Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

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Lu Frances Fangjia

2012-11-01

Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

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Dalton, George D; Dewey, William L

2006-02-01

Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous

G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

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Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

2014-08-08

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Telocinobufagin inhibits the epithelial-mesenchymal transition of breast cancer cells through the phosphoinositide 3-kinase/protein kinase B/extracellular signal-regulated kinase/Snail signaling pathway.

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Gao, Yuxue; Shi, Lihong; Cao, Zhen; Zhu, Xuetao; Li, Feng; Wang, Ruyan; Xu, Jinyuan; Zhong, Jinyi; Zhang, Baogang; Lu, Shijun

2018-05-01

Telocinobufagin (TBG), an active ingredient of Venenumbufonis , exhibits an immunomodulatory activity. However, its antimetastatic activity in breast cancer remains unknown. The present study investigated whether TBG prevents breast cancer metastasis and evaluated its regulatory mechanism. TBG inhibited the migration and invasion of 4T1 breast cancer cells. Furthermore, TBG triggered the collapse of F-actin filaments in breast cancer. The epithelial-mesenchymal transition (EMT) markers, vimentin and fibronectin, were downregulated following TBG treatment. However, E-cadherin was upregulated following TBG treatment. Snail, a crucial transcriptional factor of EMT, was downregulated following TBG treatment. Signaling pathway markers, including phosphorylated protein kinase B (P-Akt), p-mechanistic target of rapamycin (mTOR) and p-extracellular signal-regulated kinase (ERK), were decreased following TBG treatment. The same results were obtained from in vivo experiments. In conclusion, in vitro and in vivo experiments reveal that TBG inhibited migration, invasion and EMT via the phosphoinositide 3-kinase (PI3K)/Akt/ERK/Snail signaling pathway in breast cancer.

Inhibition of Vascular Smooth Muscle Growth via Signaling Crosstalk between AMP-Activated Protein Kinase and cAMP-Dependent Protein Kinase

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Joshua Daniel Stone

2012-10-01

Full Text Available Abnormal vascular smooth muscle (VSM growth is central in the pathophysiology of vascular disease yet fully effective therapies to curb this growth are lacking. Recent findings from our lab and others support growth control of VSM by adenosine monophosphate (AMP-based approaches including the metabolic sensor AMP-activated protein kinase (AMPK and cAMP-dependent protein kinase (PKA. Molecular crosstalk between AMPK and PKA has been previously suggested, yet the extent to which this occurs and its biological significance in VSM remains unclear. Considering their common AMP backbone and similar signaling characteristics, we hypothesized that crosstalk exists between AMPK and PKA in the regulation of VSM growth. Using rat primary VSM cells, the AMPK agonist AICAR increased AMPK activity and phosphorylation of the catalytic Thr172 site on AMPK. Interestingly, AICAR also phosphorylated a suspected PKA-inhibitory Ser485 site on AMPK, and these cumulative events were reversed by the PKA inhibitor PKI suggesting possible PKA-mediated regulation of AMPK. AICAR also increased PKA activity in a reversible fashion. The cAMP stimulator forskolin increased PKA activity and completely ameliorated Ser/Thr protein phosphatase-2C activity, suggesting a potential mechanism of AMPK modulation by PKA since inhibition of PKA by PKI reduced AMPK activity. Functionally, AMPK inhibited serum-stimulated cell cycle progression and cellular proliferation; however, PKA failed to do so. Moreover, AMPK and PKA reduced PDGF-β-stimulated VSM cell migration. Collectively, these results show that AMPK is capable of reducing VSM growth in both anti-proliferative and anti-migratory fashions. Furthermore, these data suggest that AMPK may be modulated by PKA and that positive feedback may exist between these two systems. These findings reveal a discrete nexus between AMPK and PKA in VSM and provide basis for metabolically-directed targets in reducing pathologic VSM growth.

Pharmacological Inhibition of Protein Kinase G1 Enhances Bone Formation by Human Skeletal Stem Cells Through Activation of RhoA-Akt Signaling

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Kermani, Abbas Jafari; Siersbaek, Majken S; Chen, Li

2015-01-01

for several malignant and nonmalignant conditions. We screened a library of kinase inhibitors to identify small molecules that enhance bone formation by human skeletal (stromal or mesenchymal) stem cells (hMSC). We identified H-8 (known to inhibit protein kinases A, C, and G) as a potent enhancer of ex vivo......Development of novel approaches to enhance bone regeneration is needed for efficient treatment of bone defects. Protein kinases play a key role in regulation of intracellular signal transduction pathways, and pharmacological targeting of protein kinases has led to development of novel treatments...

Calcium-dependent but calmodulin-independent protein kinase from soybean

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Harmon, A.C.; Putnam-Evans, C.; Cormier, M.J.

1987-01-01

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≥ 2 micromolar). The protein kinase activity was stimulated 100-fold by ≥ 10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤ 2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound 45 Ca 2+ in the presence of KCl and MgCl 2 , which indicated that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity

The cytotoxic effects of regorafenib in combination with protein kinase D inhibition in human colorectal cancer cells

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Wei, Ning; Chu, Edward; Wu, Shao-yu; Wipf, Peter; Schmitz, John C.

2015-01-01

Metastatic colorectal cancer (mCRC) remains a major public health problem, and diagnosis of metastatic disease is usually associated with poor prognosis. The multi-kinase inhibitor regorafenib was approved in 2013 in the U.S. for the treatment of mCRC patients who progressed after standard therapies. However, the clinical efficacy of regorafenib is quite limited. One potential strategy to improve mCRC therapy is to combine agents that target key cellular signaling pathways, which may lead to synergistic enhancement of antitumor efficacy and overcome cellular drug resistance. Protein kinase D (PKD), a family of serine/threonine kinases, mediates key signaling pathways implicated in multiple cellular processes. Herein, we evaluated the combination of regorafenib with a PKD inhibitor in several human CRC cells. Using the Chou-Talalay model, the combination index values for this combination treatment demonstrated synergistic effects on inhibition of cell proliferation and clonal formation. This drug combination resulted in induction of apoptosis as determined by flow cytometry, increased PARP cleavage, and decreased activation of the anti-apoptotic protein HSP27. This combination also yielded enhanced inhibition of ERK, AKT, and NF-κB signaling. Taken together, PKD inhibition in combination with regorafenib appears to be a promising strategy for the treatment of mCRC. PMID:25544765

The Haemophilus ducreyi LspA1 protein inhibits phagocytosis by using a new mechanism involving activation of C-terminal Src kinase.

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Dodd, Dana A; Worth, Randall G; Rosen, Michael K; Grinstein, Sergio; van Oers, Nicolai S C; Hansen, Eric J

2014-05-20

Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways. Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for

Roles of Apicomplexan protein kinases at each life cycle stage.

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Kato, Kentaro; Sugi, Tatsuki; Iwanaga, Tatsuya

2012-06-01

Inhibitors of cellular protein kinases have been reported to inhibit the development of Apicomplexan parasites, suggesting that the functions of protozoan protein kinases are critical for their life cycle. However, the specific roles of these protein kinases cannot be determined using only these inhibitors without molecular analysis, including gene disruption. In this report, we describe the functions of Apicomplexan protein kinases in each parasite life stage and the potential of pre-existing protein kinase inhibitors as Apicomplexan drugs against, mainly, Plasmodium and Toxoplasma. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase.

