr4-specific Polo-like kinase 1. Binding of Pol II to centrosomes does not require the CTD but may involve subunits of the non-canonical R2TP-Prefoldin-like complex, which bind to and co-localize with Pol II at centrosomes. CTD Thr4 mutants, but not Ser2 and Ser5 mutants, display severe mitosis and cytokinesis defects characterized by multipolar spindles and polyploid cells. We conclude that proper M phase progression of cells requires binding of Pol II to centrosomes to facilitate regulation of mitosis and cytokinesis in a CTD Thr4-P dependent manner.

  1. Assessment of current mass spectrometric workflows for the quantification of low abundant proteins and phosphorylation sites

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    Manuel Bauer


    Full Text Available The data described here provide a systematic performance evaluation of popular data-dependent (DDA and independent (DIA mass spectrometric (MS workflows currently used in quantitative proteomics. We assessed the limits of identification, quantification and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy labeled reference peptides, respectively, covering six orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5–10 times more sensitive than directed or DDA methods. In particular, higher stage fragmentation (MS3 of the neutral loss peak using a linear ion trap increased dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying 9 phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from non-enriched pull-down samples. The data are associated to the research article ‘Evaluation of data-dependent and data-independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites׳ (Bauer et al., 2014 [1]. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium ( via the PRIDE partner repository with the dataset identifier PXD000964.

  2. Phosphorylated ERK5/BMK1 transiently accumulates within division spindles in mouse oocytes and preimplantation embryos

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    Maria A. Ciemerych


    Full Text Available MAP kinases of the ERK family play important roles in oocyte maturation, fertilization, and early embryo development. The role of the signaling pathway involving ERK5 MAP kinase during meiotic and mitotic M-phase of the cell cycle is not well known. Here, we studied the localization of the phosphorylated, and thus potentially activated, form of ERK5 in mouse maturing oocytes and mitotically dividing early embryos. We show that phosphorylation/dephosphorylation, i.e. likely activation/inactivation of ERK5, correlates with M-phase progression. Phosphorylated form of ERK5 accumulates in division spindle of both meiotic and mitotic cells, and precisely co-localizes with spindle microtubules at metaphase. This localization changes drastically in the anaphase, when phospho-ERK5 completely disappears from microtubules and transits to the cytoplasmic granular, vesicle-like structures. In telophase oocytes it becomes incorporated into the midbody. Dynamic changes in the localization of phospho-ERK5 suggests that it may play an important role both in meiotic and mitotic division. (Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 528–534

  3. Phosphorylated Peptide Functionalization of Lanthanide Upconversion Nanoparticles for Tuning Nanomaterial-Cell Interactions. (United States)

    Yao, Chi; Wei, Caiyi; Huang, Zhi; Lu, Yiqing; El-Toni, Ahmed Mohamed; Ju, Dianwen; Zhang, Xiangmin; Wang, Wenning; Zhang, Fan


    Peptide modification of nanoparticles with high efficiency is critical in determining the properties and bioapplications of nanoparticles, but the methodology remains a challenging task. Here, by using the phosphorylated linear and cyclic peptide with the arginine-glycine-aspartic acid (RGD) targeting motifs as typical examples, the peptides binding efficiency for the inorganic metal compound nanoparticles was increased significantly after the phosphorylation treatment, and the modification allowed for improving the selectivity and signal-to-noise ratio for cancer targeting and reduced the toxicity derived from nonspecific interactions of nanoparticles with cells owing to the higher amount of phosphopeptide binding. In addition, molecular dynamics (MD) simulations of various peptides on inorganic metal compound surfaces revealed that the peptide adsorption on the surface is mainly driven by electrostatic interactions between phosphate oxygen and the polarized interfacial water layer, consistent with the experimental observation of the strong binding propensity of phosphorylated peptides. Significantly, with the RGD phosphopeptide surface modification, these nanoparticles provide a versatile tool for tuning material-cell interactions to achieve the desired level of autophagy and may prove useful for various diagnostic and therapeutic applications.

  4. Phosphorylation-dependent Trafficking of Plasma Membrane Proteins in Animal and Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Remko Offringa; and Fang Huang


    In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.

  5. Phosphorylation-dependent trafficking of plasma membrane proteins in animal and plant cells. (United States)

    Offringa, Remko; Huang, Fang


    In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.

  6. Phosphorylation and Ionic Strength Alter the LRAP-HAP Interface in the N-terminus

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Junxia; Xu, Yimin; Shaw, Wendy J.


    The conditions present during enamel crystallite development change dramatically as a function of time, including the pH, protein concentration, surface type and ionic strength. In this work, we investigate the role that two of these changing conditions, pH and ionic strength, have in modulating the interaction of amelogenin, LRAP, with hydroxyapatite (HAP). Using solid state NMR dipolar recoupling and chemical shift data, we investigate the structure, orientation and dynamics of three regions in the N-terminus of the protein, L15 to V19, V19 to L23 and K24 to S28. These regions are also near the only phosphorylated residue in the protein, pS16, therefore, changes in the LRAP-HAP interaction as a function of phosphorylation (LRAP(-P) vs. LRAP(+P)) were also investigated. All of the regions and conditions studies for the surface immobilized proteins showed restricted motion, with more mobility under all conditions for L15(+P) and K24(-P). The structure and orientation of the LRAP-HAP interaction in the N-terminus of the phosphorylated protein is very stable to changing solution conditions. From REDOR dipolar recoupling data, the structure and orientation in the region L15V19(+P) did not change significantly as a function of pH or ionic strength. The structure and orientation of the region V19L23(+P) were also stable to changes in pH, with the only significant change observed at high ionic strength, where the region becomes extended, suggesting this may be an important region in regulating mineral development. Chemical shift studies also suggest minimal changes in all three regions studied for both LRAP(-P) and LRAP(+P) as a function of pH or ionic strength. Phosphorylation also alters the LRAP-HAP interface. All of the three residues investigated (L15, V19, and K24) are closer to the surface in LRAP(+P), but K24S28 also changes structure as a result of phosphorylation, from a random coil to a largely helical structure, and V19L23 becomes more extended at high ionic

  7. Phosphorylation modulates clearance of alpha-synuclein inclusions in a yeast model of Parkinson's disease.

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    Sandra Tenreiro


    Full Text Available Alpha-synuclein (aSyn is the main component of proteinaceous inclusions known as Lewy bodies (LBs, the typical pathological hallmark of Parkinson's disease (PD and other synucleinopathies. Although aSyn is phosphorylated at low levels under physiological conditions, it is estimated that ∼ 90% of aSyn in LBs is phosphorylated at S129 (pS129. Nevertheless, the significance of pS129 in the biology of aSyn and in PD pathogenesis is still controversial. Here, we harnessed the power of budding yeast in order to assess the implications of phosphorylation on aSyn cytotoxicity, aggregation and sub-cellular distribution. We found that aSyn is phosphorylated on S129 by endogenous kinases. Interestingly, phosphorylation reduced aSyn toxicity and the percentage of cells with cytosolic inclusions, in comparison to cells expressing mutant forms of aSyn (S129A or S129G that mimic the unphosphorylated form of aSyn. Using high-resolution 4D imaging and fluorescence recovery after photobleaching (FRAP in live cells, we compared the dynamics of WT and S129A mutant aSyn. While WT aSyn inclusions were very homogeneous, inclusions formed by S129A aSyn were larger and showed FRAP heterogeneity. Upon blockade of aSyn expression, cells were able to clear the inclusions formed by WT aSyn. However, this process was much slower for the inclusions formed by S129A aSyn. Interestingly, whereas the accumulation of WT aSyn led to a marked induction of autophagy, cells expressing the S129A mutant failed to activate this protein quality control pathway. The finding that the phosphorylation state of aSyn on S129 can alter the ability of cells to clear aSyn inclusions provides important insight into the role that this posttranslational modification may have in the pathogenesis of PD and other synucleinopathies, opening novel avenues for investigating the molecular basis of these disorders and for the development of therapeutic strategies.

  8. cAMP-dependent protein kinase and c-Jun N-terminal kinase mediate stathmin phosphorylation for the maintenance of interphase microtubules during osmotic stress. (United States)

    Yip, Yan Y; Yeap, Yvonne Y C; Bogoyevitch, Marie A; Ng, Dominic C H


    Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.

  9. Making an effective switch at the kinetochore by phosphorylation and dephosphorylation. (United States)

    Funabiki, Hironori; Wynne, David J


    The kinetochore, the proteinaceous structure on the mitotic centromere, functions as a mechanical latch that hooks onto microtubules to support directional movement of chromosomes. The structure also brings in a number of signaling molecules, such as kinases and phosphatases, which regulate microtubule dynamics and cell cycle progression. Erroneous microtubule attachment is destabilized by Aurora B-mediated phosphorylation of multiple microtubule-binding protein complexes at the kinetochore, such as the KMN network proteins and the Ska/Dam1 complex, while Plk-dependent phosphorylation of BubR1 stabilizes kinetochore-microtubule attachment by recruiting PP2A-B56. Spindle assembly checkpoint (SAC) signaling, which is activated by unattached kinetochores and inhibits the metaphase-to-anaphase transition, depends on kinetochore recruitment of the kinase Bub1 through Mps1-mediated phosphorylation of the kinetochore protein KNL1 (also known as Blinkin in mammals, Spc105 in budding yeast, and Spc7 in fission yeast). Recruitment of protein phosphatase 1 to KNL1 is necessary to silence the SAC upon bioriented microtubule attachment. One of the key unsolved questions in the mitosis field is how a mechanical change at the kinetochore upon microtubule attachment is converted to these and other chemical signals that control microtubule attachment and the SAC. Rapid progress in the field is revealing the existence of an intricate signaling network created right on the kinetochore. Here we review the current understanding of phosphorylation-mediated regulation of kinetochore functions and discuss how this signaling network generates an accurate switch that turns on and off the signaling output in response to kinetochore-microtubule attachment.

  10. Phosphorylation of Lbx1 controls lateral myoblast migration into the limb. (United States)

    Masselink, Wouter; Masaki, Megumi; Sieiro, Daniel; Marcelle, Christophe; Currie, Peter D


    The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud. Copyright © 2017. Published by Elsevier Inc.

  11. Prostate Cell Specific Regulation of Androgen Receptor Phosphorylation In Vivo (United States)


    analysis indicates that the screen is preferentially isolating proteins with a known role in gene transcription and we are currently assessing the phosphorylation- dependence of the putative AR interacting proteins .

  12. The Synthesis of a Series of Phosphoryl Coumarins

    Institute of Scientific and Technical Information of China (English)


    Different hydroxy substituted coumarins were successfully phosphorylated with diisopropylphophite (DIPPH) by the Atherton-Todd reaction in 76-89% yields. Moreover, the reaction activities of different hydroxys of the coumarins in the Atherton-Todd reaction were studied.

  13. Phosphorylation: The Molecular Switch of Double-Strand Break Repair

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    K. C. Summers


    Full Text Available Repair of double-stranded breaks (DSBs is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ, homologous recombination (HR, or the inclusive DNA damage response (DDR. These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

  14. Phosphorylation of the viral coat protein regulates RNA virus infection

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    Hoover HS


    Full Text Available Haley S Hoover, C Cheng Kao Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA Abstract: Coat proteins (CPs are the most abundant protein produced during a viral infection. CPs have been shown to regulate the infection processes of RNA viruses, including RNA replication and gene expression. The numerous activities of the CP in infection are likely to require regulation, possibly through posttranslational modifications. Protein posttranslational modifications are involved in signal transduction, expanding and regulating protein function, and responding to changes in the environment. Accumulating evidence suggests that phosphorylation of viral CPs is involved in the regulation of the viral infection process from enabling virion disassembly to regulation of viral protein synthesis and replication. CP phosphorylation also affects viral trafficking and virion assembly. This review focuses on the regulatory roles that phosphorylation of CPs has in the life cycle of viruses with RNA genomes. Keywords: viral capsid protein, posttranslational modification, phosphorylation, protein–RNA interaction

  15. A Green Synthesis of Diisopropyl Phosphoryl Amino Acid

    Institute of Scientific and Technical Information of China (English)


    In this paper, we report that diisopropyl phosphoryl amino acid could be prepared with reasonable yields under solvent-free condition by adding amino acid to the mixture of diisopropyl phosphite and N-chlorodiisopropylamine.

  16. Tau Phosphorylation by GSK3 in Different Conditions (United States)

    Avila, Jesús; León-Espinosa, Gonzalo; García, Esther; García-Escudero, Vega; Hernández, Félix; DeFelipe, Javier


    Almost a 20% of the residues of tau protein are phosphorylatable amino acids: serine, threonine, and tyrosine. In this paper we comment on the consequences for tau of being a phosphoprotein. We will focus on serine/threonine phosphorylation. It will be discussed that, depending on the modified residue in tau molecule, phosphorylation could be protective, in processes like hibernation, or toxic like in development of those diseases known as tauopathies, which are characterized by an hyperphosphorylation and aggregation of tau. PMID:22675648

  17. Fission yeast Rad52 phosphorylation restrains error prone recombination pathways.

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    Angela Bellini

    Full Text Available Rad52 is a key protein in homologous recombination (HR, a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive.

  18. Identification of Phosphorylation Sites Regulating sst3 Somatostatin Receptor Trafficking. (United States)

    Lehmann, Andreas; Kliewer, Andrea; Günther, Thomas; Nagel, Falko; Schulz, Stefan


    The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst3 receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced β-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst3 antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1β as key regulators of sst3 phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, β-arrestin recruitment, and internalization of this clinically relevant receptor.

  19. Protein phosphorylation and its role in archaeal signal transduction. (United States)

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C; Albers, Sonja-Verena; Siebers, Bettina


    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies.


    Castellani, Rudy J.; Nunomura, Akihiko; Lee, Hyoung-gon; Perry, George; Smith, Mark A.


    Identification of phosphorylated tau as the major protein component of neurofibrillary tangles (NFTs) led to the concept that phosphorylated tau was inherently toxic and, as such, intimately involved in Alzheimer’s disease (AD) pathogenesis. While superficially logical, this construct ignores a number of key findings in AD, including i) that NFTs are encountered in viable neurons until late stage disease; ii) that NFTs persist within the neuronal cytoplasm for decades; iii) that NFTs are encountered, sometimes in significant numbers, in cognitively intact elderly; and iv) that neurons with NFTs contain normal content and structure of microtubules. Experimental data in transgenic animal models has further demonstrated that NFTs accumulate in neurons in spite of tau suppression and behavior normalization. These data call into question the inherent toxicity of phosphorylated tau, seemingly leaving the only viable hypothesis of the ad hoc “toxic intermediate” phosphorylated tau concept. However, since we also know that phosphorylated tau sequesters redox active heavy metals and protects against oxidative stress, here we suggest that phosphorylated tau serves a protective role against cellular toxicity. PMID:18688087

  1. Structural basis for Mep2 ammonium transceptor activation by phosphorylation. (United States)

    van den Berg, Bert; Chembath, Anupama; Jefferies, Damien; Basle, Arnaud; Khalid, Syma; Rutherford, Julian C


    Mep2 proteins are fungal transceptors that play an important role as ammonium sensors in fungal development. Mep2 activity is tightly regulated by phosphorylation, but how this is achieved at the molecular level is not clear. Here we report X-ray crystal structures of the Mep2 orthologues from Saccharomyces cerevisiae and Candida albicans and show that under nitrogen-sufficient conditions the transporters are not phosphorylated and present in closed, inactive conformations. Relative to the open bacterial ammonium transporters, non-phosphorylated Mep2 exhibits shifts in cytoplasmic loops and the C-terminal region (CTR) to occlude the cytoplasmic exit of the channel and to interact with His2 of the twin-His motif. The phosphorylation site in the CTR is solvent accessible and located in a negatively charged pocket ∼30 Å away from the channel exit. The crystal structure of phosphorylation-mimicking Mep2 variants from C. albicans show large conformational changes in a conserved and functionally important region of the CTR. The results allow us to propose a model for regulation of eukaryotic ammonium transport by phosphorylation.

  2. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    Energy Technology Data Exchange (ETDEWEB)

    Deaciuc, I.V.; Spitzer, J.A. (Louisiana State Univ. Medical Center, New Orleans (USA))


    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with (32P)H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis.

  3. Phosphorylation of the centrosomal protein, Cep169, by Cdk1 promotes its dissociation from centrosomes in mitosis. (United States)

    Mori, Yusuke; Inoue, Yoko; Taniyama, Yuki; Tanaka, Sayori; Terada, Yasuhiko


    Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle.

  4. STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes. (United States)

    Bergner, Sonja Verena; Scholz, Martin; Trompelt, Kerstin; Barth, Johannes; Gäbelein, Philipp; Steinbeck, Janina; Xue, Huidan; Clowez, Sophie; Fucile, Geoffrey; Goldschmidt-Clermont, Michel; Fufezan, Christian; Hippler, Michael


    In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of light-harvesting complex stress-related protein3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-light harvesting complex I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions. © 2015 American Society of Plant Biologists. All Rights Reserved.

  5. Thr175-phosphorylated tau induces pathologic fibril formation via GSK3β-mediated phosphorylation of Thr231 in vitro. (United States)

    Moszczynski, Alexander J; Gohar, May; Volkening, Kathryn; Leystra-Lantz, Cheryl; Strong, Wendy; Strong, Michael J


    We have previously shown that amyotrophic lateral sclerosis with cognitive impairment can be characterized by pathologic inclusions of microtubule-associated protein tau (tau) phosphorylated at Thr(175) (pThr(175)) in association with GSK3β activation. We have now examined whether pThr(175) induces GSK3β activation and whether this leads to pathologic fibril formation through Thr(231) phosphorylation. Seventy-two hours after transfection of Neuro2A cells with pseudophosphorylated green fluorescent protein-tagged 2N4R tau (Thr(175)Asp), phosphorylated kinase glycogen synthase kinase 3 beta (active GSK3β) levels were significantly increased as was pathologic fibril formation and cell death. Treatment with each of 4 GSK3β inhibitors or small hairpin RNA knockdown of GSK3β abolished fibril formation and prevented cell death. Inhibition of Thr(231) phosphorylation (Thr(231)Ala) prevented pathologic tau fibril formation, regardless of Thr(175) state, whereas Thr(231)Asp (pseudophosphorylated at Thr(231)) developed pathologic tau fibrils. Ser(235) mutations did not affect fibril formation, indicating an unprimed mechanism of Thr(231) phosphorylation. These findings suggest a mechanism of tau pathology by which pThr(175) induces GSK3β phosphorylation of Thr(231) leading to fibril formation, indicating a potential therapeutic avenue for amyotrophic lateral sclerosis with cognitive impairment. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Structural Basis for Inactivation of the Human Pyruvate Dehydrogenase Complex by Phosphorylation: Role of Disordered Phosphorylation Loops

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Masato; Wynn, R. Max; Chuang, Jacinta L.; Tso, Shih-Chia; Machius, Mischa; Li, Jun; Chuang, David T. (UTSMC)


    We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-{alpha} (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-{alpha} prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-{alpha}, which nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p, negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.