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Deprez, J; Bertrand, L; Alessi, D R; Krause, U; Hue, L; Rider, M H

2000-01-01

A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Czeta (PKCzeta). Comparison of the inhibition of WISK, PKCalpha and PKCzeta by different protein kinase inhibitors suggested that WISK was not a member of the PKC...

Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

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Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas

2009-01-01

of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites......, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found...... that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do...

Inhibition of Cartilage Acidic Protein 1 Reduces Ultraviolet B Irradiation Induced-Apoptosis through P38 Mitogen-Activated Protein Kinase and Jun Amino-Terminal Kinase Pathways

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Yinghong Ji

2016-11-01

Full Text Available Background/Aims: Ultraviolet B (UVB irradiation can easily induce apoptosis in human lens epithelial cells (HLECs and further lead to various eye diseases including cataract. Here for the first time, we investigated the role of cartilage acidic protein 1 (CRTAC1 gene in UVB irradiation induced-apoptosis in HLECs. Methods: Three groups of HLECs were employed including model group, empty vector group, and CRTAC1 interference group. Results: After UVB irradiation, the percentage of primary apoptotic cells was obviously fewer in CRTAC1 interference group. Meanwhile, inhibition of CRTAC1 also reduced both reactive oxygen species (ROS production and intracellular Ca2+ concentration, but the level of mitochondrial membrane potential (Δψm was increased in HLECs. Further studies indicated that superoxide dismutase (SOD activity and total antioxidative (T-AOC level were significantly increased in CRTAC1-inhibited cells, while the levels of malondialdehyde (MDA and lactate dehydrogenase (LDH were significantly decreased. ELISA analysis of CRTAC1-inhibited cells showed that the concentrations of tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6 were significantly decreased, but the concentration of interleukin-10 (IL-10 was significantly increased. Western blot analyses of eight apoptosis-associated proteins including Bax, Bcl-2, p38, phospho-p38 (p-p38, Jun amino-terminal kinases (JNK1/2, phospho-JNK1/2 (p-JNK1/2, calcium-sensing receptor (CasR, and Ca2+/calmodulin-dependent protein kinase II (CaMKII indicated that the inhibition of CRTAC1 alleviated oxidative stress and inflammation response, inactivated calcium-signaling pathway, p38 and JNK1/2 signal pathways, and eventually reduced UVB irradiation induced-apoptosis in HLECs. Conclusion: These results provided new insights into the mechanism of cataract development, and demonstrated that CRTAC1 could be a potentially novel target for cataract treatment.

Inhibition of Cartilage Acidic Protein 1 Reduces Ultraviolet B Irradiation Induced-Apoptosis through P38 Mitogen-Activated Protein Kinase and Jun Amino-Terminal Kinase Pathways.

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Ji, Yinghong; Rong, Xianfang; Li, Dan; Cai, Lei; Rao, Jun; Lu, Yi

2016-01-01

Ultraviolet B (UVB) irradiation can easily induce apoptosis in human lens epithelial cells (HLECs) and further lead to various eye diseases including cataract. Here for the first time, we investigated the role of cartilage acidic protein 1 (CRTAC1) gene in UVB irradiation induced-apoptosis in HLECs. Three groups of HLECs were employed including model group, empty vector group, and CRTAC1 interference group. After UVB irradiation, the percentage of primary apoptotic cells was obviously fewer in CRTAC1 interference group. Meanwhile, inhibition of CRTAC1 also reduced both reactive oxygen species (ROS) production and intracellular Ca2+ concentration, but the level of mitochondrial membrane potential (Δψm) was increased in HLECs. Further studies indicated that superoxide dismutase (SOD) activity and total antioxidative (T-AOC) level were significantly increased in CRTAC1-inhibited cells, while the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH) were significantly decreased. ELISA analysis of CRTAC1-inhibited cells showed that the concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly decreased, but the concentration of interleukin-10 (IL-10) was significantly increased. Western blot analyses of eight apoptosis-associated proteins including Bax, Bcl-2, p38, phospho-p38 (p-p38), Jun amino-terminal kinases (JNK1/2), phospho-JNK1/2 (p-JNK1/2), calcium-sensing receptor (CasR), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) indicated that the inhibition of CRTAC1 alleviated oxidative stress and inflammation response, inactivated calcium-signaling pathway, p38 and JNK1/2 signal pathways, and eventually reduced UVB irradiation induced-apoptosis in HLECs. These results provided new insights into the mechanism of cataract development, and demonstrated that CRTAC1 could be a potentially novel target for cataract treatment. © 2016 The Author(s) Published by S. Karger AG, Basel.

S -Nitrosylation inhibits the kinase activity of tomato phosphoinositide-dependent kinase 1 (PDK1)

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Liu, Jian-Zhong; Duan, Jicheng; Ni, Min; Liu, Zhen; Qiu, Wen-Li; Whitham, Steven A.; Qian, Wei-Jun

2017-09-29

It is well known that the reactive oxygen species, nitric oxide (NO), can trigger cell death in plants, but the underlying molecular mechanisms are not well understood. Here, we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicon) through inhibiting the phosphoinositide-dependent kinase 1 (PDK1) kinase activity via S-nitrosylation. Biotin-switch assays and LC-MS/MS analyses demonstrated that SlPDK1 was a target of S-nitrosylation modification, which primarily occurred on the cysteine residue at position 128 (Cys128). Accordingly, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione (GSNO) both in vitro and in vivo in a concentration-dependent manner, indicating that SlPDK1 activity is regulated by S-nitrosylation. The inhibition of SlPDK1 kinase activity by GSNO was reversible in the presence of a reducing agent but synergistically enhanced by hydrogen peroxide (H2O2). Mutation of Cys128 to serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys128 is responsible for the inhibition of the kinase activity of SlPDK1. In sum, our results established a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1, a conserved negative regulator of cell death in yeasts, mammals and plants. Nitric oxide (NO) potentiates the induction of hypersensitive cell death in soybean cells by reactive oxygen species (ROS) (1). However, the molecular mechanism of the NO-induced cell death remains an enigma. One potential mechanism is that the activity of proteins that control cell death may be altered by a post-translational modification, S-nitrosylation. S-nitrosylation is the addition of the NO moiety to thiol groups, including cysteine (Cys) residues in proteins, to form S-nitrosothiols (SNOs). S-nitrosylation is an enzyme-independent post-translational and labile modification that can function as an on/off switch of protein activity (2- 4). Thousands of diverse

LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

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Marker Daniel F

2012-11-01

Full Text Available Abstract Background Human Immunodeficiency Virus-1 (HIV-1 associated neurocognitive disorders (HANDs are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART. While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2 as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs. Methods We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i. We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons. Results We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence

[Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

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Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

2012-01-01

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy.

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Ojo, Kayode K; Dangoudoubiyam, Sriveny; Verma, Shiv K; Scheele, Suzanne; DeRocher, Amy E; Yeargan, Michelle; Choi, Ryan; Smith, Tess R; Rivas, Kasey L; Hulverson, Matthew A; Barrett, Lynn K; Fan, Erkang; Maly, Dustin J; Parsons, Marilyn; Dubey, Jitender P; Howe, Daniel K; Van Voorhis, Wesley C

2016-12-01

Sarcocystis neurona is the most frequent cause of equine protozoal myeloencephalitis, a debilitating neurological disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium-dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma gondii. SnCDPK1 shares the glycine "gatekeeper" residue of the well-characterized T. gondii enzyme, which allows the latter to be targeted by bumped kinase inhibitors. This study presents detailed molecular and phenotypic evidence that SnCDPK1 can be targeted for rational drug development. Recombinant SnCDPK1 was tested against four bumped kinase inhibitors shown to potently inhibit both T. gondii (Tg) CDPK1 and T. gondii tachyzoite growth. SnCDPK1 was inhibited by low nanomolar concentrations of these BKIs and S. neurona growth was inhibited at 40-120nM concentrations. Thermal shift assays confirmed these bumped kinase inhibitors bind CDPK1 in S. neurona cell lysates. Treatment with bumped kinase inhibitors before or after invasion suggests that bumped kinase inhibitors interfere with S. neurona mammalian host cell invasion in the 0.5-2.5μM range but interfere with intracellular division at 2.5μM. In vivo proof-of-concept experiments were performed in a murine model of S. neurona infection. The experimental infected groups treated for 30days with compound BKI-1553 (n=10 mice) had no signs of disease, while the infected control group had severe signs and symptoms of infection. Elevated antibody responses were found in 100% of control infected animals, but only 20% of BKI-1553 treated infected animals. Parasites were found in brain tissues of 100% of the control infected animals, but only in 10% of the BKI-1553 treated animals. The bumped kinase inhibitors used in these assays have been chemically optimized for potency, selectivity and pharmacokinetic properties, and hence are good candidates for treatment of equine protozoal myeloencephalitis. Copyright © 2016

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

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Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

Protein Kinase-A Inhibition Is Sufficient to Support Human Neural Stem Cells Self-Renewal.

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Georges, Pauline; Boissart, Claire; Poulet, Aurélie; Peschanski, Marc; Benchoua, Alexandra

2015-12-01

Human pluripotent stem cell-derived neural stem cells offer unprecedented opportunities for producing specific types of neurons for several biomedical applications. However, to achieve it, protocols of production and amplification of human neural stem cells need to be standardized, cost effective, and safe. This means that small molecules should progressively replace the use of media containing cocktails of protein-based growth factors. Here we have conducted a phenotypical screening to identify pathways involved in the regulation of hNSC self-renewal. We analyzed 80 small molecules acting as kinase inhibitors and identified compounds of the 5-isoquinolinesulfonamide family, described as protein kinase A (PKA) and protein kinase G inhibitors, as candidates to support hNSC self-renewal. Investigating the mode of action of these compounds, we found that modulation of PKA activity was central in controlling the choice between self-renewal or terminal neuronal differentiation of hNSC. We finally demonstrated that the pharmacological inhibition of PKA using the small molecule HA1004 was sufficient to support the full derivation, propagation, and long-term maintenance of stable hNSC in absence of any other extrinsic signals. Our results indicated that tuning of PKA activity is a core mechanism regulating hNSC self-renewal and differentiation and delineate the minimal culture media requirement to maintain undifferentiated hNSC in vitro. © 2015 AlphaMed Press.

Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition.

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Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

2017-06-14

ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition.

Hsp90 inhibition differentially destabilises MAP kinase and TGF-beta signalling components in cancer cells revealed by kinase-targeted chemoproteomics

International Nuclear Information System (INIS)

Haupt, Armin; Dahl, Andreas; Lappe, Michael; Lehrach, Hans; Gonzalez, Cayetano; Drewes, Gerard; Lange, Bodo MH; Joberty, Gerard; Bantscheff, Marcus; Fröhlich, Holger; Stehr, Henning; Schweiger, Michal R; Fischer, Axel; Kerick, Martin; Boerno, Stefan T

2012-01-01

The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level. We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ('kinobeads'). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure. We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain. We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications

Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

International Nuclear Information System (INIS)

Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo

2005-01-01

Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression

Inhibition of protein kinase C affects on mode of synaptic vesicle exocytosis due to cholesterol depletion

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Petrov, Alexey M., E-mail: fysio@rambler.ru; Zakyrjanova, Guzalija F., E-mail: guzik121192@mail.ru; Yakovleva, Anastasia A., E-mail: nastya1234qwer@mail.ru; Zefirov, Andrei L., E-mail: zefiroval@rambler.ru

2015-01-02

Highlights: • We examine the involvement of PKC in MCD induced synaptic vesicle exocytosis. • PKC inhibitor does not decrease the effect MCD on MEPP frequency. • PKC inhibitor prevents MCD induced FM1-43 unloading. • PKC activation may switch MCD induced exocytosis from kiss-and-run to a full mode. • Inhibition of phospholipase C does not lead to similar change in exocytosis. - Abstract: Previous studies demonstrated that depletion of membrane cholesterol by 10 mM methyl-beta-cyclodextrin (MCD) results in increased spontaneous exocytosis at both peripheral and central synapses. Here, we investigated the role of protein kinase C in the enhancement of spontaneous exocytosis at frog motor nerve terminals after cholesterol depletion using electrophysiological and optical methods. Inhibition of the protein kinase C by myristoylated peptide and chelerythrine chloride prevented MCD-induced increases in FM1-43 unloading, whereas the frequency of spontaneous postsynaptic events remained enhanced. The increase in FM1-43 unloading still could be observed if sulforhodamine 101 (the water soluble FM1-43 quencher that can pass through the fusion pore) was added to the extracellular solution. This suggests a possibility that exocytosis of synaptic vesicles under these conditions could occur through the kiss-and-run mechanism with the formation of a transient fusion pore. Inhibition of phospholipase C did not lead to similar change in MCD-induced exocytosis.

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface*

Science.gov (United States)

Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

2016-01-01

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. PMID:26912659

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

Science.gov (United States)

Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

2016-04-15

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

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Kumar, Priyadarsini; Walsh, Donal A

2002-03-15

We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

Protein kinase Cɛ inhibition restores megakaryocytic differentiation of hematopoietic progenitors from primary myelofibrosis patients.

Science.gov (United States)

Masselli, E; Carubbi, C; Gobbi, G; Mirandola, P; Galli, D; Martini, S; Bonomini, S; Crugnola, M; Craviotto, L; Aversa, F; Vitale, M

2015-11-01

Among the three classic Philadelphia chromosome-negative myeloproliferative neoplasms, primary myelofibrosis (PMF) is the most severe in terms of disease biology, survival and quality of life. Abnormalities in the process of differentiation of PMF megakaryocytes (MKs) are a hallmark of the disease. Nevertheless, the molecular events that lead to aberrant megakaryocytopoiesis have yet to be clarified. Protein kinase Cɛ (PKCɛ) is a novel serine/threonine kinase that is overexpressed in a variety of cancers, promoting aggressive phenotype, invasiveness and drug resistance. Our previous findings on the role of PKCɛ in normal (erythroid and megakaryocytic commitment) and malignant (acute myeloid leukemia) hematopoiesis prompted us to investigate whether it could be involved in the pathogenesis of PMF MK-impaired differentiation. We demonstrate that PMF megakaryocytic cultures express higher levels of PKCɛ than healthy donors, which correlate with higher disease burden but not with JAK2V617F mutation. Inhibition of PKCɛ function (by a negative regulator of PKCɛ translocation) or translation (by target small hairpin RNA) leads to reduction in PMF cell growth, restoration of PMF MK differentiation and inhibition of PKCɛ-related anti-apoptotic signaling (Bcl-xL). Our data suggest that targeting PKCɛ directly affects the PMF neoplastic clone and represent a proof-of-concept for PKCɛ inhibition as a novel therapeutic strategy in PMF.