  7. PP2A(Cdc55) Phosphatase Imposes Ordered Cell-Cycle Phosphorylation by Opposing Threonine Phosphorylation. (United States)

    Godfrey, Molly; Touati, Sandra A; Kataria, Meghna; Jones, Andrew; Snijders, Ambrosius P; Uhlmann, Frank


    In the quantitative model of cell-cycle control, progression from G1 through S phase and into mitosis is ordered by thresholds of increasing cyclin-dependent kinase (Cdk) activity. How such thresholds are read out by substrates that respond with the correct phosphorylation timing is not known. Here, using the budding yeast model, we show that the abundant PP2A(Cdc55) phosphatase counteracts Cdk phosphorylation during interphase and delays phosphorylation of late Cdk substrates. PP2A(Cdc55) specifically counteracts phosphorylation on threonine residues, and consequently, we find that threonine-directed phosphorylation occurs late in the cell cycle. Furthermore, the late phosphorylation of a model substrate, Ndd1, depends on threonine identity of its Cdk target sites. Our results support a model in which Cdk-counteracting phosphatases contribute to cell-cycle ordering by imposing Cdk thresholds. They also unveil a regulatory principle based on the phosphoacceptor amino acid, which is likely to apply to signaling pathways beyond cell-cycle control. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy. (United States)

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang


    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

  9. Tyrosine phosphorylation of NEDD4 activates its ubiquitin ligase activity. (United States)

    Persaud, Avinash; Alberts, Philipp; Mari, Sara; Tong, Jiefei; Murchie, Ryan; Maspero, Elena; Safi, Frozan; Moran, Michael F; Polo, Simona; Rotin, Daniela


    Ligand binding to the receptor tyrosine kinase fibroblast growth factor (FGF) receptor 1 (FGFR1) causes dimerization and activation by transphosphorylation of tyrosine residues in the kinase domain. FGFR1 is ubiquitylated by the E3 ligase NEDD4 (also known as NEDD4-1), which promotes FGFR1 internalization and degradation. Although phosphorylation of FGFR1 is required for NEDD4-dependent endocytosis, NEDD4 directly binds to a nonphosphorylated region of FGFR1. We found that activation of FGFR1 led to activation of c-Src kinase-dependent tyrosine phosphorylation of NEDD4, enhancing the ubiquitin ligase activity of NEDD4. Using mass spectrometry, we identified several FGF-dependent phosphorylated tyrosines in NEDD4, including Tyr(43) in the C2 domain and Tyr(585) in the HECT domain. Mutating these tyrosines to phenylalanine to prevent phosphorylation inhibited FGF-dependent NEDD4 activity and FGFR1 endocytosis and enhanced cell proliferation. Mutating the tyrosines to glutamic acid to mimic phosphorylation enhanced NEDD4 activity. Moreover, the NEDD4 C2 domain bound the HECT domain, and the presence of phosphomimetic mutations inhibited this interaction, suggesting that phosphorylation of NEDD4 relieves an inhibitory intra- or intermolecular interaction. Accordingly, activation of FGFR1 was not required for activation of NEDD4 that lacked its C2 domain. Activation of c-Src by epidermal growth factor (EGF) also promoted tyrosine phosphorylation and enhanced the activity of NEDD4. Thus, we identified a feedback mechanism by which receptor tyrosine kinases promote catalytic activation of NEDD4 and that may represent a mechanism of receptor crosstalk.

  10. Phosphorylation of ATPase subunits of the 26S proteasome. (United States)

    Mason, G G; Murray, R Z; Pappin, D; Rivett, A J


    The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [32P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.

  11. Integrin Ligation Results in Nephrin Tyrosine Phosphorylation In Vitro.

    Directory of Open Access Journals (Sweden)

    Rakesh Verma

    Full Text Available Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm. In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin β1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand.

  12. Specific serine-proline phosphorylation and glycogen synthase kinase 3β-directed subcellular targeting of stathmin 3/Sclip in neurons. (United States)

    Devaux, Sara; Poulain, Fabienne E; Devignot, Véronique; Lachkar, Sylvie; Irinopoulou, Theano; Sobel, André


    During nervous system development, neuronal growth, migration, and functional morphogenesis rely on the appropriate control of the subcellular cytoskeleton including microtubule dynamics. Stathmin family proteins play major roles during the various stages of neuronal differentiation, including axonal growth and branching, or dendritic development. We have shown previously that stathmins 2 (SCG10) and 3 (SCLIP) fulfill distinct, independent and complementary regulatory roles in axonal morphogenesis. Although the two proteins have been proposed to display the four conserved phosphorylation sites originally identified in stathmin 1, we show here that they possess distinct phosphorylation sites within their specific proline-rich domains (PRDs) that are differentially regulated by phosphorylation by proline-directed kinases involved in the control of neuronal differentiation. ERK2 or CDK5 phosphorylate the two proteins but with different site specificities. We also show for the first time that, unlike stathmin 2, stathmin 3 is a substrate for glycogen synthase kinase (GSK) 3β both in vitro and in vivo. Interestingly, stathmin 3 phosphorylated at its GSK-3β target site displays a specific subcellular localization at neuritic tips and within the actin-rich peripheral zone of the growth cone of differentiating hippocampal neurons in culture. Finally, pharmacological inhibition of GSK-3β induces a redistribution of stathmin 3, but not stathmin 2, from the periphery toward the Golgi region of neurons. Stathmin proteins can thus be either regulated locally or locally targeted by specific phosphorylation, each phosphoprotein of the stathmin family fulfilling distinct and specific roles in the control of neuronal differentiation.

  13. Cellular Functions Regulated by Phosphorylation of EGFR on Tyr845

    Directory of Open Access Journals (Sweden)

    Ken-ichi Sato


    Full Text Available The Src gene product (Src and the epidermal growth factor receptor (EGFR are prototypes of oncogene products and function primarily as a cytoplasmic non-receptor tyrosine kinase and a transmembrane receptor tyrosine kinase, respectively. The identification of Src and EGFR, and the subsequent extensive investigations of these proteins have long provided cutting edge research in cancer and other molecular and cellular biological studies. In 1995, we reported that the human epidermoid carcinoma cells, A431, contain a small fraction of Src and EGFR in which these two kinase were in physical association with each other, and that Src phosphorylates EGFR on tyrosine 845 (Y845 in the Src-EGFR complex. Y845 of EGFR is located in the activation segment of the kinase domain, where many protein kinases contain kinase-activating autophosphorylation sites (e.g., cAMP-dependent protein kinase, Src family kinases, transmembrane receptor type tyrosine kinases or trans-phosphorylation sites (e.g., cyclin-dependent protein kinase, mitogen-activated protein kinase, Akt protein kinase. A number of studies have demonstrated that Y845 phosphorylation serves an important role in cancer as well as normal cells. Here we compile the experimental facts involving Src phosphorylation of EGFR on Y845, by which cell proliferation, cell cycle control, mitochondrial regulation of cell metabolism, gamete activation and other cellular functions are regulated. We also discuss the physiological relevance, as well as structural insights of the Y845 phosphorylation.

  14. Determining in vivo phosphorylation sites using mass spectrometry. (United States)

    Breitkopf, Susanne B; Asara, John M


    Phosphorylation is the most studied protein post-translational modification (PTM) in biological systems, since it controls cell growth, proliferation, survival, and other processes. High-resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity, and throughput. The protocols described here focus on two common strategies: (1) identifying phosphorylation sites from individual proteins and small protein complexes, and (2) identifying global phosphorylation sites from whole-cell and tissue extracts. For the first, endogenous or epitope-tagged proteins are typically immunopurified from cell lysates, purified via gel electrophoresis or precipitation, and enzymatically digested into peptides. Samples can be optionally enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO(2)) and then analyzed by microcapillary liquid chromatography/tandem mass spectrometry (LC-MS/MS). Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time consuming and involve digesting the whole-cell lysate, followed by peptide fractionation by strong cation-exchange chromatography, phosphopeptide enrichment by IMAC or TiO(2), and LC-MS/MS. Alternatively, the protein lysate can be fractionated by SDS-PAGE, followed by digestion, phosphopeptide enrichment, and LC-MS/MS. One can also immunoprecipitate only phosphotyrosine peptides using a pTyr antibody followed by LC-MS/MS.

  15. Phosphorylation of actopaxin regulates cell spreading and migration (United States)

    Clarke, Dominic M.; Brown, Michael C.; LaLonde, David P.; Turner, Christopher E.


    Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration. PMID:15353548

  16. Tau phosphorylation affects its axonal transport and degradation (United States)

    Rodríguez-Martín, Teresa; Cuchillo-Ibáñez, Inmaculada; Noble, Wendy; Nyenya, Fanon; Anderton, Brian H.; Hanger, Diane P.


    Phosphorylated forms of microtubule-associated protein tau accumulate in neurofibrillary tangles in Alzheimer's disease. To investigate the effects of specific phosphorylated tau residues on its function, wild type or phosphomutant tau was expressed in cells. Elevated tau phosphorylation decreased its microtubule binding and bundling, and increased the number of motile tau particles, without affecting axonal transport kinetics. In contrast, reducing tau phosphorylation enhanced the amount of tau bound to microtubules and inhibited axonal transport of tau. To determine whether differential tau clearance is responsible for the increase in phosphomimic tau, we inhibited autophagy in neurons which resulted in a 3-fold accumulation of phosphomimic tau compared with wild type tau, and endogenous tau was unaffected. In autophagy-deficient mouse embryonic fibroblasts, but not in neurons, proteasomal degradation of phosphomutant tau was also reduced compared with wild type tau. Therefore, autophagic and proteasomal pathways are involved in tau degradation, with autophagy appearing to be the primary route for clearing phosphorylated tau in neurons. Defective autophagy might contribute to the accumulaton of tau in neurodegenerative diseases. PMID:23601672

  17. Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation

    CSIR Research Space (South Africa)

    Owen, GR


    Full Text Available –deuterium exchange (HX) mass spectrometry were used to investigate the changes to the stability and conformation/conformational dynamics of JNK1ß1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide...

  18. Neurofilament subunit (NFL) head domain phosphorylation regulates axonal transport of neurofilaments.

    LENUS (Irish Health Repository)

    Yates, Darran M


    Neurofilaments are the intermediate filaments of neurons and are synthesised in neuronal cell bodies and then transported through axons. Neurofilament light chain (NFL) is a principal component of neurofilaments, and phosphorylation of NFL head domain is believed to regulate the assembly of neurofilaments. However, the role that NFL phosphorylation has on transport of neurofilaments is poorly understood. To address this issue, we monitored axonal transport of phosphorylation mutants of NFL. We mutated four known phosphorylation sites in NFL head domain to either preclude phosphorylation, or mimic permanent phosphorylation. Mutation to preclude phosphorylation had no effect on transport but mutation of three sites to mimic permanent phosphorylation inhibited transport. Mutation of all four sites together to mimic permanent phosphorylation proved especially potent at inhibiting transport and also disrupted neurofilament assembly. Our results suggest that NFL head domain phosphorylation is a regulator of neurofilament axonal transport.

  19. Postsynaptic density 95 (PSD-95) serine 561 phosphorylation regulates a conformational switch and bidirectional dendritic spine structural plasticity. (United States)

    Wu, Qian; Sun, Miao; Bernard, Laura P; Zhang, Huaye


    Postsynaptic density 95 (PSD-95) is a major synaptic scaffolding protein that plays a key role in bidirectional synaptic plasticity, which is a process important for learning and memory. It is known that PSD-95 shows increased dynamics upon induction of plasticity. However, the underlying structural and functional changes in PSD-95 that mediate its role in plasticity remain unclear. Here we show that phosphorylation of PSD-95 at Ser-561 in its guanylate kinase (GK) domain, which is mediated by the partitioning-defective 1 (Par1) kinases, regulates a conformational switch and is important for bidirectional plasticity. Using a fluorescence resonance energy transfer (FRET) biosensor, we show that a phosphomimetic mutation of Ser-561 promotes an intramolecular interaction between GK and the nearby Src homology 3 (SH3) domain, leading to a closed conformation, whereas a non-phosphorylatable S561A mutation or inhibition of Par1 kinase activity decreases SH3-GK interaction, causing PSD-95 to adopt an open conformation. In addition, S561A mutation facilitates the interaction between PSD-95 and its binding partners. Fluorescence recovery after photobleaching imaging reveals that the S561A mutant shows increased stability, whereas the phosphomimetic S561D mutation increases PSD-95 dynamics at the synapse. Moreover, molecular replacement of endogenous PSD-95 with the S561A mutant blocks dendritic spine structural plasticity during chemical long-term potentiation and long-term depression. Endogenous Ser-561 phosphorylation is induced by synaptic NMDA receptor activation, and the SH3-GK domains exhibit a Ser-561 phosphorylation-dependent switch to a closed conformation during synaptic plasticity. Our results provide novel mechanistic insight into the regulation of PSD-95 in dendritic spine structural plasticity through phosphorylation-mediated regulation of protein dynamics and conformation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Identification of sennoside A as a novel inhibitor of the slingshot (SSH) family proteins related to cancer metastasis. (United States)

    Lee, Seon Young; Kim, Wooil; Lee, Young Geun; Kang, Hyo Jin; Lee, Sang-Hyun; Park, Sun Young; Min, Jeong-Ki; Lee, Sang-Rae; Chung, Sang J


    Phospho-cofilin (p-cofilin), which has a phosphate group on Ser-3, is involved in actin polymerization. Its dephosphorylated form promotes filopodia formation and cell migration by enhancing actin depolymerization. Protein phosphatase slingshot homologs (SSHs), known as dual-specificity phosphatases, catalyze hydrolytic removal of the Ser-3 phosphate group from phospho-cofilin. Aberrant SSH activity results in cancer metastasis, implicating SSHs as potential therapeutic targets for cancer metastasis. In this study, we screened 658 natural products purified from traditional oriental medicinal plants to identify three potent SSH inhibitors with submicromolar or single-digit micromolar Ki values: gossypol, hypericin, and sennoside A. The three compounds were purified from cottonseed, Saint John's wort, and rhubarb, respectively. Sennoside A markedly increased cofilin phosphorylation in pancreatic cancer cells, leading to impaired actin dynamics in pancreatic cancer cells with or without EGF stimulation and reduced motility and invasiveness in vitro and in vivo. Collaboratively, these results demonstrate that sennoside A is a novel inhibitor of SSHs and suggest that it may be valuable in the development of pharmaceutical drugs for treating cancer metastasis.

  1. Rat1p maintains RNA polymerase II CTD phosphorylation balance

    DEFF Research Database (Denmark)

    Jimeno-González, Silvia; Schmid, Manfred; Malagon, Francisco


    In S. cerevisiae, the 5'-3' exonuclease Rat1p partakes in transcription termination. Although Rat1p-mediated RNA degradation has been suggested to play a role for this activity, the exact mechanisms by which Rat1p helps release RNA polymerase II (RNAPII) from the DNA template are poorly understood....... Here we describe a function of Rat1p in regulating phosphorylation levels of the C-terminal domain (CTD) of the largest RNAPII subunit, Rpb1p, during transcription elongation. The rat1-1 mutant exhibits highly elevated levels of CTD phosphorylation as well as RNAPII distribution and transcription...... termination defects. These phenotypes are all rescued by overexpression of the CTD phosphatase Fcp1p, suggesting a functional relationship between the absence of Rat1p activity, elevated CTD phosphorylation, and transcription defects. We also demonstrate that rat1-1 cells display increased RNAPII...

  2. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.


    Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

  3. Exploring the intramolecular phosphorylation sites in human Chk2

    DEFF Research Database (Denmark)

    Olsen, Birgitte B; Larsen, Martin R; Boldyreff, Brigitte;


    A comparative biochemical analysis was performed using recombinant human protein kinase Chk2 (checkpoint kinase 2) expressed in bacteria and insect cells. Dephosphorylated, inactive, recombinant human Chk2 could be reactivated in a concentration-dependent manner. Despite distinct time....... Mass spectrometric analyses of human recombinant Chk2 isolated from bacteria and insect cells showed distinct differences. The number of phosphorylated residues in human recombinant Chk2 isolated from bacteria was 16, whereas in the case of the recombinant human Chk2 from insect cells it was 8. Except...... for phosphorylated amino acid T378 which was not found in the Chk2 isolated from bacteria, all other phosphorylated residues identified in human Chk2 from insect cells were present also in Chk2 from bacteria....

  4. Ca/calmodulin-dependent phosphorylation of endocytic scaffold ITSN1

    Directory of Open Access Journals (Sweden)

    Morderer D. Ye.


    Full Text Available ITSN1 is an endocytic scaffold protein with a prominent function in synaptic transmission. It is known that Ca signaling is crucial for the regulation of synaptic proteins functioning. Aim. Checking the possibility of Ca/calmodulin-dependent phosphorylation of ITSN1. Methods. Affinity chromatography, in vitro kinase reaction, Western blotting, gel staining with fluorescent stains. Results. We show that the fraction of calmodulin-binding proteins is able to phosphorylate the recombinant fragments encoding the coiled-coil region and the SH3 domain-containing region of ITSN1 in the presence of Ca ions and calmodulin. Conclusions. The coiled-coil region and the SH3 domain-containing region of ITSN1 undergo Ca/calmodulin-dependent phosphorylation in vitro, suggesting a possible regulation of ITSN1 by Ca signaling.

  5. Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase (United States)

    Zhang, Jian; Ishkhanian, Rita; Uckun, Fatih M.


    Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros. PMID:23977012

  6. Regulatory phosphorylation of Ikaros by Bruton's tyrosine kinase.

    Directory of Open Access Journals (Sweden)

    Hong Ma

    Full Text Available Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4 within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros.

  7. Crystal Structure of a Phosphorylation-coupled Saccharide Transporter

    Energy Technology Data Exchange (ETDEWEB)

    Y Cao; X Jin; E Levin; H Huang; Y Zong; W Hendrickson; J Javitch; K Rajashankar; M Zhou; et al.


    Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

  8. Selective phosphorylation during early macrophage differentiation

    KAUST Repository

    Zhang, Huoming


    The differentiation of macrophages from monocytes is a tightly controlled and complex biological process. Although numerous studies have been conducted using biochemical approaches or global gene/gene profiling, the mechanisms of the early stages of differentiation remain unclear. Here we used SILAC-based quantitative proteomics approach to perform temporal phosphoproteome profiling of early macrophage differentiation. We identified a large set of phosphoproteins and grouped them as PMA-regulated and non-regulated phosphoproteins in the early stages of differentiation. Further analysis of the PMA-regulated phosphoproteins revealed that transcriptional suppression, cytoskeletal reorganization and cell adhesion were among the most significantly activated pathways. Some key involved regulators of these pathways are mTOR, MYB, STAT1 and CTNNB. Moreover, we were able to classify the roles and activities of several transcriptional factors during different differentiation stages and found that E2F is likely to be an important regulator during the relatively late stages of differentiation. This study provides the first comprehensive picture of the dynamic phosphoproteome during myeloid cells differentiation, and identifies potential molecular targets in leukemic cells.

  9. Surface-phosphorylated copolymer promotes direct bone bonding. (United States)

    Gopalakrishnanchettiyar, Sailaja S; Mohanty, Mira; Kumary, Thrikkovil V; Valappil, Mohanan P; Parameshwaran, Ramesh; Varma, Harikrishna K


    The bone bonding potential of surface-phosphorylated poly (2-hydroxyethyl methacrylate-co-methyl methacrylate) [poly (HEMA-co-MMA)] has been investigated and compared with commercially available poly (methyl methacrylate) bone cement (CMW1 radiopaque, Depuy; Johnson & Johnson, Blackpool, Lancashire, England, United Kingdom) as control. Poly (HEMA-co-MMA) is synthesized by free radical-initiated copolymerization and surface functionalized by phosphorylation. The X-ray photoelectron spectroscopy confirms the presence of surface-bound phosphate groups on poly (HEMA-co-MMA). The surface-phosphorylated poly (HEMA-co-MMA) promotes in vitro biomineralization, cell viability, cell adhesion, and expression of bone-specific markers such as osteocalcin and alkaline phosphatase. The bone implantation study performed in rabbits as per ISO 10993-6; 1994 (E) shows that surface-phosphorylated poly (HEMA-co-MMA) elicits bone bonding and new bone formation. New woven bone trabeculae are formed at the defect site of surface-phosphorylated poly (HEMA-co-MMA) within 1 week, while for control sample, inflammatory cells--predominantly, macrophages, fibroblasts, and fibrocytes--are present at the cortical margins around the defect. The 4 and 12 weeks postimplantation results show that the major part of the defects around the surface-phosphorylated poly (HEMA-co-MMA) implant is bridged with new woven bone, with significant remodeling (evident from resorption bays) along both the margins of the defect, but for control implants, the defects are only partially closed, with slight remodeling along the margins, but most of them are separated by fibrous tissue.

  10. The phosphorylation pattern of bovine heart complex I subunits

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Sardanelli, Anna Maria; Signorile, Anna;


    The phosphoproteome of bovine heart complex I of the respiratory chain has been analysed with a procedure based on nondenaturing gel electrophoretic separation of complex I from small quantities of mitochondria samples, in-gel digestion, in combination with phosphopeptide enrichment by titanium...... dioxide and MS. The results, complemented by analyses of purified samples of complex I, showed phosphorylation of five subunits of the complex, 42 kDa (human gene NDUFA10), ESSS, B14.5a (human gene NDUFA7), B14.5b (human gene NDUFC2) and B16.6 (GRIM-19). MS also revealed the presence of phosphorylated...

  11. Kinase-specific prediction of protein phosphorylation sites

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Blom, Nikolaj


    -substrate specificity. Here, we briefly describe the available resources for predicting kinase-specific phosphorylation from sequence properties. We address the strengths and weaknesses of these resources, which are based on methods ranging from simple consensus patterns to more advanced machine-learning algorithms....... Furthermore, a protocol for the use of the artificial neural network based predictors, NetPhos and NetPhosK, is provided. Finally, we point to possible developments with the intention of providing the community with improved and additional phosphorylation predictors for large-scale modeling of cellular...... signaling networks....

  12. Chemical phosphorylation of deoxyribonucleosides and thermolytic DNA oligonucleotides. (United States)

    Ausín, Cristina; Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L


    The phosphorylating reagent bis[S-(4,4'-dimethoxytrityl)-2-mercaptoethyl]-N,N-diisopropylphosphoramidite is prepared in three steps from commercial methyl thioglycolate and diisopropylphosphoramidous dichloride. The phosphorylating reagent has been used successfully in the solid-phase synthesis of deoxyribonucleoside 5'-/3'-phosphate or -thiophosphate monoesters and oligonucleotide 5'-phosphate/-thiophosphate monoesters. Bis[S-(4,4'-dimethoxytrityl)-2-mercaptoethyl]-N,N-diisopropylphosphoramidite has also been employed in the construction of a thermolytic dinucleotide prodrug model to evaluate the ability of the reagent to produce thermosentive oligonucleotide prodrugs under mild temperature conditions ( approximately 25 degrees C) for potential therapeutic applications.

  13. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G


    to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic...... phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP...... of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  14. Phosphorylation of the actin binding protein Drebrin at S647 is regulated by neuronal activity and PTEN.

    Directory of Open Access Journals (Sweden)

    Patricia Kreis

    Full Text Available Defects in actin dynamics affect activity-dependent modulation of synaptic transmission and neuronal plasticity, and can cause cognitive impairment. A salient candidate actin-binding protein linking synaptic dysfunction to cognitive deficits is Drebrin (DBN. However, the specific mode of how DBN is regulated at the central synapse is largely unknown. In this study we identify and characterize the interaction of the PTEN tumor suppressor with DBN. Our results demonstrate that PTEN binds DBN and that this interaction results in the dephosphorylation of a site present in the DBN C-terminus--serine 647. PTEN and pS647-DBN segregate into distinct and complimentary compartments in neurons, supporting the idea that PTEN negatively regulates DBN phosphorylation at this site. We further demonstrate that neuronal activity increases phosphorylation of DBN at S647 in hippocampal neurons in vitro and in ex vivo hippocampus slices exhibiting seizure activity, potentially by inducing rapid dissociation of the PTEN:DBN complex. Our results identify a novel mechanism by which PTEN is required to maintain DBN phosphorylation at dynamic range and signifies an unusual regulation of an actin-binding protein linked to cognitive decline and degenerative conditions at the CNS synapse.

  15. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

    Energy Technology Data Exchange (ETDEWEB)

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik


    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

  16. Hyper-O-GlcNAcylation of YB-1 affects Ser102 phosphorylation and promotes cell proliferation in hepatocellular carcinoma. (United States)

    Liu, Qingqing; Tao, Tao; Liu, Fang; Ni, Runzhou; Lu, Cuihua; Shen, Aiguo


    As an essential post-translational modification, O-GlcNAcylation has been thought to be able to modulate various nuclear and cytoplasmic proteins and is emerging as a key regulator of multiple biological processes, such as transcription, cell growth, signal transduction, and cell motility. Recently, authoritative glycomics analyses have reported extensive crosstalk between O-GlcNAcylation and phosphorylation, which always dynamically interplay with each other and regulate signaling, transcription, and other cellular processes. Also, plentiful studies have shown close correlation between YB-1 phosphorylation and tumorigenesis. Therefore, our study aimed to determine whether YB-1 was O-GlcNAc modified and whether such modification could interact with its phosphorylation during the process of HCC development. Western blot and immunohistochemistry were firstly conducted to reveal obvious up-regulation of YB-1, OGT and O-GlcNAc modification in HCC tissues. What is more, not only YB-1 was identified to be O-GlcNAcylated but hyper-O-GlcNAcylation was demonstrated to facilitate HCC cell proliferation in a YB-1 dependent manner. Moreover, we detected four specific O-GlcNAc sites and confirmed T126A to be the most effective mutant in HCC cell proliferation via close O-GlcNAcylation-phosphorylation interaction. Even more interestingly, we discovered that T126A-induced HCC cell retardation and subdued transcriptional activity of YB-1 could be partially reversed by T126A/S102E mutant. From all above, it is not difficult to find that glycosylated-YB-1 mainly enhanced cell proliferation through congenerous actions with YB-1 phosphorylation and thus played indispensable roles in fine-tuning cell proliferation and procession of HCC.

  17. Phosphorylation of the Synaptonemal Complex Protein Zip1 Regulates the Crossover/Noncrossover Decision during Yeast Meiosis. (United States)

    Chen, Xiangyu; Suhandynata, Ray T; Sandhu, Rima; Rockmill, Beth; Mohibullah, Neeman; Niu, Hengyao; Liang, Jason; Lo, Hsiao-Chi; Miller, Danny E; Zhou, Huilin; Börner, G Valentin; Hollingsworth, Nancy M


    Interhomolog crossovers promote proper chromosome segregation during meiosis and are formed by the regulated repair of programmed double-strand breaks. This regulation requires components of the synaptonemal complex (SC), a proteinaceous structure formed between homologous chromosomes. In yeast, SC formation requires the "ZMM" genes, which encode a functionally diverse set of proteins, including the transverse filament protein, Zip1. In wild-type meiosis, Zmm proteins promote the biased resolution of recombination intermediates into crossovers that are distributed throughout the genome by interference. In contrast, noncrossovers are formed primarily through synthesis-dependent strand annealing mediated by the Sgs1 helicase. This work identifies a conserved region on the C terminus of Zip1 (called Zip1 4S), whose phosphorylation is required for the ZMM pathway of crossover formation. Zip1 4S phosphorylation is promoted both by double-strand breaks (DSBs) and the meiosis-specific kinase, MEK1/MRE4, demonstrating a role for MEK1 in the regulation of interhomolog crossover formation, as well as interhomolog bias. Failure to phosphorylate Zip1 4S results in meiotic prophase arrest, specifically in the absence of SGS1. This gain of function meiotic arrest phenotype is suppressed by spo11Δ, suggesting that it is due to unrepaired breaks triggering the meiotic recombination checkpoint. Epistasis experiments combining deletions of individual ZMM genes with sgs1-md zip1-4A indicate that Zip1 4S phosphorylation functions prior to the other ZMMs. These results suggest that phosphorylation of Zip1 at DSBs commits those breaks to repair via the ZMM pathway and provides a mechanism by which the crossover/noncrossover decision can be dynamically regulated during yeast meiosis.

  18. Elucidation of O-Phosphoryl and N-Phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

    Institute of Scientific and Technical Information of China (English)

    ZHANG,Jian-Chen(张建臣); CAO,Shu-Xiaa(曹书霞); XU,Juna(徐军); LIAO,Xin-Cheng(廖新成); ZHAO,Yu-Fen(赵玉芬)


    Mass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). Besides N-diisopropyloxyphosphoryl amino acids (N-DIPP-AA), O-phospho- and O-diisopropyloxyphosphoryl amino acids (O-DIPP-AA) were studied and compared to N-DIPP-AA. The fragmentation pathways of [M+H]+, [M+Na]+ and [M-H]- ions of phosphoryl amino acids were summarized. In addition to several similar patterns,each of them showed its characteristic fragmention.

  19. Phytophthora infestans specific phosphorylation patterns and new putative control targets. (United States)

    Frades, Itziar; Andreasson, Erik


    In this study we applied biomathematical searches of gene regulatory mechanisms to learn more about oomycete biology and to identify new putative targets for pesticides or biological control against Phytophthora infestans. First, oomycete phylum-specific phosphorylation motifs were found by discriminative n-gram analysis. We found 11.600 P. infestans specific n-grams, mapping 642 phosphoproteins. The most abundant group among these related to phosphatidylinositol metabolism. Due to the large number of possible targets found and our hypothesis that multi-level control is a sign of usefulness as targets for intervention, we identified overlapping targets with a second screen. This was performed to identify proteins dually regulated by small RNA and phosphorylation. We found 164 proteins to be regulated by both sRNA and phosphorylation and the dominating functions where phosphatidylinositol signalling/metabolism, endocytosis, and autophagy. Furthermore we performed a similar regulatory study and discriminative n-gram analysis of proteins with no clear orthologs in other species and proteins that are known to be unique to P. infestans such as the RxLR effectors, Crinkler (CRN) proteins and elicitins. We identified CRN proteins with specific phospho-motifs present in all life stages. PITG_12626, PITG_14042 and PITG_23175 are CRN proteins that have species-specific phosphorylation motifs and are subject to dual regulation.

  20. Construction and Analysis of N-phosphoryl Peptide Libraries

    Institute of Scientific and Technical Information of China (English)

    Shu Xia CAO; Jian Chen ZHANG; Ming Yu NIU; Kui LU; Xin Cheng LIAO; Yu Fen ZHAO


    N-Phosphoryl peptide libraries were constructed by transformation from homo-oligopeptide libraries, which was synthesized by self-assembly of amino acids with the assistance of phosphorus oxychloride. Electrospray ionization mass spectrometry (ESI-MS) was used to monitor the reaction.

  1. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Adi, Y. A., E-mail: [Department of Mathematic Faculty of MIPA Universitas Ahmad Dahlan (Indonesia); Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Kusumo, F. A.; Aryati, L. [Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Hardianti, M. S. [Department of Internal Medicine, Faculty of Medicine, Universitas Gadjah Mada (Indonesia)


    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  2. Genetic defects in the oxidative phosphorylation (OXPHOS) system.

    NARCIS (Netherlands)

    Janssen, R.J.R.J.; Heuvel, L.P.W.J. van den; Smeitink, J.A.M.


    The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual gene

  3. Spatial separation of Plk1 phosphorylation and activity

    Directory of Open Access Journals (Sweden)

    Wytse eBruinsma


    Full Text Available Polo-like kinase 1 (Plk1 is one of the major kinases controlling mitosis and cell division. Plk1 is first recruited to the centrosome in S phase, then appears on the kinetochores in late G2 and at the end of mitosis it translocates to the central spindle. Activation of Plk1 requires phosphorylation of T210 by Aurora A, an event that critically depends on the co-factor Bora. However, conflicting reports exist as to where Plk1 is first activated. Phosphorylation of T210 is first observed at the centrosomes, but kinase activity seems to be restricted to the nucleus in the earlier phases of G2. Here we demonstrate that Plk1 activity manifests itself first in the nucleus using a nuclear FRET-based biosensor for Plk1 activity. However, we find that Bora is restricted to the cytoplasm and that Plk1 is phosphorylated on T210 at the centrosomes. Our data demonstrate that while Plk1 activation occurs on centrosomes, downstream target phosphorylation by Plk1 first occurs in the nucleus. We discuss several explanations for this surprising separation of activation and function.

  4. Phosphorylation of formate dehydrogenase in potato tuber mitochondria

    DEFF Research Database (Denmark)

    Bykova, N.V.; Stensballe, A.; Egsgaard, H.


    Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha...

  5. Histone 3 s10 phosphorylation: "caught in the R loop!". (United States)

    Skourti-Stathaki, Konstantina; Proudfoot, Nicholas J


    In this issue of Molecular Cell, Castellano-Pozo et al. (2013) describe a connection between R loop structures and histone 3 S10 phosphorylation (H3S10P), a mark of chromatin compaction. Their results constitute a significant advance in our understanding of the role of R loops in genomic instability.

  6. Annealing properties of potato starches with different degrees of phosphorylation

    DEFF Research Database (Denmark)

    Muhrbeck, Per; Svensson, E


    Changes in the gelatinization temperature interval and gelatinization enthalpy with annealing time at 50 degrees C were followed for a number of potato starch samples, with different degrees of phosphorylation, using differential scanning calorimetry. The gelatinization temperature increased with...... and crystalline structure of amylopectin helices. (C) 1997 Elsevier Science Ltd....

  7. Genetic defects in the oxidative phosphorylation (OXPHOS) system.

    NARCIS (Netherlands)

    Janssen, R.J.R.J.; Heuvel, L.P.W.J. van den; Smeitink, J.A.M.


    The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual

  8. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation (United States)

    Jena, Ananta Kumar


    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  9. Enteric GFAP expression and phosphorylation in Parkinson's disease

    NARCIS (Netherlands)

    Clairembault, Thomas; Kamphuis, W.; Leclair-Visonneau, Laurène; Rolli-Derkinderen, Malvyne; Coron, Emmanuel; Neunlist, Michel; Hol, Elly M; Derkinderen, Pascal


    Enteric glial cells (EGCs) are in many respects similar to astrocytes of the central nervous system and express similar proteins including glial fibrillary acidic protein (GFAP). Changes in GFAP expression and/or phosphorylation have been reported during brain damage or central nervous system degene

  10. Phosphorylation of as1-casein is regulated by different genes

    NARCIS (Netherlands)

    Bijl, E.; Valenberg, van H.J.F.; Huppertz, T.; Hooijdonk, van A.C.M.; Bovenhuis, H.