Non-degradative Ubiquitination of Protein Kinases.

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K Aurelia Ball

2016-06-01

Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

The antitumor natural product tanshinone IIA inhibits protein kinase C and acts synergistically with 17-AAG.

Science.gov (United States)

Lv, Chao; Zeng, Hua-Wu; Wang, Jin-Xin; Yuan, Xing; Zhang, Chuang; Fang, Ting; Yang, Pei-Ming; Wu, Tong; Zhou, Yu-Dong; Nagle, Dale G; Zhang, Wei-Dong

2018-02-07

Tanshinone IIA (Tan IIA), the primary bioactive compound derived from the traditional Chinese medicine (TCM) Salvia miltiorrhiza Bunge, has been reported to possess antitumor activity. However, its antitumor mechanisms are not fully understood. To resolve the potential antitumor mechanism(s) of Tan IIA, its gene expression profiles from our database was analyzed by connectivity map (CMAP) and the CMAP-based mechanistic predictions were confirmed/validated in further studies. Specifically, Tan IIA inhibited total protein kinase C (PKC) activity and selectively suppressed the expression of cytosolic and plasma membrane PKC isoforms ζ and ε. The Ras/MAPK pathway that is closely regulated by the PKC signaling is also inhibited by Tan IIA. While Tan IIA did not inhibit heat shock protein 90 (Hsp90), it synergistically enhanced the antitumor efficacy of the Hsp90 inhibitors 17-AAG and ganetespib in human breast cancer MCF-7 cells. In addition, Tan IIA significantly inhibited PI3K/Akt/mTOR signaling, and induced both cell cycle arrest and autophagy. Collectively, these studies provide new insights into the molecular mechanisms responsible for antitumor activity of Tan IIA.

Pharmacological Inhibition of the Protein Kinase MRK/ZAK Radiosensitizes Medulloblastoma.

Science.gov (United States)

Markowitz, Daniel; Powell, Caitlin; Tran, Nhan L; Berens, Michael E; Ryken, Timothy C; Vanan, Magimairajan; Rosen, Lisa; He, Mingzu; Sun, Shan; Symons, Marc; Al-Abed, Yousef; Ruggieri, Rosamaria

2016-08-01

Medulloblastoma is a cerebellar tumor and the most common pediatric brain malignancy. Radiotherapy is part of the standard care for this tumor, but its effectiveness is accompanied by significant neurocognitive sequelae due to the deleterious effects of radiation on the developing brain. We have previously shown that the protein kinase MRK/ZAK protects tumor cells from radiation-induced cell death by regulating cell-cycle arrest after ionizing radiation. Here, we show that siRNA-mediated MRK depletion sensitizes medulloblastoma primary cells to radiation. We have, therefore, designed and tested a specific small molecule inhibitor of MRK, M443, which binds to MRK in an irreversible fashion and inhibits its activity. We found that M443 strongly radiosensitizes UW228 medulloblastoma cells as well as UI226 patient-derived primary cells, whereas it does not affect the response to radiation of normal brain cells. M443 also inhibits radiation-induced activation of both p38 and Chk2, two proteins that act downstream of MRK and are involved in DNA damage-induced cell-cycle arrest. Importantly, in an animal model of medulloblastoma that employs orthotopic implantation of primary patient-derived UI226 cells in nude mice, M443 in combination with radiation achieved a synergistic increase in survival. We hypothesize that combining radiotherapy with M443 will allow us to lower the radiation dose while maintaining therapeutic efficacy, thereby minimizing radiation-induced side effects. Mol Cancer Ther; 15(8); 1799-808. ©2016 AACR. ©2016 American Association for Cancer Research.

Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

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Muchir, Antoine, E-mail: a.muchir@institut-myologie.org [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Wu, Wei [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Sera, Fusako; Homma, Shunichi [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Worman, Howard J., E-mail: hjw14@columbia.edu [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States)

2014-10-03

Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna{sup H222P/H222P} mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna{sup H222P/H222P} mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna{sup H222P/H222P} mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna{sup H222P/H222P} mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left

Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

International Nuclear Information System (INIS)

Muchir, Antoine; Wu, Wei; Sera, Fusako; Homma, Shunichi; Worman, Howard J.

2014-01-01

Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna H222P/H222P mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna H222P/H222P mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna H222P/H222P mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna H222P/H222P mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left ventricular fractional

Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition

Science.gov (United States)

Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

2017-01-01

ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition. DOI: http://dx.doi.org/10.7554/eLife.24998.001 PMID:28613156

Resorufin: a lead for a new protein kinase CK2 inhibitor

DEFF Research Database (Denmark)

Sandholt, Iben Skjøth; Olsen, Birgitte Brinkmann; Guerra, Barbara

2009-01-01

Screening a natural compound library led to the identification of resorufin as a highly selective and potent inhibitor of protein kinase CK2. Out of 52 kinases tested, only CK2 was inhibited, in contrast to emodin, a structurally related, known CK2 inhibitor that, in addition to CK2, inhibited te...

Phorbol-ester-induced down-regulation of protein kinase C in mouse pancreatic islets. Potentiation of phase 1 and inhibition of phase 2 of glucose-induced insulin secretion

DEFF Research Database (Denmark)

Thams, P; Capito, K; Hedeskov, C J

1990-01-01

and potentiated phase 1 of glucose-induced secretion. Furthermore, perifusion of islets in the presence of staurosporine (1 microM), an inhibitor of protein kinase C, potentiated phase 1 and inhibited phase 2 of glucose-induced secretion. In addition, down-regulation of protein kinase C potentiated phase 1...

Isolation of a novel protein, P12-from adult Drosophila melanogaster that inhibits deoxyribonucleoside and protein kinase activities and activates 3'-5'-exonuclease activity

DEFF Research Database (Denmark)

Christiansen, Louise Slot; Zanten, Gabriella van; Berenstein, Dvora

2016-01-01

We have previously found that Drosophila melanogaster only has one deoxyribonucleoside kinase, Dm-dNK, however, capable to phosphorylate all four natural deoxyribonucleosides. Dm-dNK was originally isolated from an embryonic cell line. We wanted to study the expression of Dm-dNK during development......-dNK, also inhibited the two protein kinases to the same degree. Furthermore, testing P12 in a DNA polymerase based assay we found that the 3'-5'-exonuclease part of the DNA polymerase (Klenow polymerase) was activated....

Inhibition of the neutrophil oxidative burst by sphingoid long-chain bases: role of protein kinase C in the activation of the burst

International Nuclear Information System (INIS)

Wilson, E.; Olcott, M.C.; Bell, R.M.; Merrill, A.H.; Lambeth, J.D.

1986-01-01

The neutrophil oxidative burst is triggered by a variety of both particulate (opsonized zymosan) and soluble agonists [formylmethionylleucylphenylalanine (FMLP), arachidonate, short-chained diacylglycerols (DAG) and phorbol myristate acetate (PMA)]. The authors show that the long-chain lipid bases sphinganine and sphingosine block activation of the burst in human neutrophils. Inhibition is reversible, does not alter cell viability, and does not affect phagocytosis. The inhibition affects the activation mechanism rather than the NADPH-oxidase enzyme. The structural requirements for inhibition include a hydrophobic carbon chain and an amino-containing headgroup, and the naturally occurring erythro sphinganine was more potent than the threo isomer. Activation of the oxidative burst by a variety of agonists was blocked by the same concentration of sphinganine indicating a common inhibited step. The authors suggest that the common step is protein kinase C, as evidenced by the following: 1) long-chain bases inhibit PKC in a micelle reconstituted system, 2) PMA-induced phophorylation is inhibited by sphinganine, and 3) sphinganine competes with ( 3 H)-phorbol dibutyrate for its cytosolic receptor (i.e. protein kinase C). The authors suggest that sphingoid long-chain bases play a role in the cellular regulations

MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Bregenholt, S; Johansen, B

1999-01-01

B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h...... post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine...

The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

International Nuclear Information System (INIS)

Yang, Se-Ran; Cho, Sung-Dae; Ahn, Nam-Shik; Jung, Ji-Won; Park, Joon-Suk; Jo, Eun-Hye; Hwang, Jae-Woong; Kim, Sung-Hoon; Lee, Bong-Hee; Kang, Kyung-Sun; Lee, Yong-Soon

2005-01-01

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK. These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis

Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

Science.gov (United States)

Santafé, M M; Garcia, N; Lanuza, M A; Tomàs, M; Tomàs, J

2009-02-15

We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

Science.gov (United States)

Chen, Kung-Yen; Lin, Jui-An; Yao, Han-Yun; Hsu, An-Chih; Tai, Yu-Ting; Chen, Jui-Tai; Hsieh, Mao-Chih; Shen, Tang-Long; Hsu, Ren-Yi; Wu, Hong-Tan; Wang, Guey Horng; Ho, Bing-Ying; Chen, Yu-Pei

2018-04-01

Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1β, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species

International Nuclear Information System (INIS)

Haase, Hajo; Engelhardt, Gabriela; Hebel, Silke; Rink, Lothar

2011-01-01

Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg 2+ ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn 2+ ); and (3) by inducing reactive oxygen species (ROS). Hg 2+ causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn 2+ release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn 2+ or Hg 2+ . Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg 2+ -induced oxidation, because phosphatase activity is inhibited at concentrations of Hg 2+ that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

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Weatherford Wendy

2005-05-01

Full Text Available Abstract Background High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Results Using a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP, Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1. Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4. Enzyme inhibition yielded IC50 values that were comparable to those obtained using

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

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Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

2005-05-31

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that

Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

International Nuclear Information System (INIS)

Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

2007-01-01

Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe

Src protein-tyrosine kinase structure and regulation

International Nuclear Information System (INIS)

Roskoski, Robert

2004-01-01

Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPα displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the αD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu 4 Tyr)

Insulin utilizes the PI 3-kinase pathway to inhibit SP-A gene expression in lung epithelial cells

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Snyder Jeanne M

2002-10-01

Full Text Available Abstract Background It has been proposed that high insulin levels may cause delayed lung development in the fetuses of diabetic mothers. A key event in lung development is the production of adequate amounts of pulmonary surfactant. Insulin inhibits the expression of surfactant protein A (SP-A, the major surfactant-associated protein, in lung epithelial cells. In the present study, we investigated the signal transduction pathways involved in insulin inhibition of SP-A gene expression. Methods H441 cells, a human lung adenocarcinoma cell line, or human fetal lung explants were incubated with or without insulin. Transcription run-on assays were used to determine SP-A gene transcription rates. Northern blot analysis was used to examine the effect of various signal transduction inhibitors on SP-A gene expression. Immunoblot analysis was used to evaluate the levels and phosphorylation states of signal transduction protein kinases. Results Insulin decreased SP-A gene transcription in human lung epithelial cells within 1 hour. Insulin did not affect p44/42 mitogen-activated protein kinase (MAPK phosphorylation and the insulin inhibition of SP-A mRNA levels was not affected by PD98059, an inhibitor of the p44/42 MAPK pathway. In contrast, insulin increased p70 S6 kinase Thr389 phosphorylation within 15 minutes. Wortmannin or LY294002, both inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase, or rapamycin, an inhibitor of the activation of p70 S6 kinase, a downstream effector in the PI 3-kinase pathway, abolished or attenuated the insulin-induced inhibition of SP-A mRNA levels. Conclusion Insulin inhibition of SP-A gene expression in lung epithelial cells probably occurs via the rapamycin-sensitive PI 3-kinase signaling pathway.

Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

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Cursio, R; Filippa, N; Miele, C; Van Obberghen, E; Gugenheim, J

2006-06-01

This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

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Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Chloride sensing by WNK1 kinase involves inhibition of autophosphorylation

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Piala, Alexander T.; Moon, Thomas M.; Akella, Radha; He, Haixia; Cobb, Melanie H.; Goldsmith, Elizabeth J.

2014-01-01

WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. Here, we found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation. PMID:24803536

Effects of Src Kinase Inhibition on Expression of Protein Tyrosine Phosphatase 1B after Brain Hypoxia in a Piglet Animal Model

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Dimitrios Angelis

2017-01-01

Full Text Available Background. Protein tyrosine phosphatases (PTPs in conjunction with protein tyrosine kinases (PTKs regulate cellular processes by posttranslational modifications of signal transduction proteins. PTP nonreceptor type 1B (PTP-1B is an enzyme of the PTP family. We have previously shown that hypoxia induces an increase in activation of a class of nonreceptor PTK, the Src kinases. In the present study, we investigated the changes that occur in the expression of PTP-1B in the cytosolic component of the brain of newborn piglets acutely after hypoxia as well as long term for up to 2 weeks. Methods. Newborn piglets were divided into groups: normoxia, hypoxia, hypoxia followed by 1 day and 15 days in FiO2 0.21, and hypoxia pretreated with Src kinase inhibitor PP2, prior to hypoxia followed by 1 day and 15 days. Hypoxia was achieved by providing 7% FiO2 for 1 hour and PTP-1B expression was measured via immunoblotting. Results. PTP-1B increased posthypoxia by about 30% and persisted for 2 weeks while Src kinase inhibition attenuated the expected PTP-1B-increased expression. Conclusions. Our study suggests that Src kinase mediates a hypoxia-induced increased PTP-1B expression.

Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

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Li, H.; Roux, S. J.

1992-01-01

Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

Energy Technology Data Exchange (ETDEWEB)

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

2012-03-02

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

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Kobayashi, Soushi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

2015-07-01

Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ribosomal protein mutations induce autophagy through S6 kinase inhibition of the insulin pathway.

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Harry F Heijnen

Full Text Available Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA, for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS. The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS. We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies.

The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response.