    Casein phosphorylation is a posttranslational modification catalyzed by kinase enzymes that attach phosphate groups to specific AA in the protein sequence. This modification is one of the key factors responsible for the stabilization of calcium phosphate nanoclusters in casein micelles and for the i

  11. Eph-mediated tyrosine phosphorylation of citron kinase controls abscission (United States)

    Jungas, Thomas; Perchey, Renaud T.; Fawal, Mohamad; Callot, Caroline; Froment, Carine; Burlet-Schiltz, Odile; Besson, Arnaud


    Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis. PMID:27551053

  12. ACTH, cyclic nucleotides, and brain protein phosphorylation in vitro

    NARCIS (Netherlands)

    Zwiers, H; Veldhuis, H D; Schotman, P; Gispen, W H


    Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated(32)P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide

  13. The phosphatase calcineurin regulates pathological TDP-43 phosphorylation. (United States)

    Liachko, Nicole F; Saxton, Aleen D; McMillan, Pamela J; Strovas, Timothy J; Currey, Heather N; Taylor, Laura M; Wheeler, Jeanna M; Oblak, Adrian L; Ghetti, Bernardino; Montine, Thomas J; Keene, C Dirk; Raskind, Murray A; Bird, Thomas D; Kraemer, Brian C


    Detergent insoluble inclusions of TDP-43 protein are hallmarks of the neuropathology in over 90 % of amyotrophic lateral sclerosis (ALS) cases and approximately half of frontotemporal dementia (FTLD-TDP) cases. In TDP-43 proteinopathy disorders, lesions containing aggregated TDP-43 protein are extensively post-translationally modified, with phosphorylated TDP-43 (pTDP) being the most consistent and robust marker of pathological TDP-43 deposition. Abnormally phosphorylated TDP-43 has been hypothesized to mediate TDP-43 toxicity in many neurodegenerative disease models. To date, several different kinases have been implicated in the genesis of pTDP, but no phosphatases have been shown to reverse pathological TDP-43 phosphorylation. We have identified the phosphatase calcineurin as an enzyme binding to and catalyzing the removal of pathological C-terminal phosphorylation of TDP-43 in vitro. In C. elegans models of TDP-43 proteinopathy, genetic elimination of calcineurin results in accumulation of excess pTDP, exacerbated motor dysfunction, and accelerated neurodegenerative changes. In cultured human cells, treatment with FK506 (tacrolimus), a calcineurin inhibitor, results in accumulation of pTDP species. Lastly, calcineurin co-localizes with pTDP in degenerating areas of the central nervous system in subjects with FTLD-TDP and ALS. Taken together, these findings suggest calcineurin acts on pTDP as a phosphatase in neurons. Furthermore, patient treatment with calcineurin inhibitors may have unappreciated adverse neuropathological consequences.

  14. Studies on the synthesis of phosphorylated and alanylated cytokinins.

    NARCIS (Netherlands)

    Shadid, B.


    New approaches are described in this thesis towards the syntheses of phosphorylated and alanylated cytokinins.In chapter 1 a general picture of the stucture of cytokinins, their occurence in nature, their biological synthesis, their effects on plants and their chemical synthesis is described.A liter

  15. Stem rust spores elicit rapid RPG1 phosphorylation (United States)

    Stem rust threatens cereal production worldwide. Understanding the mechanism by which durable resistance genes, such as Rpg1, function is critical. We show that the RPG1 protein is phosphorylated within 5 min by exposure to spores from avirulent but not virulent races of stem rust. Transgenic mutant...

  16. One-Pot Synthesis of N-Phosphoryl Amino Acids

    Institute of Scientific and Technical Information of China (English)

    GUO Xin; FU Hua; LIN Chang-Xue; ZHAO Yu-Fen


    @@ Phosphoramidates have been considered as an important class of rationally designed therapeutics especially asoligonucleotide analogs employed as antisene and antigene agents. [1] N-Phosphoryl amino acids are of biological andpharmaceutical interest, [2] and can be used as the building blocks in synthesis of polypeptides. [3

  17. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia (United States)

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.


    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  18. Decoding the phosphorylation code in Hedgehog signal transduction

    Institute of Scientific and Technical Information of China (English)

    Yongbin Chen; Jin Jiang


    Hedgehog (Hh) signaling plays pivotal roles in embryonic development and adult tissue homeostasis,and its deregulation leads to numerous human disorders including cancer.Binding of Hh to Patched (Ptc),a twelve-transmembrane protein,alleviates its inhibition of Smoothened (Smo),a seven-transmembrane protein related to G-proteincoupled receptors (GPCRs),leading to Smo phosphorylation and activation.Smo acts through intracellular signaling complexes to convert the latent transcription factor Cubitus interruptus (Ci)/Gli from a truncated repressor to a fulllength activator,leading to derepression/activation of Hh target genes.Increasing evidence suggests that phosphorylation participates in almost every step in the signal relay from Smo to Ci/Gli,and that differential phosphorylation of several key pathway components may be crucial for translating the Hh morphogen gradient into graded pathway activities.In this review,we focus on the multifaceted roles that phosphorylation plays in Hh signal transduction,and discuss the conservation and difference between Drosophila and mammalian Hh signaling mechanisms.

  19. Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain

    DEFF Research Database (Denmark)

    Oh, E S; Couchman, J R; Woods, A


    Protein kinase C (PKC) is involved in cell-matrix and cell-cell adhesion, and the cytoplasmic domain of syndecan-2 contains two serines (residues 197 and 198) which lie in a consensus sequence for phosphorylation by PKC. Other serine and threonine residues are present but not in a consensus seque...

  20. Bioinformatics Study of Cancer-Related Mutations within p53 Phosphorylation Site Motifs

    Directory of Open Access Journals (Sweden)

    Xiaona Ji


    Full Text Available p53 protein has about thirty phosphorylation sites located at the N- and C-termini and in the core domain. The phosphorylation sites are relatively less mutated than other residues in p53. To understand why and how p53 phosphorylation sites are rarely mutated in human cancer, using a bioinformatics approaches, we examined the phosphorylation site and its nearby flanking residues, focusing on the consensus phosphorylation motif pattern, amino-acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer mutations observed within the phosphorylation motifs. Many p53 phosphorylation sites are targets for several kinases. The phosphorylation sites match 17 consensus sequence motifs out of the 29 classified. In addition to proline, which is common in kinase specificity-determining sites, we found high propensity of acidic residues to be adjacent to phosphorylation sites. Analysis of human cancer mutations in the phosphorylation motifs revealed that motifs with adjacent acidic residues generally have fewer mutations, in contrast to phosphorylation sites near proline residues. p53 phosphorylation motifs are mostly disordered. However, human cancer mutations within phosphorylation motifs tend to decrease the disorder propensity. Our results suggest that combination of acidic residues Asp and Glu with phosphorylation sites provide charge redundancy which may safe guard against loss-of-function mutations, and that the natively disordered nature of p53 phosphorylation motifs may help reduce mutational damage. Our results further suggest that engineering acidic amino acids adjacent to potential phosphorylation sites could be a p53 gene therapy strategy.

  1. PR65A phosphorylation regulates PP2A complex signaling.

    Directory of Open Access Journals (Sweden)

    Kumar Kotlo

    Full Text Available Serine-threonine Protein phosphatase 2 A (PP2A, a member of the PPP family of phosphatases, regulates a variety of essential cellular processes, including cell-cycling, DNA replication, transcription, translation, and secondary signaling pathways. In the heart, increased PP2A activity/signaling has been linked to cardiac remodeling, contractile dysfunction and, in failure, arrythmogenicity. The core PP2A complex is a hetero-trimeric holoenzyme consisting of a 36 kDa catalytic subunit (PP2Ac; a regulatory scaffold subunit of 65 kDa (PR65A or PP2Aa; and one of at least 18 associated variable regulatory proteins (B subunits classified into 3 families. In the present study, three in vivo sites of phosphorylation in cardiac PR65A are identified (S303, T268, S314. Using HEK cells transfected with recombinant forms of PR65A with phosphomimetic (P-PR65A and non-phosphorylated (N-PR65A amino acid substitutions at these sites, these phosphorylations were shown to inhibit the interaction of PR65A with PP2Ac and PP2A holoenzyme signaling. Forty-seven phospho-proteins were increased in abundance in HEK cells transfected with P-PR65A versus N-PR65A by phospho-protein profiling using 2D-DIGE analysis on phospho-enriched whole cell protein extracts. Among these proteins were elongation factor 1α (EF1A, elongation factor 2, heat shock protein 60 (HSP60, NADPH-dehydrogenase 1 alpha sub complex, annexin A, and PR65A. Compared to controls, failing hearts from the Dahl rat had less phosphorylated PR65A protein abundance and increased PP2A activity. Thus, PR65A phosphorylation is an in vivo mechanism for regulation of the PP2A signaling complex and increased PP2A activity in heart failure.

  2. A redox-regulated tyrosine phosphorylation cascade in rat spermatozoa. (United States)

    Lewis, B; Aitken, R J


    Rat spermatozoa from both the caput and cauda epididymidis were shown to generate superoxide anion (O2-.) both spontaneously and following stimulation with NAD(P)H. Caput spermatozoa gave a significantly greater O2- response to NADPH stimulation than caudal cells, whereas in both cell types the responses to exogenous NADPH and NADH were approximately equivalent. Analysis of H2O2 production revealed that this oxidant was generated only by caudal epididymal cells and only in these cells did the stimulation of reactive oxygen species (ROS) production with NADPH lead to an increase in tyrosine phosphorylation. Stimulation of ROS production with NADPH increased intracellular cyclic adenosine monophosphate (cAMP) levels in both caput and caudal epididymal cells, but only in caudal cells did cAMP stimulate tyrosine phosphorylation, in keeping with the NADPH results. On the basis of these findings we propose that tyrosine phosphorylation in rat spermatozoa is driven by ROS acting via 2 different but complementary mechanisms; O2-. stimulates tyrosine kinase activity indirectly through the elevation of intracellular cAMP while H2O2 acts directly on the kinase/phosphatase system, stimulating the former and inhibiting the latter. Zinc was examined as a potential regulator of this signal transduction cascade and was shown to suppress tyrosine phosphorylation in caput cells but to promote this activity in caudal spermatozoa, possibly through an inhibitory effect on tyrosine phosphatase activity. These results reveal the maturation of a redox-regulated, cAMP-mediated, signal transduction cascade during epididymal transit in the rat that is sensitive to zinc and plays a key role in the control of tyrosine phosphorylation events associated with capacitation.

  3. Disruption of GluA2 phosphorylation potentiates stress responsivity. (United States)

    Ellis, Alexandra S; Fosnocht, Anne Q; Lucerne, Kelsey E; Briand, Lisa A


    Cocaine addiction is characterized by persistent craving and addicts frequently relapse even after long periods of abstinence. Exposure to stress can precipitate relapse in humans and rodents. Stress and drug use can lead to common alterations in synaptic plasticity and these commonalities may contribute to the ability of stress to elicit relapse. These common changes in synaptic plasticity are mediated, in part, by alterations in the trafficking and stabilization of AMPA receptors. Exposure to both cocaine and stress can lead to alterations in protein kinase C-mediated phosphorylation of GluA2 AMPA subunits and thus alter the trafficking of GluA2-containing AMPARs. However, it is not clear what role AMPAR trafficking plays in the interactions between stress and cocaine. The current study utilized a mouse with a point mutation within the GluA2 subunit c-terminus resulting in a disruption of PKC-mediated GluA2 phosphorylation to examine stress responsivity. Although no differences were seen in the response to a forced swim stress in naïve mice, GluA2 K882A knock-in mice exhibited an increased stress response following cocaine self-administration. Furthermore, we demonstrated that disrupting GluA2 phosphorylation increases vulnerability to stress-induced reinstatement of both cocaine seeking and cocaine-conditioned reward. Finally, GluA2 K882A knock-in mice exhibit an increased vulnerability to social defeat as indicated by increased social avoidance. Taken together these results indicate that disrupting GluA2 phosphorylation leads to increased responsivity to acute stress following cocaine exposure and increased vulnerability to chronic stress. These results highlight the GluA2 phosphorylation site as a novel target for the stress-related disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Phosphorylation of the chromatin binding domain of KSHV LANA.

    Directory of Open Access Journals (Sweden)

    Crystal Woodard

    Full Text Available The Kaposi sarcoma associated herpesvirus (KSHV latency associated nuclear antigen (LANA is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1-329 that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3-21. Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.

  5. Phosphorylation regulates NCC stability and transporter activity in vivo. (United States)

    Yang, Sung-Sen; Fang, Yu-Wei; Tseng, Min-Hua; Chu, Pei-Yi; Yu, I-Shing; Wu, Han-Chung; Lin, Shu-Wha; Chau, Tom; Uchida, Shinichi; Sasaki, Sei; Lin, Yuh-Feng; Sytwu, Huey-Kang; Lin, Shih-Hua


    A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc(T58M/T58M) mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4(D561A/+) mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with Ncc(T58M/T58M) mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.

  6. Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells. (United States)

    Yan, Yitang; Cummings, Connie A; Sutton, Deloris; Yu, Linda; Castro, Lysandra; Moore, Alicia B; Gao, Xiaohua; Dixon, Darlene


    Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells.

  7. Phosphorylation of acidic ribosomal proteins from rabbit reticulocytes by a ribosome-associated casein kinase

    DEFF Research Database (Denmark)

    Issinger, O G


    Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate polyacryl......Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate...

  8. KNL1 facilitates phosphorylation of outer kinetochore proteins by promoting Aurora B kinase activity. (United States)

    Caldas, Gina V; DeLuca, Keith F; DeLuca, Jennifer G


    Aurora B kinase phosphorylates kinetochore proteins during early mitosis, increasing kinetochore–microtubule (MT) turnover and preventing premature stabilization of kinetochore–MT attachments. Phosphorylation of kinetochore proteins during late mitosis is low, promoting attachment stabilization, which is required for anaphase onset. The kinetochore protein KNL1 recruits Aurora B–counteracting phosphatases and the Aurora B–targeting factor Bub1, yet the consequences of KNL1 depletion on Aurora B phospho-regulation remain unknown. Here, we demonstrate that the KNL1 N terminus is essential for Aurora B activity at kinetochores. This region of KNL1 is also required for Bub1 kinase activity at kinetochores, suggesting that KNL1 promotes Aurora B activity through Bub1-mediated Aurora B targeting. However, ectopic targeting of Aurora B to kinetochores does not fully rescue Aurora B activity in KNL1-depleted cells, suggesting KNL1 influences Aurora B activity through an additional pathway. Our findings establish KNL1 as a requirement for Aurora B activity at kinetochores and for wild-type kinetochore–MT attachment dynamics.

  9. A phosphorylation-deubiquitination cascade regulates the BRCA2-RAD51 axis in homologous recombination. (United States)

    Luo, Kuntian; Li, Lei; Li, Yunhui; Wu, Chenming; Yin, Yujiao; Chen, Yuping; Deng, Min; Nowsheen, Somaira; Yuan, Jian; Lou, Zhenkun


    Homologous recombination (HR) is one of the major DNA double-strand break (DSB) repair pathways in mammalian cells. Defects in HR trigger genomic instability and result in cancer predisposition. The defining step of HR is homologous strand exchange directed by the protein RAD51, which is recruited to DSBs by BRCA2. However, the regulation of the BRCA2-RAD51 axis remains unclear. Here we report that ubiquitination of RAD51 hinders RAD51-BRCA2 interaction, while deubiquitination of RAD51 facilitates RAD51-BRCA2 binding and RAD51 recruitment and thus is critical for proper HR. Mechanistically, in response to DNA damage, the deubiquitinase UCHL3 is phosphorylated and activated by ATM. UCHL3, in turn, deubiquitinates RAD51 and promotes the binding between RAD51 and BRCA2. Overexpression of UCHL3 renders breast cancer cells resistant to radiation and chemotherapy, while depletion of UCHL3 sensitizes cells to these treatments, suggesting a determinant role of UCHL3 in cancer therapy. Overall, we identify UCHL3 as a novel regulator of DNA repair and reveal a model in which a phosphorylation-deubiquitination cascade dynamically regulates the BRCA2-RAD51 pathway. © 2016 Luo et al.; Published by Cold Spring Harbor Laboratory Press.

  10. c-Jun N-terminal kinase phosphorylation of MARCKSL1 determines actin stability and migration in neurons and in cancer cells. (United States)

    Björkblom, Benny; Padzik, Artur; Mohammad, Hasan; Westerlund, Nina; Komulainen, Emilia; Hollos, Patrik; Parviainen, Lotta; Papageorgiou, Anastassios C; Iljin, Kristiina; Kallioniemi, Olli; Kallajoki, Markku; Courtney, Michael J; Mågård, Mats; James, Peter; Coffey, Eleanor T


    Cell migration is a fundamental biological function, critical during development and regeneration, whereas deregulated migration underlies neurological birth defects and cancer metastasis. MARCKS-like protein 1 (MARCKSL1) is widely expressed in nervous tissue, where, like Jun N-terminal protein kinase (JNK), it is required for neural tube formation, though the mechanism is unknown. Here we show that MARCKSL1 is directly phosphorylated by JNK on C-terminal residues (S120, T148, and T183). This phosphorylation enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers and dynamics, and retard migration in neurons. Conversely, when MARCKSL1 phosphorylation is inhibited, actin mobility increases and filopodium formation is compromised whereas lamellipodium formation is enhanced, as is cell migration. We find that MARCKSL1 mRNA is upregulated in a broad range of cancer types and that MARCKSL1 protein is strongly induced in primary prostate carcinomas. Gene knockdown in prostate cancer cells or in neurons reveals a critical role for MARCKSL1 in migration that is dependent on the phosphorylation state; phosphomimetic MARCKSL1 (MARCKSL1(S120D,T148D,T183D)) inhibits whereas dephospho-MARCKSL1(S120A,T148A,T183A) induces migration. In summary, these data show that JNK phosphorylation of MARCKSL1 regulates actin homeostasis, filopodium and lamellipodium formation, and neuronal migration under physiological conditions and that, when ectopically expressed in prostate cancer cells, MARCKSL1 again determines cell movement.

  11. Molecular mechanism of APC/C activation by mitotic phosphorylation. (United States)

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David


    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  12. HILI destabilizes microtubules by suppressing phosphorylation and Gigaxonin-mediated degradation of TBCB (United States)

    Tan, Hao; Liao, Hua; Zhao, Lianfang; Lu, Yilu; Jiang, Siyuan; Tao, Dachang; Liu, Yunqiang; Ma, Yongxin


    Human PIWIL2, aka HILI, is a member of PIWI protein family and overexpresses in various tumors. However, the underlying mechanisms of HILI in tumorigenesis remain largely unknown. TBCB has a critical role in regulating microtubule dynamics and is overexpressed in many cancers. Here we report that HILI inhibits Gigaxonin-mediated TBCB ubiquitination and degradation by interacting with TBCB, promoting the binding between HSP90 and TBCB, and suppressing the interaction between Gigaxonin and TBCB. Meanwhile, HILI can also reduce phosphorylation level of TBCB induced by PAK1. Our results showed that HILI suppresses microtubule polymerization and promotes cell proliferation, migration and invasion via TBCB for the first time, revealing a novel mechanism for HILI in tumorigenesis. PMID:28393858

  13. Phosphorylation of rat aquaporin-4 at Ser(111) is not required for channel gating

    DEFF Research Database (Denmark)

    Assentoft, Mette; Kaptan, Shreyas; Fenton, Robert A


    of a phosphorylation of AQP4.Ser(111) recorded no phosphorylation-induced change in water permeability. A phospho-specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser(111) , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation...... is therefore of therapeutic interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser(111) has been reported to increase the water permeability of AQP4 expressed in an astrocytic...... cell line. This possibility was, however, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser(111) was indeed a site involved in phosphorylation-mediated gating of AQP4. The water permeability of AQP4-expressing Xenopus oocytes was not altered by a range...