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Jan Karlseder

2004-08-01

Full Text Available The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Here we show that overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogated the cell cycle arrest after ionizing radiation and diminished several other readouts of the DNA damage response, including phosphorylation of Nbs1, induction of p53, and upregulation of p53 targets. TRF2 inhibited autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 was found to directly interact with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. We propose that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage.

Action of mercurials on activity of partially purified soluble protein kinase C from mice brain

International Nuclear Information System (INIS)

Inoue, Y.; Saijoh, K.; Sumino, K.

1988-01-01

The enzymatic activity of soluble protein kinase C from mice brain was inhibited by mercuric chloride (II) (HgCl 2 ) and organic mercurials, i.e. methyl mercury, phenyl mercury and p-chloromercuribenzoic acid (PCMB). The IC50 was 0.08 μM for HgCl 2 and about 1 μM for organic mercurials. Sulfhydryl blocking reagents such as 5.5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM) were less potent but nevertheless inhibited the enzymic activity of protein kinase C. The Hill coefficients of HgCl 2 , DTNB and NEM were close to unity whereas the values for organic mercurials were 1.3 to 1.5. The inhibition was of a non-competitive type with respect to Hl histone. 3 H-PDBu binding activity was also inhibited by all of the reagents in a non-competitive manner. Mercurials apparently bind to sulfhydryl groups of protein kinase C to inhibit the enzymatic activity. (author)

Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent Protein Kinase PKR

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Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Lokugamage, Nandadeva; Head, Jennifer A.; Ikegami, Tetsuro

2012-01-01

Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general tr...

Calcitonin causes a sustained inhibition of protein kinase C-stimulated bone resorption in contrast to the transient inhibition of parathyroid hormone-induced bone resorption

International Nuclear Information System (INIS)

Ransjoe, M.; Lerner, U.H.

1990-01-01

Calcitonin is a well known inhibitor of osteoclastic bone resortion, both in vivo and in vitro. However, it is also known that calcitonin has only a transient inhibitory effect on bone resorption. The mechanism for this so-called ''escape from inhibition'' phenomenon is not clear. In the present study, the inhibitory effect of calcitonin on phorbol ester-induced bone resorption was examined in cultured neonatal mouse calvaria. Bone resorption was assessed as the release of radioactivity from bones prelabelled in vivo with 45 Ca. Two proteon kinase C-activating phorbol esters, phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate, both stimulated 45 Ca release in 120-h cultures at a concentration of 10 nmul/l. Calcitonin (30 nmol/l) inhibited phorbol esterstimulated bone resorption without any ''escape from inhibition''. This was in contrast to the transient inhibitory effect of calcitonin on bone resorption stimulated by parathyroid hormone (10 nmol/l), prostaglandin E 2 (2 μmol/l), and bradykinin (1 μmol/l). Our results suggest that activation of protein kinase C produces a sustained inhibitory effect of calcitonin on bone resorption. (author)

Bacterial Protein-Tyrosine Kinases

DEFF Research Database (Denmark)

Shi, Lei; Kobir, Ahasanul; Jers, Carsten

2010-01-01

in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

[Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].

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Zhao, Song; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Pu, Feifei; Ma, Dongchu

2016-10-01

Objective To investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1 (S6K1) on the polyploidization of megakaryocytes. Methods SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and H-89, a cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells separately or in combination. With propidium iodide (PI) to dye DNA in the treated cells, the relative DNA content was detected by flow cytometry, and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1 (S6K1), an important mammalian target of rapamycin (mTOR) downstream target molecule, was analyzed by Western blotting. Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time, it increased the phosphorylation of S6K1 at Thr421/Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization, but also decreased the phosphorylation of S6K1 at Thr421/Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably, both H-89 and SP600125 inhibited the activity of PKA. Moreover, the two drugs further inhibited the activity of PKA when used together. Therefore, these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state, rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

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Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

An inhibition of p38 mitogen activated protein kinase delays the platelet storage lesion.

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Andrey Skripchenko

Full Text Available BACKGROUND AND OBJECTIVES: Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK. Another MAPK, extracellular signal-related kinase (ERK, is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors. MATERIALS AND METHODS: A single Trima apheresis platelet unit (n = 12 was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20-24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters. RESULTS: Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties. CONCLUSION: Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.

Effective intracellular inhibition of the cAMP-dependent protein kinase by microinjection of a modified form of the specific inhibitor peptide PKi in living fibroblasts.

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Fernandez, A; Mery, J; Vandromme, M; Basset, M; Cavadore, J C; Lamb, N J

1991-08-01

In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.

Lycopene depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase C in rat cerebrocortical nerve terminals.

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Lu, Cheng-Wei; Hung, Chi-Feng; Jean, Wei-Horng; Lin, Tzu-Yu; Huang, Shu-Kuei; Wang, Su-Jane

2018-05-01

Lycopene is a natural dietary carotenoid that was reported to exhibit a neuroprotective profile. Considering that excitotoxicity and cell death induced by glutamate are involved in many brain disorders, the effect of lycopene on glutamate release in rat cerebrocortical nerve terminals and the possible mechanism involved in such effect was investigated. We observed here that lycopene inhibited 4-aminopyridine (4-AP)-evoked glutamate release and intrasynaptosomal Ca 2+ concentration elevation. The inhibitory effect of lycopene on 4-AP-evoked glutamate release was markedly reduced in the presence of the Ca v 2.2 (N-type) and Ca v 2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but was insensitive to the intracellular Ca 2+ -release inhibitors dantrolene and CGP37157. Furthermore, in the presence of the protein kinase C inhibitors GF109203X and Go6976, the action of lycopene on evoked glutamate release was prevented. These results are the first to suggest that lycopene inhibits glutamate release from rat cortical synaptosomes by suppressing presynaptic Ca 2+ entry and protein kinase C activity.

Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration.

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Qian-Shi Zhang

Full Text Available Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1, also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5, after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1, the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93, or the downstream target, c-Jun N-terminal kinase (SP600125 also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/β isoforms, (SB203580 had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration.

Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration

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Zhang, Qian-Shi; Kurpad, Deepa S.; Mahoney, My G.; Steinbeck, Marla J.

2017-01-01

Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO) or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1), the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93), or the downstream target, c-Jun N-terminal kinase (SP600125) also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/β isoforms, (SB203580) had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration. PMID:29045420

A historical overview of protein kinases and their targeted small molecule inhibitors.