  14. The Self-catalytic Esterification Reaction of O-Phosphoryl Serine Derivative

    Institute of Scientific and Technical Information of China (English)

    Jin Tang DU; Yan Mei LI; Zhong Zhou CHEN; Shi Zhong LUO; Yu Fen ZHAO


    O-Phosphoryl serine derivative can perform self-catalytic esterification reaction in the mixture of CH3OH and CHCl3 at the room temperature. The phosphoryl group participation was the key step of the esterification. This type of reactions were proposed through an intermediate of mixed phosphoric-carboxylic anhydride that might provide a clue to the function of the phosphoryl group in the phosphorylated enzymes and in the prebiotic synthesis of protein.

  15. Phospho.ELM: A database of experimentally verified phosphorylation sites in eukaryotic proteins

    DEFF Research Database (Denmark)

    Diella, F.; Cameron, S.; Gemund, C.


    need for an accurate database dedicated to phosphorylation to provide easily retrievable information on phosphoproteins. Description: Phospho. ELM is a new resource containing experimentally verified phosphorylation sites manually curated from the literature and is developed...... to be phosphorylated by cellular kinases. Additional annotation includes literature references, subcellular compartment, tissue distribution, and information about the signaling pathways involved as well as links to the molecular interaction database MINT. Phospho. ELM version 2.0 contains 1703 phosphorylation site...

  16. Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan. (United States)

    Moraz, Marie-Laurence; Pythoud, Christelle; Turk, Rolf; Rothenberger, Sylvia; Pasquato, Antonella; Campbell, Kevin P; Kunz, Stefan


    The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.

  17. Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation. (United States)

    Brunelle, Joslyn K; Bell, Eric L; Quesada, Nancy M; Vercauteren, Kristel; Tiranti, Valeria; Zeviani, Massimo; Scarpulla, Richard C; Chandel, Navdeep S


    Mammalian cells detect decreases in oxygen concentrations to activate a variety of responses that help cells adapt to low oxygen levels (hypoxia). One such response is stabilization of the protein HIF-1 alpha, a component of the transcription factor HIF-1. Here we show that a small interfering RNA (siRNA) against the Rieske iron-sulfur protein of mitochondrial complex III prevents the hypoxic stabilization of HIF-1 alpha protein. Fibroblasts from a patient with Leigh's syndrome, which display residual levels of electron transport activity and are incompetent in oxidative phosphorylation, stabilize HIF-1 alpha during hypoxia. The expression of glutathione peroxidase or catalase, but not superoxide dismutase 1 or 2, prevents the hypoxic stabilization of HIF-1 alpha. These findings provide genetic evidence that oxygen sensing is dependent on mitochondrial-generated reactive oxygen species (ROS) but independent of oxidative phosphorylation.

  18. Random-coil chemical shifts of phosphorylated amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Bienkiewicz, Ewa A.; Lumb, Kevin J. [Colorado State University, Department of Biochemistry and Molecular Biology (United States)


    The {sup 1}H, {sup 13}C, {sup 15}N and {sup 31} P random-coil chemical shifts and phosphate pK{sub a} values of the phosphorylated amino acids pSer, pThr and pTyr in the protected peptide Ac-Gly-Gly-X-Gly-Gly-NH{sub 2} have been obtained in water at 25 deg. C over the pH range 2 to 9. Analysis of ROESY spectra indicates that the peptides are unstructured. Phosphorylation induces changes in random-coil chemical shifts, some of which are comparable to those caused by secondary structure formation, and are therefore significant in structural analyses based on the chemical shift.

  19. GABAB receptor phosphorylation regulates KCTD12-induced K+ current desensitization

    DEFF Research Database (Denmark)

    Adelfinger, L; Turecek, R; Ivankova, K


    released from the G-protein. Receptor-activated K+ currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K+ currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which...... reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G......-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast...

  20. ERK phosphorylation regulates sleep and plasticity in Drosophila.

    Directory of Open Access Journals (Sweden)

    William M Vanderheyden

    Full Text Available Given the relationship between sleep and plasticity, we examined the role of Extracellular signal-regulated kinase (ERK in regulating baseline sleep, and modulating the response to waking experience. Both sleep deprivation and social enrichment increase ERK phosphorylation in wild-type flies. The effects of both sleep deprivation and social enrichment on structural plasticity in the LNvs can be recapitulated by expressing an active version of ERK (UAS-ERK(SEM pan-neuronally in the adult fly using GeneSwitch (Gsw Gsw-elav-GAL4. Conversely, disrupting ERK reduces sleep and prevents both the behavioral and structural plasticity normally induced by social enrichment. Finally, using transgenic flies carrying a cAMP response Element (CRE-luciferase reporter we show that activating ERK enhances CRE-Luc activity while disrupting ERK reduces it. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep.

  1. Reactions of. cap alpha. -phosphorylated carbonyl compounds with amino alcohols

    Energy Technology Data Exchange (ETDEWEB)

    Moskva, V.V.; Sitdikova, T.Sh.; Zykova, T.V.; Alparova, M.V.; Shagvaleev, F.Sh.


    2-Aminoethanol reacts with carbonyl compounds with the formation, depending on the structure of the latter, either of a mixture of azomethines and oxazolidines, or of only azomethines. In the development of investigations on the reactivity of ..cap alpha..-phosphorylated carbonyl compounds the authors studied the reactions of a number of amino alcohols with phosphorylated acetaldehyde and acetone. In both cases they observed the formation of compounds of enamine structure, oxazolidines and azomethines were not observed. By means of NMR spectroscopy they established clearly the formation of the E-isomeric products. The /sup 1/H, /sup 31/P, and /sup 13/C NMR spectra were recorded on a WP-80 spectrometer. Chemical shifts of protons and /sup 13/C nuclei are given relative to TMS, and phosphorus nuclei relative to orthophosphoric acid.

  2. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase.



    The structure of isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC] from Escherichia coli has been solved and refined at 2.5 A resolution and is topologically different from that of any other dehydrogenase. This enzyme, a dimer of identical 416-residue subunits, is inactivated by phosphorylation at Ser-113, which lies at the edge of an interdomain pocket that also contains many residues conserved between isocitrate dehydrogenase and isopropylma...

  3. Snapshot of a phosphorylated substrate intermediate by kinetic crystallography


    Käck, Helena; Gibson, Katharine J.; Lindqvist, Ylva; Schneider, Gunter


    The ATP-dependent enzyme dethiobiotin synthetase from Escherichia coli catalyses the formation of dethiobiotin from CO2 and 7,8-diaminopelargonic acid. The reaction is initiated by the formation of a carbamate and proceeds through a phosphorylated intermediate, a mixed carbamic phosphoric anhydride. Here, we report the crystal structures at 1.9- and 1.6-Å resolution, respectively, of the enzyme–MgATP–diaminopelargonic acid and enzyme–MgADP–carbamic–phosphoric acid anhydride complexes, observe...

  4. Regulation of PCNA Function by Tyrosine Phosphorylation in Prostate Cancer (United States)


    prostate cancer. 15. SUBJECT TERMS PCNA, Tyrosine Phosphorylation, Prostate Cancer 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT...directed peptide is a rational strategy to target proliferation-competent PCNA, limitations associated with peptides as a therapeutic agent, particularly...radical prostatectomy: Where have we been? Where are we going? Urol Oncol 2007;25:11–8. 2. Moldovan GL, Pfander B, Jentsch S. PCNA, the maestro of the

  5. Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation. (United States)

    Spät, Philipp; Maček, Boris; Forchhammer, Karl


    Cyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria.

  6. A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells. (United States)

    Bakkenist, Christopher J; Czambel, R Kenneth; Hershberger, Pamela A; Tawbi, Hussein; Beumer, Jan H; Schmitz, John C


    Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy.

  7. Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins

    DEFF Research Database (Denmark)

    Rao, R Shyama Prasad; Møller, Ian Max


    maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins....

  8. Effect of some organic solvents on oxidative phosphorylation in rat liver mitochondria

    DEFF Research Database (Denmark)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré


    The effect of acetone, acetonitrile, dimethyl sulfoxide (DMSO), ethanol and methanol on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria has been studied. All the organic solvents inhibited the oxidative phosphorylation in a concentration dependent manner, but with differences...... on oxidative phosphorylation in mitochondria should therefore include the use of relevant concentrations of the organic solvent in order to validate the contribution....

  9. Absolute Phosphorylation Stoichiometry Analysis by Motif-Targeting Quantitative Mass Spectrometry. (United States)

    Tsai, Chia-Feng; Ku, Wei-Chi; Chen, Yu-Ju; Ishihama, Yasushi


    Direct measurement of site-specific phosphorylation stoichiometry can unambiguously distinguish whether the degree of phosphorylation is regulated by upstream kinase/phosphatase activity or by transcriptional regulation to alter protein expression level. Here, we describe a motif-targeting quantitative proteomic approach that integrates dephosphorylation, isotope tag labeling, and enzymatic kinase reaction for large-scale phosphorylation stoichiometry measurement of the human proteome.

  10. Detection of tyrosine phosphorylated proteins in trichinella spiralis L1 larvae

    Directory of Open Access Journals (Sweden)

    Allegretti S.


    Full Text Available Western-blotting analysis showed the presence of tyrosine phosphorylated proteins in crude extracts of T. spiralis larvae and these phosphorylated proteins were located by immunofluorescence on the striations of the larval cuticle. The patterns of phosphorylated proteins were modified when larvae were incubated with bile.

  11. NetPhosYeast: prediction of protein phosphorylation sites in yeast

    DEFF Research Database (Denmark)

    Ingrell, C.R.; Miller, Martin Lee; Jensen, O.N.


    We here present a neural network-based method for the prediction of protein phosphorylation sites in yeast-an important model organism for basic research. Existing protein phosphorylation site predictors are primarily based on mammalian data and show reduced sensitivity on yeast phosphorylation s...

  12. Spatial proximity statistics suggest a regulatory role of protein phosphorylation on compound binding. (United States)

    Korkuć, Paula; Walther, Dirk


    Phosphorylation is an important post-translational modification that regulates protein function by the attachment of negatively charged phosphate groups to phosphorylatable amino acid residues. As a mode of action, an influence of phosphorylation on the binding of compounds to proteins has been discussed and described for a number of proteins in the literature. However, a systematic statistical survey probing for enriched phosphorylation sites close to compound binding sites in support of this notion and with properly chosen random reference distributions has not been presented yet. Using high-resolution protein structures from the Protein Data Bank including their co-crystallized non-covalently bound compounds and experimentally determined phosphorylation sites, we analyzed the pairwise distance distributions of phosphorylation and compound binding sites on protein surfaces. We found that phosphorylation sites are indeed located at significantly closer distances to compounds than expected by chance holding true specifically also for the subset of compound binding sites serving as catalytic sites of metabolic reactions. This tendency was particularly evident when treating phosphorylation sites as collective sets supporting the relevance of phosphorylation hotspots. Interestingly, phosphorylation sites were found to be closer to negatively charged than to positively charged compounds suggesting a stronger modulation of the binding of negatively charged compounds in dependence on phosphorylation status than on positively charged compounds. The enrichment of phosphorylation sites near compound binding sites confirms a regulatory role of phosphorylation in compound binding and provides a solid statistical basis for the literature-reported selected events.

  13. Phosphorylation of the Na+,K+-ATPase and the H+,K+-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Morth, Jens Preben; Jensen, Jan Egebjerg;


    Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na(+),K(+)- and H(+),K(+)-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two...... as supported by electrophysiological results presented here. We further review the other proposed pump phosphorylations....

  14. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains (United States)

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald


    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  15. Coilin phosphorylation mediates interaction with SMN and SmB'. (United States)

    Toyota, Cory G; Davis, Misty D; Cosman, Angela M; Hebert, Michael D


    Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB' binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB' than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.

  16. Coilin phosphorylation mediates interaction with SMN and SmB′ (United States)

    Toyota, Cory G.; Davis, Misty D.; Cosman, Angela M.; Hebert, Michael D.


    Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB′ binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB′ than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB. PMID:19997741

  17. Glucose phosphorylation is required for Mycobacterium tuberculosis persistence in mice.

    Directory of Open Access Journals (Sweden)

    Joeli Marrero


    Full Text Available Mycobacterium tuberculosis (Mtb is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. (13C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt Mtb. When both glucokinase genes, ppgK and glkA, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose in vivo and relies on glucose phosphorylation to survive during chronic mouse infections.

  18. TTBK2: A Tau Protein Kinase beyond Tau Phosphorylation

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    Jung-Chi Liao


    Full Text Available Tau tubulin kinase 2 (TTBK2 is a kinase known to phosphorylate tau and tubulin. It has recently drawn much attention due to its involvement in multiple important cellular processes. Here, we review the current understanding of TTBK2, including its sequence, structure, binding sites, phosphorylation substrates, and cellular processes involved. TTBK2 possesses a casein kinase 1 (CK1 kinase domain followed by a ~900 amino acid segment, potentially responsible for its localization and substrate recruitment. It is known to bind to CEP164, a centriolar protein, and EB1, a microtubule plus-end tracking protein. In addition to autophosphorylation, known phosphorylation substrates of TTBK2 include tau, tubulin, CEP164, CEP97, and TDP-43, a neurodegeneration-associated protein. Mutations of TTBK2 are associated with spinocerebellar ataxia type 11. In addition, TTBK2 is essential for regulating the growth of axonemal microtubules in ciliogenesis. It also plays roles in resistance of cancer target therapies and in regulating glucose and GABA transport. Reported sites of TTBK2 localization include the centriole/basal body, the midbody, and possibly the mitotic spindles. Together, TTBK2 is a multifunctional kinase involved in important cellular processes and demands augmented efforts in investigating its functions.

  19. Raf activation is regulated by tyrosine 510 phosphorylation in Drosophila.

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    Fan Xia


    Full Text Available The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf, which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved.

  20. Rat1p maintains RNA polymerase II CTD phosphorylation balance (United States)

    Jimeno-González, Silvia; Schmid, Manfred; Malagon, Francisco; Haaning, Line Lindegaard; Jensen, Torben Heick


    In S. cerevisiae, the 5′-3′ exonuclease Rat1p partakes in transcription termination. Although Rat1p-mediated RNA degradation has been suggested to play a role for this activity, the exact mechanisms by which Rat1p helps release RNA polymerase II (RNAPII) from the DNA template are poorly understood. Here we describe a function of Rat1p in regulating phosphorylation levels of the C-terminal domain (CTD) of the largest RNAPII subunit, Rpb1p, during transcription elongation. The rat1-1 mutant exhibits highly elevated levels of CTD phosphorylation as well as RNAPII distribution and transcription termination defects. These phenotypes are all rescued by overexpression of the CTD phosphatase Fcp1p, suggesting a functional relationship between the absence of Rat1p activity, elevated CTD phosphorylation, and transcription defects. We also demonstrate that rat1-1 cells display increased RNAPII transcription kinetics, a feature that may contribute to the cellular phenotypes of the mutant. Consistently, the rat1-1 allele is synthetic lethal with the rpb1-E1103G mutation, causing increased RNAPII speed, and is suppressed by the rpb2-10 mutation, causing slowed transcription. Thus, Rat1p plays more complex roles in controlling transcription than previously thought. PMID:24501251

  1. Catalytic constants enable the emergence of bistability in dual phosphorylation. (United States)

    Conradi, Carsten; Mincheva, Maya


    Dual phosphorylation of proteins is a principal component of intracellular signalling. Bistability is considered an important property of such systems and its origin is not yet completely understood. Theoretical studies have established parameter values for multistationarity and bistability for many types of proteins. However, up to now no formal criterion linking multistationarity and bistability to the parameter values characterizing dual phosphorylation has been established. Deciding whether an unclassified protein has the capacity for bistability, therefore requires careful numerical studies. Here, we present two general algebraic conditions in the form of inequalities. The first employs the catalytic constants, and if satisfied guarantees multistationarity (and hence the potential for bistability). The second involves the catalytic and Michaelis constants, and if satisfied guarantees uniqueness of steady states (and hence absence of bistability). Our method also allows for the direct computation of the total concentration values such that multistationarity occurs. Applying our results yields insights into the emergence of bistability in the ERK-MEK-MKP system that previously required a delicate numerical effort. Our algebraic conditions present a practical way to determine the capacity for bistability and hence will be a useful tool for examining the origin of bistability in many models containing dual phosphorylation.

  2. Phosphorylation regulates proteolytic efficiency of TEV protease detected by a 5(6)-carboxyfluorescein-pyrene based fluorescent sensor. (United States)

    He, Yao-Hui; Li, Yan-Mei; Chen, Yong-Xiang


    TEV protease is of great importance for in vitro and in vivo site-specific cleavage of proteins. The proteolytic efficiency of TEV protease is often regulated by mutation of the substrate, which is irreversible and hard to be modulated. Herein, a facile and reversible method, based on phosphorylation in the substrate, is developed to regulate the cleavage capability of TEV protease. Phosphorylation at P3 tyrosine hinders the recognition of TEV protease to the substrate by using a robust fluorescent protease sensor. Moreover, the phosphate group can be easily removed by alkaline phosphatases for recovering the proteolytic efficiency of TEV protease. Additionally, 5(6)-carboxyfluorescein and pyrene have been used as high-efficiency mutual fluorophore-quencher pair in the peptide-based protease sensor for the first time, which provides a chance to simultaneously monitor the cleavage process in two respective fluorescence channels. Further studies indicated both dynamic and static components contributing to the mutual quenching system. The phosphorylation-regulated TEV protease proteolysis system can be used in conditional cleavage of protein or peptide tag.

  3. Mass Spectrometric-Based Selected Reaction Monitoring of Protein Phosphorylation during Symbiotic Signaling in the Model Legume, Medicago truncatula.

    Directory of Open Access Journals (Sweden)

    Lori K Van Ness

    Full Text Available Unlike the major cereal crops corn, rice, and wheat, leguminous plants such as soybean and alfalfa can meet their nitrogen requirement via endosymbiotic associations with soil bacteria. The establishment of this symbiosis is a complex process playing out over several weeks and is facilitated by the exchange of chemical signals between these partners from different kingdoms. Several plant components that are involved in this signaling pathway have been identified, but there is still a great deal of uncertainty regarding the early events in symbiotic signaling, i.e., within the first minutes and hours after the rhizobial signals (Nod factors are perceived at the plant plasma membrane. The presence of several protein kinases in this pathway suggests a mechanism of signal transduction via posttranslational modification of proteins in which phosphate is added to the hydroxyl groups of serine, threonine and tyrosine amino acid side chains. To monitor the phosphorylation dynamics and complement our previous untargeted 'discovery' approach, we report here the results of experiments using a targeted mass spectrometric technique, Selected Reaction Monitoring (SRM that enables the quantification of phosphorylation targets with great sensitivity and precision. Using this approach, we confirm a rapid change in the level of phosphorylation in 4 phosphosites of at least 4 plant phosphoproteins that have not been previously characterized. This detailed analysis reveals aspects of the symbiotic signaling mechanism in legumes that, in the long term, will inform efforts to engineer this nitrogen-fixing symbiosis in important non-legume crops such as rice, wheat and corn.

  4. Mass Spectrometric-Based Selected Reaction Monitoring of Protein Phosphorylation during Symbiotic Signaling in the Model Legume, Medicago truncatula. (United States)

    Van Ness, Lori K; Jayaraman, Dhileepkumar; Maeda, Junko; Barrett-Wilt, Gregory A; Sussman, Michael R; Ané, Jean-Michel


    Unlike the major cereal crops corn, rice, and wheat, leguminous plants such as soybean and alfalfa can meet their nitrogen requirement via endosymbiotic associations with soil bacteria. The establishment of this symbiosis is a complex process playing out over several weeks and is facilitated by the exchange of chemical signals between these partners from different kingdoms. Several plant components that are involved in this signaling pathway have been identified, but there is still a great deal of uncertainty regarding the early events in symbiotic signaling, i.e., within the first minutes and hours after the rhizobial signals (Nod factors) are perceived at the plant plasma membrane. The presence of several protein kinases in this pathway suggests a mechanism of signal transduction via posttranslational modification of proteins in which phosphate is added to the hydroxyl groups of serine, threonine and tyrosine amino acid side chains. To monitor the phosphorylation dynamics and complement our previous untargeted 'discovery' approach, we report here the results of experiments using a targeted mass spectrometric technique, Selected Reaction Monitoring (SRM) that enables the quantification of phosphorylation targets with great sensitivity and precision. Using this approach, we confirm a rapid change in the level of phosphorylation in 4 phosphosites of at least 4 plant phosphoproteins that have not been previously characterized. This detailed analysis reveals aspects of the symbiotic signaling mechanism in legumes that, in the long term, will inform efforts to engineer this nitrogen-fixing symbiosis in important non-legume crops such as rice, wheat and corn.

  5. Roles of LIM kinases in central nervous system function and dysfunction. (United States)

    Cuberos, H; Vallée, B; Vourc'h, P; Tastet, J; Andres, C R; Bénédetti, H


    LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) regulate actin dynamics by phosphorylating cofilin. In this review, we outline studies that have shown an involvement of LIMKs in neuronal function and we detail some of the pathways and molecular mechanisms involving LIMKs in neurodevelopment and synaptic plasticity. We also review the involvement of LIMKs in neuronal diseases and emphasize the differences in the regulation of LIMKs expression and mode of action. We finally present the existence of a cofilin-independent pathway also involved in neuronal function. A better understanding of the differences between both LIMKs and of the precise molecular mechanisms involved in their mode of action and regulation is now required to improve our understanding of the physiopathology of the neuronal diseases associated with LIMKs.

  6. Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization. (United States)

    Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan


    Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

  7. Mcm2 phosphorylation and the response to replicative stress

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    Stead Brent E


    Full Text Available Abstract Background The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm proteins 2 through 7 (Mcm2-7 and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK. In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress. Results We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2AA is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU and to the base analogue 5-fluorouracil (5-FU but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2AA and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2AA strain compared to wild type, whereas with a phosphomimetic allele (mcm2EE the mutation rate was decreased. These results led to the idea that the mcm2AA strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2AA. Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2AA including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate. Conclusions Together these observations point to a role for DDK-mediated phosphorylation

  8. The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation. (United States)

    Beavis, A D; Lehninger, A L


    Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.

  9. PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan; Zheng, Wei; Wu, Haochen [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Lu, Yan, E-mail: [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Shen, Pingping, E-mail: [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Model Animal Research Center (MARC), Nanjing University, Nanjing (China)


    Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPARγ1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPARγ1{sup S84A}, PPARγ1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPARγ1 transcriptional activity. • Phosphorylated PPARγ1 promotes HT1080 cells proliferation. • PPARγ1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPARγ1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPARγ1 phosphorylation as a critical event in HT1080

  10. Btk29A-mediated tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in Drosophila oogenesis. (United States)

    Hamada-Kawaguchi, Noriko; Nishida, Yasuyoshi; Yamamoto, Daisuke


    Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150) and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

  11. Btk29A-mediated tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in Drosophila oogenesis.

    Directory of Open Access Journals (Sweden)

    Noriko Hamada-Kawaguchi

    Full Text Available Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150 and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

  12. Phosphoproteome analysis of E-coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

    DEFF Research Database (Denmark)

    Macek, B.; Gnad, F.; Soufi, Boumediene


    we use a recently developed proteomics approach based on phosphopeptide enrichment and high accuracy MS to analyze the phosphoproteome of the model Gram-negative bacterium Escherichia coli. We report 81 phosphorylation sites on 79 E. coli proteins, with distribution of Ser/Thr/Tyr phosphorylation...... sites 68%/23%/9%. Despite their phylogenetic distance, phosphoproteomes of E. coli and B. subtilis show striking similarity in size, classes of phosphorylated proteins, and distribution of Ser/Thr/Tyr phosphorylation sites. By combining the two datasets, we created the largest phosphorylation site...

  13. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

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    Lei eShi


    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  14. dbPPT: a comprehensive database of protein phosphorylation in plants. (United States)

    Cheng, Han; Deng, Wankun; Wang, Yongbo; Ren, Jian; Liu, Zexian; Xue, Yu


    As one of the most important protein post-translational modifications, the reversible phosphorylation is critical for plants in regulating a variety of biological processes such as cellular metabolism, signal transduction and responses to environmental stress. Numerous efforts especially large-scale phosphoproteome profiling studies have been contributed to dissect the phosphorylation signaling in various plants, while a large number of phosphorylation events were identified. To provide an integrated data resource for further investigations, here we present a comprehensive database of dbPPT (database of Phosphorylation site in PlanTs, at, which contains experimentally identified phosphorylation sites in proteins from plants. The phosphorylation sites in dbPPT were manually curated from the literatures, whereas datasets in other public databases were also integrated. In total, there were 82,175 phosphorylation sites in 31,012 proteins from 20 plant organisms in dbPPT, presenting a larger quantity of phosphorylation sites and a higher coverage of plant species in comparison with other databases. The proportions of residue types including serine, threonine and tyrosine were 77.99, 17.81 and 4.20%, respectively. All the phosphoproteins and phosphorylation sites in the database were critically annotated. Since the phosphorylation signaling in plants attracted great attention recently, such a comprehensive resource of plant protein phosphorylation can be useful for the research community. Database URL: http://dbppt.biocuckoo.or

  15. The Impact of Phosphorylation on Electron Capture Dissociation of Proteins: A Top-Down Perspective (United States)

    Chen, Bifan; Guo, Xiao; Tucholski, Trisha; Lin, Ziqing; McIlwain, Sean; Ge, Ying


    Electron capture dissociation (ECD) is well suited for the characterization of phosphoproteins, with which labile phosphate groups are generally preserved during the fragmentation process. However, the impact of phosphorylation on ECD fragmentation of intact proteins remains unclear. Here, we have performed a systematic investigation of the phosphorylation effect on ECD of intact proteins by comparing the ECD cleavages of mono-phosphorylated α-casein, multi-phosphorylated β-casein, and immunoaffinity-purified phosphorylated cardiac troponin I with those of their unphosphorylated counterparts, respectively. In contrast to phosphopeptides, phosphorylation has significantly reduced deleterious effects on the fragmentation of intact proteins during ECD. On a global scale, the fragmentation patterns are highly comparable between unphosphorylated and phosphorylated precursors under the same ECD conditions, despite a slight decrease in the number of fragment ions observed for the phosphorylated forms. On a local scale, single phosphorylation of intact proteins imposes minimal effects on fragmentation near the phosphorylation sites, but multiple phosphorylations in close proximity result in a significant reduction of ECD bond cleavages. [Figure not available: see fulltext.

  16. The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system. (United States)

    Robinson, Kelly A; Ou, Wei-Lin; Guan, Xinyu; Sugamori, Kim S; Bandyopadhyay, Abhishek; Ernst, Oliver P; Mitchell, Jane


    Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation.

  17. Association between intrinsic disorder and serine/threonine phosphorylation in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Gajinder Pal Singh


    Full Text Available Serine/threonine phosphorylation is an important mechanism that is involved in the regulation of protein function. In eukaryotes, phosphorylation occurs predominantly in intrinsically disordered regions of proteins. Though serine/threonine phosphorylation and protein disorder are much less prevalent in prokaryotes, some bacteria have high levels of serine/threonine phosphorylation and disorder, including the medically important M. tuberculosis. Here I show that serine/threonine phosphorylation sites in M. tuberculosis are highly enriched in intrinsically disordered regions, indicating similarity in the substrate recognition mechanisms of eukaryotic and M. tuberculosis kinases. Serine/threonine phosphorylation has been linked to the pathogenicity and survival of M. tuberculosis. Thus, a better understanding of how its kinases recognize their substrates could have important implications in understanding and controlling the biology of this deadly pathogen. These results also indicate that the association between serine/threonine phosphorylation and disorder is not a feature restricted to eukaryotes.

  18. Effect of phosphorylation on antioxidant activities of pumpkin (Cucurbita pepo, Lady godiva) polysaccharide. (United States)

    Song, Yi; Ni, Yuanying; Hu, Xiaosong; Li, Quanhong


    Phosphorylated derivatives of pumpkin polysaccharide with different degree of substitution were synthesized using POCl3 and pyridine. Antioxidant activities and cytoprotective effects of unmodified polysaccharide and phosphorylated derivatives were investigated employing various in vitro systems. Results showed that high ratio of POCl3/pyridine could increase the degree of substitution and no remarkable degradation occurred in the phosphorylation process. Characteristic absorption of phosphorylation appeared both in the IR and (31)P NMR spectrum. The df values between 2.27 and 2.55 indicated the relatively expanded conformation of the phosphorylated derivatives. All the phosphorylated polysaccharides exhibited higher antioxidant activities. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by the derivatives. In general, phosphorylation could improve the antioxidant activities of pumpkin polysaccharide both in vitro and in a cell system.

  19. STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions, and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes1 (United States)

    Bergner, Sonja Verena; Scholz, Martin; Trompelt, Kerstin; Barth, Johannes; Gäbelein, Philipp; Steinbeck, Janina; Xue, Huidan; Clowez, Sophie; Fucile, Geoffrey; Goldschmidt-Clermont, Michel; Fufezan, Christian; Hippler, Michael


    In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-LIGHT HARVESTING COMPLEX I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions. PMID:25858915

  20. Injectable hydrogels derived from phosphorylated alginic acid calcium complexes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Han-Sem; Song, Minsoo, E-mail:; Lee, Eun-Jung; Shin, Ueon Sang, E-mail:


    Phosphorylation of sodium alginate salt (NaAlg) was carried out using H{sub 3}PO{sub 4}/P{sub 2}O{sub 5}/Et{sub 3}PO{sub 4} followed by acid–base reaction with Ca(OAc){sub 2} to give phosphorylated alginic acid calcium complexes (CaPAlg), as a water dispersible alginic acid derivative. The modified alginate derivatives including phosphorylated alginic acid (PAlg) and CaPAlg were characterized by nuclear magnetic resonance spectroscopy for {sup 1}H, and {sup 31}P nuclei, high resolution inductively coupled plasma optical emission spectroscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. CaPAlg hydrogels were prepared simply by mixing CaPAlg solution (2 w/v%) with NaAlg solution (2 w/v%) in various ratios (2:8, 4:6, 6:4, 8:2) of volume. No additional calcium salts such as CaSO{sub 4} or CaCl{sub 2} were added externally. The gelation was completed within about 3–40 min indicating a high potential of hydrogel delivery by injection in vivo. Their mechanical properties were tested to be ≤ 6.7 kPa for compressive strength at break and about 8.4 kPa/mm for elastic modulus. SEM analysis of the CaPAlg hydrogels showed highly porous morphology with interconnected pores of width in the range of 100–800 μm. Cell culture results showed that the injectable hydrogels exhibited comparable properties to the pure alginate hydrogel in terms of cytotoxicity and 3D encapsulation of cells for a short time period. The developed injectable hydrogels showed suitable physicochemical and mechanical properties for injection in vivo, and could therefore be beneficial for the field of soft tissue engineering. - Highlights: • Preparation of water-soluble alginic acid complexes with calcium phosphate • Self-assembly of the phosphorylated alginic acid calcium complexes with sodium alginate • Preparation of injectable hydrogels with diverse gelation times within about 3–40 min.

  1. Glycogen Repletion in Brown Adipose Tissue upon Refeeding Is Primarily Driven by Phosphorylation-Independent Mechanisms.

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    Christopher M Carmean

    Full Text Available Glycogen storage in brown adipose tissue (BAT is generally thought to take place through passive, substrate-driven activation of glycogenesis rather than programmatic shifts favoring or opposing the storage and/or retention of glycogen. This perception exists despite a growing body of evidence suggesting that BAT glycogen storage is actively regulated by covalent modification of key glycogen-metabolic enzymes, protein turnover, and endocrine hormone signaling. Members of one such class of covalent-modification regulators, glycogen-binding Phosphoprotein Phosphatase-1 (PP1-regulatory subunits (PPP1Rs, targeting PP1 to glycogen-metabolic enzymes, were dynamically regulated in response to 24 hr of starvation and/or 24 hr of starvation followed by ad libitum refeeding. Over-expression of the PPP1R Protein Targeting to Glycogen (PTG, under the control of the aP2 promoter in mice, inactivated glycogen phosphorylase (GP and enhanced basal- and starvation-state glycogen storage. Total interscapular BAT glycogen synthase and the constitutive activity of GS were conditionally affected. During starvation, glucose-6-phosphate (G-6-P levels and the relative phosphorylation of Akt (p-Ser-473-Akt were both increased in PTG-overexpressing (Tg mice, suggesting that elevated glycogen storage during starvation modifies broader cellular metabolic pathways. During refeeding, Tg and WT mice reaccumulated glycogen similarly despite altered GS and GP activities. All observations during refeeding suggest that the phosphorylation states of GS and GP are not physiologically rate-controlling, despite there being a clear balance of endogenous kinase- and phosphatase activities. The studies presented here reveal IBAT glycogen storage to be a tightly-regulated process at all levels, with potential effects on nutrient sensing in vivo.

  2. Phosphorylation of the Polarity Protein BASL Differentiates Asymmetric Cell Fate through MAPKs and SPCH. (United States)

    Zhang, Ying; Guo, Xiaoyu; Dong, Juan


    Cell polarization is commonly used for the regulation of stem cell asymmetric division in both animals and plants. Stomatal development in Arabidopsis, a process that produces breathing pores in the epidermis, requires asymmetric cell division to differentiate highly specialized guard cells while maintaining a stem cell population [1, 2]. The BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) protein exhibits a polarized localization pattern in the cell and is required for differential cell fates resulting from asymmetric cell division [3]. The polarization of BASL is made possible by a positive feedback loop with a canonical mitogen-activated protein kinase (MAPK) pathway that recruits the MAPKK kinase YODA (YDA) and MAPK 6 (MPK6) to the cortical polarity site [4]. Here, we study BASL intracellular dynamics and show that the membrane-associated BASL is slowly replenished at the cortical polarity site and that the mobility is tightly linked to its phosphorylation status. Because BASL polarity is only exhibited by one daughter cell after an asymmetric cell division, we study how BASL differentially functions in the two daughter cells. The YDA MAPK cascade transduces upstream ligand-receptor signaling [5-13] to the transcription factor SPEECHLESS (SPCH), which controls stomatal initiation and is directly suppressed by MPK3/6-mediated phosphorylation [14, 15]. We show that BASL polarization leads to elevated nuclear MPK6 signaling and lowered SPCH abundance in one of the two daughter cells. Therefore, two daughter cells are differentiated by BASL polarity-mediated differential suppression of SPCH, which may provide developmental plasticity in plant stem cell asymmetric cell division (ACD).

  3. A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica (United States)

    Mansuri, M. Shahid; Bhattacharya, Sudha; Bhattacharya, Alok


    Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics. PMID:25299184

  4. The cellular distribution and Ser262 phosphorylation of tau protein are regulated by BDNF in vitro.

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    Qian Chen

    Full Text Available The brain-enriched microtubule-associated protein tau, a critical regulator of cytoskeletal dynamics, forms insoluble aggregates in a number of neurodegenerative diseases termed tauopathies, including Alzheimer's disease (AD. Hyperphosphorylation of tau protein is an important mechanism for aggregation, so many studies on the pathogenesis of AD and other tauopathies have focused on regulation of tau phosphorylation by kinases and phosphatases. Less studied are mechanisms of tau transcriptional and post-transcriptional regulation by extracellular signals such as BDNF and how such changes alter neuronal function. Previously, we reported that tau is required for morphological plasticity induced by BDNF. Here, we further explore tau modification during BDNF-induced changes in neuronal cell morphology. In undifferentiated SH-SY5Y cells lacking neurites, tau formed a sphere within the soma as revealed by immunocytochemistry. In contrast, tau was enriched in the neurites and sparse in the soma of SH-SY5Y cells induced to differentiate by retinoic acid (RA. Treatment with RA also increased total tau protein levels but decreased expression of tau phosphorylated at Ser262 as determined by Western blot. Both effects were further enhanced by subsequent BDNF treatment. Upregulation of tau protein and downregulation of p-Ser262 tau were correlated with total neurite length (R = .94 and R = -.98, respectively. When primary E18 hippocampal neurons were treated with nocodazole, a blocker of microtubule polymerization, nascent neurites were lost and tau shifted to the soma. This process of retrograde tau movement away from neurites was reversed by BDNF. These results indicate that tau is redistributed to neurites and dephosphorylated during RA- and BDNF-mediated differentiation.