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Roskoski, Robert

2015-10-01

catalytic subunits. PKA and all other protein kinase domains have a small amino-terminal lobe and large carboxyterminal lobe as determined by X-ray crystallography. The N-lobe and C-lobe form a cleft that serves as a docking site for MgATP. Nearly all active protein kinases contain a K/E/D/D signature sequence that plays important structural and catalytic roles. Protein kinases contain hydrophobic catalytic and regulatory spines and collateral shell residues that are required to assemble the active enzyme. There are two general kinds of conformational changes associated with most protein kinases. The first conformational change involves the formation of an intact regulatory spine to form an active enzyme. The second conformational change occurs in active kinases as they toggle between open and closed conformations during their catalytic cycles. Because mutations and dysregulation of protein kinases play causal roles in human disease, this family of enzymes has become one of the most important drug targets over the past two decades. Imatinib was approved by the United States FDA for the treatment of chronic myelogenous leukemia in 2001; this small molecule inhibits the BCR-Abl protein kinase oncoprotein that results from the formation of the Philadelphia chromosome. More than two dozen other orally effective mechanism-based small molecule protein kinase inhibitors have been subsequently approved by the FDA. These drugs bind to the ATP-binding site of their target enzymes and extend into nearby hydrophobic pockets. Most of these protein kinase inhibitors prolong survival in cancer patients only weeks or months longer than standard cytotoxic therapies. In contrast, the clinical effectiveness of imatinib against chronic myelogenous leukemia is vastly superior to that of any other targeted protein kinase inhibitor with overall survival lasting a decade or more. However, the near universal and expected development of drug resistance in the treatment of neoplastic disorders

Protein kinase substrate identification on functional protein arrays

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Zhou Fang

2008-02-01

Full Text Available Abstract Background Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. Results To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. Conclusion Functional protein microarrays are an

The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells.

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Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K

2014-05-01

G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (sotrastaurin) and PI3K/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11-mutant cells with AEB071 versus no activity in wild-type cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of myristoylated alanine-rich C-kinase substrate, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal antiproliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11-mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ- and GNA11-mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ-mutant model. These findings suggest a new therapy treatment option for G-protein-mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

DEFF Research Database (Denmark)

Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

2013-01-01

knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1)

Czech Academy of Sciences Publication Activity Database

Petrvalská, Olivia; Košek, Dalibor; Kukačka, Zdeněk; Tošner, Z.; Man, Petr; Večeř, J.; Herman, P.; Obšilová, Veronika; Obšil, Tomáš

2016-01-01

Roč. 291, č. 39 (2016), s. 20753-20765 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA14-10061S Institutional support: RVO:67985823 ; RVO:61388971 Keywords : 14-3-3 protein * apoptosis signal-regulating kinase 1 (ASK1) * fluorescence * nuclear magnetic resonance (NMR) * protein cross-linking * small-angle x-ray scattering (SAXS) Subject RIV: CE - Biochemistry Impact factor: 4.125, year: 2016

The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs.

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Sunita, S; Schwartz, Samantha L; Conn, Graeme L

2015-11-20

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain and a C-terminal kinase domain interposed by an unstructured ∼80-residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR) and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries and that each exhibits comparable polyinosinic-cytidylic (poly(rI:rC)) dsRNA activation profiles and binding affinities for adenoviral virus-associated RNA I (VA RNAI) and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain-swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rat PKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

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Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Lokugamage, Nandadeva; Head, Jennifer A; Ikegami, Tetsuro

2013-01-20

Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

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Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen (Sanofi); (Michigan); (Texas)

2010-07-19

G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase

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Guan Kunliang

2003-06-01

Full Text Available Abstract Background G protein-coupled receptors (GPCRs interact with heterotrimeric GTP-binding proteins (G proteins to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways. Results Here we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT2C receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1 on the 5-HT2C receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT2C receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT2C receptor abolished the effect of insulin. Conclusion These results show that insulin signaling can inhibit 5-HT2C receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR.

Inhibition of stress-activated MAP kinases induces clinical improvement in moderate to severe Crohn's disease

NARCIS (Netherlands)

Hommes, Daan; van den Blink, Bernt; Plasse, Terry; Bartelsman, Joep; Xu, Cuiping; Macpherson, Bret; Tytgat, Guido; Peppelenbosch, Mailkel; van Deventer, Sander

2002-01-01

Background & Aims: We investigated if inhibition of mitogen-activated protein kinases (MAPKs) was beneficial in Crohn's disease. Methods: Inhibition of JNK and p38 MAPK activation with CNI-1493, a guanylhydrazone, was tested in vitro. Twelve patients with severe Crohn's disease (mean baseline, CDAI

Curcumin Attenuates Opioid Tolerance and Dependence by Inhibiting Ca2+/Calmodulin-Dependent Protein Kinase II α Activity

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Hu, Xiaoyu; Huang, Fang; Szymusiak, Magdalena

2015-01-01

Chronic use of opioid analgesics has been hindered by the development of opioid addiction and tolerance. We have reported that curcumin, a natural flavonoid from the rhizome of Curcuma longa, attenuated opioid tolerance, although the underlying mechanism remains unclear. In this study, we tested the hypothesis that curcumin may inhibit Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα), a protein kinase that has been previously proposed to be critical for opioid tolerance and dependence. In this study, we used state-of-the-art polymeric formulation technology to produce poly(lactic-co-glycolic acid) (PLGA)-curcumin nanoparticles (nanocurcumin) to overcome the drug’s poor solubility and bioavailability, which has made it extremely difficult for studying in vivo pharmacological actions of curcumin. We found that PLGA-curcumin nanoparticles reduced the dose requirement by 11- to 33-fold. Pretreatment with PLGA-curcumin (by mouth) prevented the development of opioid tolerance and dependence in a dose-dependent manner, with ED50 values of 3.9 and 3.2 mg/kg, respectively. PLGA-curcumin dose-dependently attenuated already-established opioid tolerance (ED50 = 12.6 mg/kg p.o.) and dependence (ED50 = 3.1 mg/kg p.o.). Curcumin or PLGA-curcumin did not produce antinociception by itself or affect morphine (1–10 mg/kg) antinociception. Moreover, we found that the behavioral effects of curcumin on opioid tolerance and dependence correlated with its inhibition of morphine-induced CaMKIIα activation in the brain. These results suggest that curcumin may attenuate opioid tolerance and dependence by suppressing CaMKIIα activity. PMID:25515789

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

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Varley, C L; Royds, J A; Brown, B L; Dobson, P R

2001-01-01

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

Inhibition of protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression.

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Liang, Xiaonan; Yong, Zheng; Su, Ruibin

2018-06-11

Opioid-induced respiratory depression is a major obstacle to improving the clinical management of moderate to severe chronic pain. Opioids inhibit neuronal activity via various pathways, including calcium channels, adenylyl cyclase, and potassium channels. Currently, the underlying molecular pathway of opioid-induced respiratory depression is only partially understood. This study aimed to investigate the mechanisms of opioid-induced respiratory depression in vivo by examining the effects of different pharmacological agents on fentanyl-induced respiratory depression. Respiratory parameters were detected using whole body plethysmography in conscious rats. We show that pre-treatment with the protein kinase A (PKA) inhibitor H89 reversed the fentanyl-related effects on respiratory rate, inspiratory time, and expiratory time. Pre-treatment with the G protein-gated inwardly rectifying potassium (GIRK) channel blocker Tertiapin-Q dose-dependently reversed the fentanyl-related effects on respiratory rate and inspiratory time. A phosphodiesterase 4 (PDE4) inhibitor and cyclic adenosine monophosphate (cAMP) analogs did not affect fentanyl-induced respiratory depression. These findings suggest that PKA and GIRK may be involved in fentanyl-induced respiratory depression and could represent useful therapeutic targets for the treatment of fentanyl-induced ventilatory depression. Copyright © 2018 Elsevier B.V. All rights reserved.

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

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Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-08-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

AJUBA LIM Proteins Limit Hippo Activity in Proliferating Cells by Sequestering the Hippo Core Kinase Complex in the Cytosol.