  5. Myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping protein CapZ. (United States)

    Lin, Ying-Hsi; Warren, Chad M; Li, Jieli; McKinsey, Timothy A; Russell, Brenda


    The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCɛ (dnPKCɛ) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy. Copyright © 2016. Published by Elsevier Inc.

  6. Mitochondrial ATP synthases cluster as discrete domains that reorganize with the cellular demand for oxidative phosphorylation. (United States)

    Jimenez, Laure; Laporte, Damien; Duvezin-Caubet, Stephane; Courtout, Fabien; Sagot, Isabelle


    Mitochondria are double membrane-bounded organelles that form a dynamic tubular network. Mitochondria energetic functions depend on a complex internal architecture. Cristae, inner membrane invaginations that fold into the matrix space, are proposed to be the site of oxidative phosphorylation, reactions by which ATP synthase produces ATP. ATP synthase is also thought to have a role in crista morphogenesis. To date, the exploration of the processes regulating mitochondrial internal compartmentalization have been mostly limited to electron microscopy. Here, we describe ATP synthase localization in living yeast cells and show that it clusters as discrete inner membrane domains. These domains are dynamic within the mitochondrial network. They are impaired in mutants defective in crista morphology and partially overlap with the crista-associated MICOS-MINOS-MITOS complex. Finally, ATP synthase occupancy increases with the cellular demand for OXPHOS. Overall our data suggest that domains in which ATP synthases are clustered correspond to mitochondrial cristae. Being able to follow mitochondrial sub-compartments in living yeast cells opens new avenues to explore the mechanisms involved in inner membrane remodeling, an architectural feature crucial for mitochondrial activities.

  7. Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation. (United States)

    Llorente-Garcia, Isabel; Lenn, Tchern; Erhardt, Heiko; Harriman, Oliver L; Liu, Lu-Ning; Robson, Alex; Chiu, Sheng-Wen; Matthews, Sarah; Willis, Nicky J; Bray, Christopher D; Lee, Sang-Hyuk; Shin, Jae Yen; Bustamante, Carlos; Liphardt, Jan; Friedrich, Thorsten; Mullineaux, Conrad W; Leake, Mark C


    Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100-200nm membrane domains containing tens to hundreds of complexes are indicated. Central to our results is that domains of different functional OXPHOS complexes do not co-localize, but ubiquinone diffusion in the membrane is rapid and long-range, consistent with a mobile carrier shuttling electrons between islands of different complexes. Our results categorically demonstrate that electron transport and proton circuitry in this model bacterium are spatially delocalized over the cell membrane, in stark contrast to mitochondrial bioenergetic supercomplexes. Different organisms use radically different strategies for OXPHOS membrane organization, likely depending on the stability of their environment. Copyright © 2014. Published by Elsevier B.V.

  8. Cdk1 phosphorylation of the kinetochore protein Nsk1 prevents error-prone chromosome segregation. (United States)

    Chen, Jun-Song; Lu, Lucy X; Ohi, Melanie D; Creamer, Kevin M; English, Chauca; Partridge, Janet F; Ohi, Ryoma; Gould, Kathleen L


    Cdk1 controls many aspects of mitotic chromosome behavior and spindle microtubule (MT) dynamics to ensure accurate chromosome segregation. In this paper, we characterize a new kinetochore substrate of fission yeast Cdk1, Nsk1, which promotes proper kinetochore-MT (k-MT) interactions and chromosome movements in a phosphoregulated manner. Cdk1 phosphorylation of Nsk1 antagonizes Nsk1 kinetochore and spindle localization during early mitosis. A nonphosphorylatable Nsk1 mutant binds prematurely to kinetochores and spindle, cementing improper k-MT attachments and leading to high rates of lagging chromosomes that missegregate. Accordingly, cells lacking nsk1 exhibit synthetic growth defects with mutations that disturb MT dynamics and/or kinetochore structure, and lack of proper phosphoregulation leads to even more severe defects. Intriguingly, Nsk1 is stabilized by binding directly to the dynein light chain Dlc1 independently of the dynein motor, and Nsk1-Dlc1 forms chainlike structures in vitro. Our findings establish new roles for Cdk1 and the Nsk1-Dlc1 complex in regulating the k-MT interface and chromosome segregation.

  9. Posthoc phosphorylation of proteins derived from ischemic rat hippocampus, striatum and neocortex. (United States)

    Kirschenbaum, B; Pulsinelli, W A


    Disruption of the brain's protein phosphorylation system by ischemia may cause irreversible metabolic and structural alterations leading eventually to cell death. To examine the effect of ischemia on the phosphorylation state of brain proteins, tissue homogenates derived from the hippocampus, striatum and neocortex of normal rats and rats subjected to severe forebrain ischemia were phosphorylated with [gamma-32P]ATP. The phosphorylated proteins were separated by two-dimensional polyacrylamide gel electrophoresis and changes were assessed by autoradiography. Cerebral ischemia caused marked alterations of the phosphorylation state of many brain proteins; phosphorylation of some proteins was increased while phosphorylation of others was decreased. Despite differences in the sensitivity of the hippocampus, striatum and neocortex to ischemic injury the direction and approximate magnitude of protein phosphorylation changes caused by ischemia were similar in all three regions. Since the pattern of protein phosphorylation in the ischemia-vulnerable hippocampus was identical to that in the ischemia-resistant paramedian neocortex we conclude that abnormalities of protein phosphorylation may be necessary for ischemic injury to neurons but none are sufficient to explain the selective vulnerability of certain brain regions to ischemic damage.

  10. Ovarian hormones and prolactin increase renal NaCl cotransporter phosphorylation. (United States)

    Rojas-Vega, Lorena; Reyes-Castro, Luis A; Ramírez, Victoria; Bautista-Pérez, Rocío; Rafael, Chloe; Castañeda-Bueno, María; Meade, Patricia; de Los Heros, Paola; Arroyo-Garza, Isidora; Bernard, Valérie; Binart, Nadine; Bobadilla, Norma A; Hadchouel, Juliette; Zambrano, Elena; Gamba, Gerardo


    Unique situations in female physiology require volume retention. Accordingly, a dimorphic regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) has been reported, with a higher activity in females than in males. However, little is known about the hormones and mechanisms involved. Here, we present evidence that estrogens, progesterone, and prolactin stimulate NCC expression and phosphorylation. The sex difference in NCC abundance, however, is species dependent. In rats, NCC phosphorylation is higher in females than in males, while in mice both NCC expression and phosphorylation is higher in females, and this is associated with increased expression and phosphorylation of full-length STE-20 proline-alanine-rich kinase (SPAK). Higher expression/phosphorylation of NCC was corroborated in humans by urinary exosome analysis. Ovariectomy in rats resulted in decreased expression and phosphorylation of the cotransporter and promoted the shift of SPAK isoforms toward the short inhibitory variant SPAK2. Conversely, estradiol or progesterone administration to ovariectomized rats restored NCC phosphorylation levels and shifted SPAK expression and phosphorylation towards the full-length isoform. Estradiol administration to male rats induced a significant increase in NCC phosphorylation. NCC is also modulated by prolactin. Administration of this peptide hormone to male rats induced increased phosphorylation of NCC, an effect that was observed even using the ex vivo kidney perfusion strategy. Our results indicate that estradiol, progesterone, and prolactin, the hormones that are involved in sexual cycle, pregnancy and lactation, upregulate the activity of NCC.

  11. Casein kinase II phosphorylates lens connexin 45.6 and is involved in its degradation. (United States)

    Yin, X; Jedrzejewski, P T; Jiang, J X


    Connexin (Cx) 45.6, an avian counterpart of rodent Cx50, is phosphorylated in vivo, but the sites and function of the phosphorylation have not been elucidated. Our peptide mapping experiments showed that the Ser(363) site in the carboxyl (COOH) terminus of Cx45.6 was phosphorylated and that this site is within casein kinase (CK) II consensus sequence, although showing some similarity to CKI sequence. The peptide containing Ser(363) could be phosphorylated in vitro by CKII, but not by CKI. Furthermore, CKII phosphorylated Cx45.6 in embryonic lens membrane and the fusion protein containing the COOH terminus of Cx45.6. Two-dimensional peptide mapping experiments showed that one of the Cx45.6 peptides phosphorylated in vivo migrated to the same spot as one of those phosphorylated by CKII in vitro. Furthermore, CKII activity could be detected in lens lysates. To assess the function of this phosphorylation event, exogenous wild type and mutant Cx45.6 (Ser(363) --> Ala) were expressed in lens primary cultures by retroviral infection. The mutant Cx45.6 was shown to be more stable having a longer half-life compared with wild type Cx45.6. Together, the evidence suggests that CKII is likely a kinase responsible for the Ser(363) phosphorylation, leading to the destablization and degradation of Cx45.6. The connexin degradation induced by phosphorylation has a broad functional significance in the regulation of gap junctions in vivo.

  12. Vasopressin regulation of multisite phosphorylation of UT-A1 in the inner medullary collecting duct. (United States)

    Hoban, Carol A; Black, Lauren N; Ordas, Ronald J; Gumina, Diane L; Pulous, Fadi E; Sim, Jae H; Sands, Jeff M; Blount, Mitsi A


    Vasopressin signaling is critical for the regulation of urea transport in the inner medullary collecting duct (IMCD). Increased urea permeability is driven by a vasopressin-mediated elevation of cAMP that results in the direct phosphorylation of urea transporter (UT)-A1. The identification of cAMP-sensitive phosphorylation sites, Ser(486) and Ser(499), in the rat UT-A1 sequence was the first step in understanding the mechanism of vasopressin action on the phosphorylation-dependent modulation of urea transport. To investigate the significance of multisite phosphorylation of UT-A1 in response to elevated cAMP, we used highly specific and sensitive phosphosite antibodies to Ser(486) and Ser(499) to determine cAMP action at each phosphorylation site. We found that phosphorylation at both sites was rapid and sustained. Furthermore, the rate of phosphorylation of the two sites was similar in both mIMCD3 cells and rat inner medullary tissue. UT-A1 localized to the apical membrane in response to vasopressin was phosphorylated at Ser(486) and Ser(499). We confirmed that elevated cAMP resulted in increased phosphorylation of both sites by PKA but not through the vasopressin-sensitive exchange protein activated by cAMP pathway. These results elucidate the multisite phosphorylation of UT-A1 in response to cAMP, thus providing the beginning of understanding the intracellular factors underlying vasopressin stimulation of urea transport in the IMCD.

  13. Insulin Induces Phosphorylation of Serine Residues of Translationally Controlled Tumor Protein in 293T Cells

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    Jeehye Maeng


    Full Text Available Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation.

  14. Auto-phosphorylation Represses Protein Kinase R Activity (United States)

    Wang, Die; de Weerd, Nicole A.; Willard, Belinda; Polekhina, Galina; Williams, Bryan R. G.; Sadler, Anthony J.


    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity. PMID:28281686

  15. Adriamycin induces H2AX phosphorylation in human spermatozoa

    Institute of Scientific and Technical Information of China (English)

    Zhong-Xiang Li; Ting-Ting Wang; Yan-Ting Wu; Chen-Ming Xu; Min-Yue Dong; Jian-Zhong Sheng; He-Feng Huang


    Aim: To investigate whether adriamycin induces DNA damage and the formation of γH2AX (the phosphorylated form of histone H2AX) foci in mature spermatozoa. Methods: Human spermatozoa were treated with adriamycin at different concentrations. γH2AX was analyzed by immunofluorescent staining and flow cytometry and double- strand breaks (DSB) were detected by the comet assay. Results: The neutral comet assay revealed that the treatment with adriamycin at 2 μg/mL for different times (0.5, 2, 8 and 24 h), or for 8 h at different concentrations (0.4, 2 and 10 μg/mL), induced significant DSB in spermatozoa. Immunofluorent staining and flow cytometry showed that the expression of γH2AX was increased in a dose-dependent and time-dependant manner after the treatment of adriamycin. Adriamycin also induced the concurrent appearance of DNA maintenance/repair proteins RAD50 and 53BP1 with γH2AX in spermatozoa. Wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, abolished the co-appearance of these two proteins with γH2AX. Conclusion: Human mature spermatozoa have the same response to DSB-induced H2AX phosphorylation and subsequent recruitment of DNA maintenance/repair proteins as somatic cells.

  16. Integrating phosphorylation network with transcriptional network reveals novel functional relationships.

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    Lin Wang

    Full Text Available Phosphorylation and transcriptional regulation events are critical for cells to transmit and respond to signals. In spite of its importance, systems-level strategies that couple these two networks have yet to be presented. Here we introduce a novel approach that integrates the physical and functional aspects of phosphorylation network together with the transcription network in S.cerevisiae, and demonstrate that different network motifs are involved in these networks, which should be considered in interpreting and integrating large scale datasets. Based on this understanding, we introduce a HeRS score (hetero-regulatory similarity score to systematically characterize the functional relevance of kinase/phosphatase involvement with transcription factor, and present an algorithm that predicts hetero-regulatory modules. When extended to signaling network, this approach confirmed the structure and cross talk of MAPK pathways, inferred a novel functional transcription factor Sok2 in high osmolarity glycerol pathway, and explained the mechanism of reduced mating efficiency upon Fus3 deletion. This strategy is applicable to other organisms as large-scale datasets become available, providing a means to identify the functional relationships between kinases/phosphatases and transcription factors.

  17. Gas-Phase Acidities of Phosphorylated Amino Acids. (United States)

    Stover, Michele L; Plummer, Chelsea E; Miller, Sean R; Cassady, Carolyn J; Dixon, David A


    Gas-phase acidities and heats of formation have been predicted at the G3(MP2)/SCRF-COSMO level of theory for 10 phosphorylated amino acids and their corresponding amides, including phospho-serine (pSer), -threonine (pThr), and -tyrosine (pTyr), providing the first reliable set of these values. The gas-phase acidities (GAs) of the three named phosphorylated amino acids and their amides have been determined using proton transfer reactions in a Fourier transform ion cyclotron mass spectrometer. Excellent agreement was found between the experimental and predicted GAs. The phosphate group is the deprotonation site for pSer and pThr and deprotonation from the carboxylic acid generated the lowest energy anion for pTyr. The infrared spectra were calculated for six low energy anions of pSer, pThr, and pTyr. For deprotonated pSer and pThr, good agreement is found between the experimental IRMPD spectra and the calculated spectra for our lowest energy anion structure. For pTyr, the IR spectra for a higher energy phosphate deprotonated structure is in good agreement with experiment. Additional experiments tested electrospray ionization (ESI) conditions for pTyr and determined that variations in solvent, temperature, and voltage can result in a different experimental GA value, indicating that ESI conditions affect the conformation of the pTyr anion.

  18. Occludin S471 Phosphorylation Contributes to Epithelial Monolayer Maturation. (United States)

    Bolinger, Mark T; Ramshekar, Aniket; Waldschmidt, Helen V; Larsen, Scott D; Bewley, Maria C; Flanagan, John M; Antonetti, David A


    Multiple organ systems require epithelial barriers for normal function, and barrier loss is a hallmark of diseases ranging from inflammation to epithelial cancers. However, the molecular processes regulating epithelial barrier maturation are not fully elucidated. After contact, epithelial cells undergo size-reductive proliferation and differentiate, creating a dense, highly ordered monolayer with high resistance barriers. We provide evidence that the tight junction protein occludin contributes to the regulation of epithelial cell maturation upon phosphorylation of S471 in its coiled-coil domain. Overexpression of a phosphoinhibitory occludin S471A mutant prevents size-reductive proliferation and subsequent tight junction maturation in a dominant manner. Inhibition of cell proliferation in cell-contacted but immature monolayers recapitulated this phenotype. A kinase screen identified G-protein-coupled receptor kinases (GRKs) targeting S471, and GRK inhibitors delayed epithelial packing and junction maturation. We conclude that occludin contributes to the regulation of size-reductive proliferation and epithelial cell maturation in a phosphorylation-dependent manner. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. (Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation (United States)

    Fiesel, Fabienne C; Ando, Maya; Hudec, Roman; Hill, Anneliese R; Castanedes-Casey, Monica; Caulfield, Thomas R; Moussaud-Lamodière, Elisabeth L; Stankowski, Jeannette N; Bauer, Peter O; Lorenzo-Betancor, Oswaldo; Ferrer, Isidre; Arbelo, José M; Siuda, Joanna; Chen, Li; Dawson, Valina L; Dawson, Ted M; Wszolek, Zbigniew K; Ross, Owen A; Dickson, Dennis W; Springer, Wolfdieter


    Mutations in PINK1 and PARKIN cause recessive, early-onset Parkinson’s disease (PD). Together, these two proteins orchestrate a protective mitophagic response that ensures the safe disposal of damaged mitochondria. The kinase PINK1 phosphorylates ubiquitin (Ub) at the conserved residue S65, in addition to modifying the E3 ubiquitin ligase Parkin. The structural and functional consequences of Ub phosphorylation (pS65-Ub) have already been suggested from in vitro experiments, but its (patho-)physiological significance remains unknown. We have generated novel antibodies and assessed pS65-Ub signals in vitro and in cells, including primary neurons, under endogenous conditions. pS65-Ub is dependent on PINK1 kinase activity as confirmed in patient fibroblasts and postmortem brain samples harboring pathogenic mutations. We show that pS65-Ub is reversible and barely detectable under basal conditions, but rapidly induced upon mitochondrial stress in cells and amplified in the presence of functional Parkin. pS65-Ub accumulates in human brain during aging and disease in the form of cytoplasmic granules that partially overlap with mitochondrial, lysosomal, and total Ub markers. Additional studies are now warranted to further elucidate pS65-Ub functions and fully explore its potential for biomarker or therapeutic development. PMID:26162776

  20. Functional Analysis of PKC Phosphorylation Sites on Myelin Protein Zero

    Institute of Scientific and Technical Information of China (English)

    GangXu; MichaelShy; JohnKamhoz; JanneBalsamo


    Objective To analyze the function of Protein kinase C(PKC) phosphorylation sites on mylelin protein zero (P0) at adhesion and myelination.Methods Mutations of p0 cyto-plasmic domain motif (RSTK) and adjacent sequence which are targeted by PKC were studied.Results The point mutations in this region or an adjacent serine residue could abolish P0 adhe-sion function. PKCα,along with the PKC binding protein RACK1,were associated with wild type P0.Inhibition of PKC activity abolished the P0 mediated adhesion.Point mutation in the RSTKtarget site that abolished adhesion did not alter the association of PKC with P0,but deletion of a 14 amino acid region,which included the PSTK motif,could abolish the association.Conclusion PKC mediated phosphorylation of specific residues within the cytoplasmic domain of P0 is neces-sary for P0 mediated adhesion.The alteration of this phoporylation can cause demyelinating neu-ropathy in human.

  1. Auto-phosphorylation Represses Protein Kinase R Activity. (United States)

    Wang, Die; de Weerd, Nicole A; Willard, Belinda; Polekhina, Galina; Williams, Bryan R G; Sadler, Anthony J


    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.

  2. Immunohistochemistry of colorectal cancer biomarker phosphorylation requires controlled tissue fixation.

    Directory of Open Access Journals (Sweden)

    Abbey P Theiss

    Full Text Available Phosphorylated signaling molecules are biomarkers of cancer pathophysiology and resistance to therapy, but because phosphoprotein analytes are often labile, poorly controlled clinical laboratory practices could prevent translation of research findings in this area from the bench to the bedside. We therefore compared multiple biomarker and phosphoprotein immunohistochemistry (IHC results in 23 clinical colorectal carcinoma samples after either a novel, rapid tissue fixation protocol or a standard tissue fixation protocol employed by clinical laboratories, and we also investigated the effect of a defined post-operative "cold" ischemia period on these IHC results. We found that a one-hour cold ischemia interval, allowed by ASCO/CAP guidelines for certain cancer biomarker assays, is highly deleterious to certain phosphoprotein analytes, specifically the phosphorylated epidermal growth factor receptor (pEGFR, but shorter ischemic intervals (less than 17 minutes facilitate preservation of phosphoproteins. Second, we found that a rapid 4-hour, two temperature, formalin fixation yielded superior staining in several cases with select markers (pEGFR, pBAD, pAKT compared to a standard overnight room temperature fixation protocol, despite taking less time. These findings indicate that the future research and clinical utilities of phosphoprotein IHC for assessing colorectal carcinoma pathophysiology absolutely depend upon attention to preanalytical factors and rigorously controlled tissue fixation protocols.

  3. Phosphorylation of psyllium seed polysaccharide and its characterization. (United States)

    Rao, Monica R P; Warrier, Deepa U; Gaikwad, Snehal R; Shevate, Prachi M


    Psyllium is widely used as a medicinally active natural polysaccharide for treating conditions like constipation, diarrhea, and irritable bowel syndrome, inflammatory bowel disease, ulcerative colitis and colon cancer. Studies have been performed to characterize and modify the polysaccharide obtained from psyllium seed husk and to evaluate its use as a pharmaceutical excipient, but no studies have been performed to evaluate the properties of the polysaccharide present in psyllium seeds. The present study focuses on phosphorylation of psyllium seed polysaccharide (PPS) using sodium tri-meta phosphate as the cross-linking agent. The modified phosphorylated psyllium seed polysaccharide was then evaluated for physicochemical properties, rheological properties, spectral analysis, thermal analysis, crosslinking density and acute oral toxicity studies. The modified polysaccharide (PhPPS) has a high swelling index due to which it can be categorized as a hydrogel. The percent increase in swelling of PhPPS as compared to PPS was found to be 90.26%. The PPS & PhPPS mucilages of all strengths were found to have shear thinning properties. These findings are suggestive of the potential use of PhPPS as gelling & suspending agent. PhPPS was found to have a mucoadhesive property which was comparable with carbopol.

  4. Akt Phosphorylation and PI (3, 4, 5) P3 Binding Coordinately Inhibit the Tumor Suppressive Activity of Merlin (United States)


    Johnson KC, Eckman MS, & Jacks T (2002) Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin localization. J...J. L., Johnson, K. C., Eckman , M. S. & Jacks, T. Merlin phosphorylation by p21-activated kinase 2 and effects of phosphorylation on merlin

  5. Ligand-induced Ordering of the C-terminal Tail Primes STING for Phosphorylation by TBK1

    Directory of Open Access Journals (Sweden)

    Yuko Tsuchiya


    Full Text Available The innate immune protein Stimulator of interferon genes (STING promotes the induction of interferon beta (IFN-β production via the phosphorylation of its C-terminal tail (CTT by TANK-binding kinase 1 (TBK1. Potent ligands of STING are, therefore, promising candidates for novel anti-cancer drugs or vaccine adjuvants. However, the intrinsically flexible CTT poses serious problems in in silico drug discovery. Here, we performed molecular dynamics simulations of the STING fragment containing the CTT in ligand-bound and unbound forms and observed that the binding of a potent ligand cyclic GMP-AMP (cGAMP induced a local structure in the CTT, reminiscent of the known structure of a TBK1 substrate. The subsequent molecular biological experiments confirmed the observed dynamics of the CTT and identified essential residues for the activation of the IFN-β promoter, leading us to propose a new mechanism of STING activation.

  6. Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond. (United States)

    Zhou, Guo-Lei; Zhang, Haitao; Field, Jeffrey


    Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.

  7. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking. (United States)

    Pal Sharma, C; Goldmann, Wolfgang H


    Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

  8. Optogenetics to target actin-mediated synaptic loss in Alzheimer's (United States)

    Zahedi, Atena; DeFea, Kathryn; Ethell, Iryna


    Numerous studies in Alzheimer's Disease (AD) animal models show that overproduction of Aβ peptides and their oligomerization can distort dendrites, damage synapses, and decrease the number of dendritic spines and synapses. Aβ may trigger synapse loss by modulating activity of actin-regulating proteins, such as Rac1 and cofilin. Indeed, Aβ1-42 oligomers can activate actin severing protein cofilin through calcineurin-mediated activation of phosphatase slingshot and inhibit an opposing pathway that suppresses cofilin phosphorylation through Rac-mediated activation of LIMK1. Excessive activation of actin-severing protein cofilin triggers the formation of a non-dynamic actin bundles, called rods that are found in AD brains and cause loss of synapses. Hence, regulation of these actin-regulating proteins in dendritic spines could potentially provide useful tools for preventing the synapse/spine loss associated with earlier stages of AD neuropathology. However, lack of spatiotemporal control over their activity is a key limitation. Recently, optogenetic advancements have provided researchers with convenient light-activating proteins such as photoactivatable Rac (PARac). Here, we transfected cultured primary hippocampal neurons and human embryonic kidney (HEK) cells with a PARac/ mCherry-containing plasmid and the mCherry-positive cells were identified and imaged using an inverted fluorescence microscope. Rac1 activation was achieved by irradiation with blue light (480nm) and live changes in dendritic spine morphology were observed using mCherry (587nm). Rac activation was confirmed by immunostaining for phosphorylated form of effector proteinP21 protein-activated kinase 1 (PAK1) and reorganization of actin. Thus, our studies confirm the feasibility of using the PA-Rac construct to trigger actin re-organization in the dendritic spines.

  9. Identification and quantification of the phosphorylated ovalbumin by high resolution mass spectrometry under dry-heating treatment. (United States)

    Wang, Hui; Tu, Zong-Cai; Liu, Guang-Xian; Zhang, Lu; Chen, Yuan


    The specific phosphorylation sites and degree of phosphorylation (DP) at each site are directly related to protein's structure and functional properties. Thus, characterizing the introduced phosphate groups is of great importance. This study was to monitor the phosphorylation sites, DP and the number of phosphorylation sites in P-Oval achieved by dry heating in the presence of pyrophosphate for 1, 2 and 5days by using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Two phosphorylation sites were found in natural ovalbumin, but the number of phosphorylation sites increased to 8, 8 and 10 after dry-heating phosphorylation for 1, 2 and 5days, respectively. In addition, dual-phosphorylated peptides were detected for samples without extensive heating. The phosphorylation sites were found to be mainly on Ser residues, which could be the preferred phosphorylation site for dry heating in the presence of pyrophosphate.

  10. Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries. (United States)

    Miller, Chad J; Turk, Benjamin E


    Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors.

  11. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.


    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  12. High-accuracy identification and bioinformatic analysis of in vivo protein phosphorylation sites in yeast

    DEFF Research Database (Denmark)

    Gnad, Florian; de Godoy, Lyris M F; Cox, Jürgen


    mapped to 1118 proteins, representatively covering the yeast kinome and a multitude of transcription factors. We show that a single false discovery rate for all peptide identifications significantly overestimates occurrence of rare modifications, such as tyrosine phosphorylation in yeast. The identified...... phosphorylation sites are predominantly located on irregularly structured and accessible protein regions. We found high evolutionary conservation of phosphorylated proteins and a large overlap of significantly over-represented motifs with the human phosphoproteome. Nevertheless, phosphorylation events at the site...... level were not highly conserved between yeast and higher eukaryotes, which points to metazoan-specific kinase and substrate families. We constructed a yeast-specific phosphorylation sites predictor on the basis of a support vector machine, which - together with the yeast phosphorylation data...

  13. Changes in phosphorylation of myofibrillar proteins during postmortem development of porcine muscle

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin Røssel; Lametsch, Rene


    phosphorylated protein bands with the highest scores. The results indicate that the phosphorylation pattern of myofibrillar proteins in PM muscle is mainly changed with PM time, but only to a minor extent influenced by the rate of pH decline, suggesting that the phosphorylation of myofibrillar proteins may......A gel-based phosphoproteomic study was performed to investigate the postmortem (PM) changes in protein phosphorylation of the myofibrillar proteins in three groups of pigs with different pH decline rates, from PM 1 h to 24 h. The global phosphorylation level in the group with a fast pH decline rate...... was higher than that in the slow and intermediate groups at early PM time, but became the lowest at 24 h. The protein phosphorylation level of seven individual protein bands was only significantly (ptime, and two protein bands were subjected to a synergy effect between PM time and p...

  14. Phosphorylation of FADD at serine 194 by CKIalpha regulates its nonapoptotic activities. (United States)

    Alappat, Elizabeth C; Feig, Christine; Boyerinas, Benjamin; Volkland, Jörg; Samuels, Martin; Murmann, Andrea E; Thorburn, Andrew; Kidd, Vincent J; Slaughter, Clive A; Osborn, Stephanie L; Winoto, Astar; Tang, Wei-Jen; Peter, Marcus E


    FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis.

  15. Histone H1 phosphorylation occurs site-specifically during interphase and mitosis: identification of a novel phosphorylation site on histone H1. (United States)

    Sarg, Bettina; Helliger, Wilfried; Talasz, Heribert; Förg, Barbara; Lindner, Herbert H


    H1 histones, isolated from logarithmically growing and mitotically enriched human lymphoblastic T-cells (CCRF-CEM), were fractionated by reversed phase and hydrophilic interaction liquid chromatography, subjected to enzymatic digestion, and analyzed by amino acid sequencing and mass spectrometry. During interphase the four H1 subtypes present in these cells differ in their maximum phosphorylation levels: histone H1.5 is tri-, H1.4 di-, and H1.3 and H1.2, only monophosphorylated. The phosphorylation is site-specific and occurs exclusively on serine residues of SP(K/A)K motifs. The phosphorylation sites of histone H1.5 from mitotically enriched cells were also examined. In contrast to the situation in interphase, at mitosis there were additional phosphorylations, exclusively at threonine residues. Whereas the tetraphosphorylated H1.5 arises from the triphosphosphorylated form by phosphorylation of one of two TPKK motifs in the C-terminal domain, namely Thr137 and Thr154, the pentaphosphorylated H1.5 was the result of phosphorylation of one of the tetraphosphorylated forms at a novel nonconsensus motif at Thr10 in the N-terminal tail. Despite the fact that histone H1.5 has five (S/T)P(K/A)K motifs, all of these motifs were never found to be phosphorylated simultaneously. Our data suggest that phosphorylation of human H1 variants occurs nonrandomly during both interphase and mitosis and that distinct serine- or threonine-specific kinases are involved in different cell cycle phases. The order of increased phosphorylation and the position of modification might be necessary for regulated chromatin decondensation, thus facilitating processes of replication and transcription as well as of mitotic chromosome condensation.

  16. Phosphorylation-dependent and Phosphorylation-independent Regulation of Helicobacter pylori Acid Acclimation by the ArsRS Two-component System. (United States)

    Marcus, Elizabeth A; Sachs, George; Wen, Yi; Scott, David R


    The pH-sensitive Helicobacter pylori ArsRS two-component system (TCS) aids survival of this neutralophile in the gastric environment by directly sensing and responding to environmental acidity. ArsS is required for acid-induced trafficking of urease and its accessory proteins to the inner membrane, allowing rapid, urea-dependent cytoplasmic and periplasmic buffering. Expression of ArsR, but not its phosphorylation, is essential for bacterial viability. The aim of this study was to characterize the roles of ArsS and ArsR in the response of H. pylori to acid. Wild-type H. pylori and an arsR(D52N) phosphorylation-deficient strain were incubated at acidic or neutral pH. Gene and protein expression, survival, membrane trafficking of urease proteins, urease activity, and internal pH were studied. Phosphorylation of ArsR is not required for acid survival. ArsS-driven trafficking of urease proteins to the membrane in acid, required for recovery of internal pH, is independent of ArsR phosphorylation. ArsR phosphorylation increases expression of the urease gene cluster, and the loss of negative feedback in a phosphorylation-deficient mutant leads to an increase in total urease activity. ArsRS has a dual function in acid acclimation: regulation of urease trafficking to UreI at the cytoplasmic membrane, driven by ArsS, and regulation of urease gene cluster expression, driven by phosphorylation of ArsR. ArsS and ArsR work through phosphorylation-dependent and phosphorylation-independent regulatory mechanisms to impact acid acclimation and allow gastric colonization. Furthering understanding of the intricacies of acid acclimation will impact the future development of targeted, nonantibiotic treatment regimens. © 2015 John Wiley & Sons Ltd.

  17. Tau phosphorylation in human, primate, and rat brain: evidence that a pool of tau is highly phosphorylated in vivo and is rapidly dephosphorylated in vitro. (United States)

    Garver, T D; Harris, K A; Lehman, R A; Lee, V M; Trojanowski, J Q; Billingsley, M L


    The extent of tau phosphorylation is thought to regulate the binding of tau to microtubules: Highly phosphorylated tau does not bind to tubules, whereas dephosphorylated tau can bind to microtubules. It is interesting that the extent of tau phosphorylation in vivo has not been accurately determined. Tau was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that tau isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope-specific monoclonal and polyclonal antibodies. This highly phosphorylated tau was dephosphorylated during a 120-min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow-mobility forms of tau were shifted to faster-mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of tau in slow-mobility, highly phosphorylated forms. Approximately 25% of immunoreactive tau was present as slow-mobility (66- and 68-kDa) forms of tau. The percentage of immunoreactive tau in faster-mobility pools (42-54 kDa) increased in proportion to the decrease in content of 66-68-kDa tau as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on tau is rapid and depends on neuronal phosphatases. Furthermore, tau is highly phosphorylated in normal-appearing human, primate, and rodent brain.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. (United States)

    Hasegawa, Masato; Arai, Tetsuaki; Nonaka, Takashi; Kametani, Fuyuki; Yoshida, Mari; Hashizume, Yoshio; Beach, Thomas G; Buratti, Emanuele; Baralle, Francisco; Morita, Mitsuya; Nakano, Imaharu; Oda, Tatsuro; Tsuchiya, Kuniaki; Akiyama, Haruhiko


    TAR DNA-binding protein of 43kDa (TDP-43) is deposited as cytoplasmic and intranuclear inclusions in brains of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Previous studies reported that abnormal phosphorylation takes place in deposited TDP-43. The aim of this study was to identify the phosphorylation sites and responsible kinases, and to clarify the pathological significance of phosphorylation of TDP-43. We generated multiple antibodies specific to phosphorylated TDP-43 by immunizing phosphopeptides of TDP-43, and analyzed FTLD-U and ALS brains by immunohistochemistry, immunoelectron microscopy, and immunoblots. In addition, we performed investigations aimed at identifying the responsible kinases, and we assessed the effects of phosphorylation on TDP-43 oligomerization and fibrillization. We identified multiple phosphorylation sites in carboxyl-terminal regions of deposited TDP-43. Phosphorylation-specific antibodies stained more inclusions than antibodies to ubiquitin and, unlike existing commercially available anti-TDP-43 antibodies, did not stain normal nuclei. Ultrastructurally, these antibodies labeled abnormal fibers of 15nm diameter and on immunoblots recognized hyperphosphorylated TDP-43 at 45kDa, with additional 18 to 26kDa fragments in sarkosyl-insoluble fractions from FTLD-U and ALS brains. The phosphorylated epitopes were generated by casein kinase-1 and -2, and phosphorylation led to increased oligomerization and fibrillization of TDP-43. These results suggest that phosphorylated TDP-43 is a major component of the inclusions, and that abnormal phosphorylation of TDP-43 is a critical step in the pathogenesis of FTLD-U and ALS. Phosphorylation-specific antibodies will be powerful tools for the investigation of these disorders.

  19. In vitro phosphorylation as tool for modification of silk and keratin fibrous materials


    Volkov, Vadim; Cavaco-Paulo, Artur


    An overview is given of the recent work on in vitro enzymatic phosphorylation of silk fibroin and human hair keratin. Opposing to many chemical "conventional" approaches, enzymatic phosphorylation is in fact a mild reaction and the treatment falls within "green chemistry" approach. Silk and keratin are not phosphorylated in vivo, but in vitro. This enzyme-driven modification is a major technological breakthrough. Harsh chemical chemicals are avoided, and mild conditions make enzymatic phospho...

  20. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Ikuma; Ibuki, Yuko, E-mail:


    Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an