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Jagannathan, Radhika; Schimizzi, Gregory V; Zhang, Kun; Loza, Andrew J; Yabuta, Norikazu; Nojima, Hitoshi; Longmore, Gregory D

2016-10-15

The Hippo pathway controls organ growth and is implicated in cancer development. Whether and how Hippo pathway activity is limited to sustain or initiate cell growth when needed is not understood. The members of the AJUBA family of LIM proteins are negative regulators of the Hippo pathway. In mammalian epithelial cells, we found that AJUBA LIM proteins limit Hippo regulation of YAP, in proliferating cells only, by sequestering a cytosolic Hippo kinase complex in which LATS kinase is inhibited. At the plasma membranes of growth-arrested cells, AJUBA LIM proteins do not inhibit or associate with the Hippo kinase complex. The ability of AJUBA LIM proteins to inhibit YAP regulation by Hippo and to associate with the kinase complex directly correlate with their capacity to limit Hippo signaling during Drosophila wing development. AJUBA LIM proteins did not influence YAP activity in response to cell-extrinsic or cell-intrinsic mechanical signals. Thus, AJUBA LIM proteins limit Hippo pathway activity in contexts where cell proliferation is needed. Copyright © 2016 Jagannathan et al.

Mururins A-C, three new lignoids from Brosimum acutifolium and their protein kinase inhibitory activity.

Science.gov (United States)

Takashima, Junko; Asano, Shoichi; Ohsaki, Ayumi

2002-07-01

Two new flavonolignans, mururins A and B ( 1 and 2), and a new lignan, mururin C ( 3), were isolated from the bark of Brosimum acutifolium Huber together with three known lignans. Their structures were elucidated by spectroscopic means and chemical modifications. They were tested for protein kinase A (PKA) and protein kinase C (PKC) inhibitory activity. Mururin A showed 3 % and 63 % inhibition to PKA and PKC, respectively, at 20 microM. Mururin B showed 58 % and 38 % inhibition, respectively. Mururin C did not have significant activity.

Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

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Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

2012-02-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

Exploiting the MDM2-CK1α Protein-Protein Interface to Develop Novel Biologics That Induce UBL-Kinase-Modification and Inhibit Cell Growth

Science.gov (United States)

Huart, Anne-Sophie; MacLaine, Nicola J.; Narayan, Vikram; Hupp, Ted R.

2012-01-01

Protein-protein interactions forming dominant signalling events are providing ever-growing platforms for the development of novel Biologic tools for controlling cell growth. Casein Kinase 1 α (CK1α) forms a genetic and physical interaction with the murine double minute chromosome 2 (MDM2) oncoprotein resulting in degradation of the p53 tumour suppressor. Pharmacological inhibition of CK1 increases p53 protein level and induces cell death, whilst small interfering RNA-mediated depletion of CK1α stabilizes p53 and induces growth arrest. We mapped the dominant protein-protein interface that stabilizes the MDM2 and CK1α complex in order to determine whether a peptide derived from the core CK1α-MDM2 interface form novel Biologics that can be used to probe the contribution of the CK1-MDM2 protein-protein interaction to p53 activation and cell viability. Overlapping peptides derived from CK1α were screened for dominant MDM2 binding sites using (i) ELISA with recombinant MDM2; (ii) cell lysate pull-down towards endogenous MDM2; (iii) MDM2-CK1α complex-based competition ELISA; and (iv) MDM2-mediated ubiquitination. One dominant peptide, peptide 35 was bioactive in all four assays and its transfection induced cell death/growth arrest in a p53-independent manner. Ectopic expression of flag-tagged peptide 35 induced a novel ubiquitin and NEDD8 modification of CK1α, providing one of the first examples whereby NEDDylation of a protein kinase can be induced. These data identify an MDM2 binding motif in CK1α which when isolated as a small peptide can (i) function as a dominant negative inhibitor of the CK1α-MDM2 interface, (ii) be used as a tool to study NEDDylation of CK1α, and (iii) reduce cell growth. Further, this approach provides a technological blueprint, complementing siRNA and chemical biology approaches, by exploiting protein-protein interactions in order to develop Biologics to manipulate novel types of signalling pathways such as cross-talk between

Exploiting the MDM2-CK1α protein-protein interface to develop novel biologics that induce UBL-kinase-modification and inhibit cell growth.

Directory of Open Access Journals (Sweden)

Anne-Sophie Huart

Full Text Available Protein-protein interactions forming dominant signalling events are providing ever-growing platforms for the development of novel Biologic tools for controlling cell growth. Casein Kinase 1 α (CK1α forms a genetic and physical interaction with the murine double minute chromosome 2 (MDM2 oncoprotein resulting in degradation of the p53 tumour suppressor. Pharmacological inhibition of CK1 increases p53 protein level and induces cell death, whilst small interfering RNA-mediated depletion of CK1α stabilizes p53 and induces growth arrest. We mapped the dominant protein-protein interface that stabilizes the MDM2 and CK1α complex in order to determine whether a peptide derived from the core CK1α-MDM2 interface form novel Biologics that can be used to probe the contribution of the CK1-MDM2 protein-protein interaction to p53 activation and cell viability. Overlapping peptides derived from CK1α were screened for dominant MDM2 binding sites using (i ELISA with recombinant MDM2; (ii cell lysate pull-down towards endogenous MDM2; (iii MDM2-CK1α complex-based competition ELISA; and (iv MDM2-mediated ubiquitination. One dominant peptide, peptide 35 was bioactive in all four assays and its transfection induced cell death/growth arrest in a p53-independent manner. Ectopic expression of flag-tagged peptide 35 induced a novel ubiquitin and NEDD8 modification of CK1α, providing one of the first examples whereby NEDDylation of a protein kinase can be induced. These data identify an MDM2 binding motif in CK1α which when isolated as a small peptide can (i function as a dominant negative inhibitor of the CK1α-MDM2 interface, (ii be used as a tool to study NEDDylation of CK1α, and (iii reduce cell growth. Further, this approach provides a technological blueprint, complementing siRNA and chemical biology approaches, by exploiting protein-protein interactions in order to develop Biologics to manipulate novel types of signalling pathways such as cross

Alterations in brain Protein Kinase A activity and reversal of morphine tolerance by two fragments of native Protein Kinase A inhibitor peptide (PKI).

Science.gov (United States)

Dalton, George D; Smith, Forrest L; Smith, Paul A; Dewey, William L

2005-04-01

Two peptide fragments of native Protein Kinase A inhibitor (PKI), PKI-(6-22)-amide and PKI-(Myr-14-22)-amide, significantly reversed low-level morphine antinociceptive tolerance in mice. The inhibition of Protein Kinase A (PKA) activity by both peptide fragments was then measured in specific brain regions (thalamus, periaqueductal gray (PAG), and medulla) and in lumbar spinal cord (LSC), which in previous studies have been shown to play a role in morphine-induced analgesia. In drug naive animals, cytosolic PKA activity was greater than particulate PKA activity in each region, while cytosolic and particulate PKA activities were greater in thalamus and PAG compared to medulla and LSC. The addition of both peptides to homogenates from each region completely abolished cytosolic and particulate PKA activities in vitro. Following injection into the lateral ventricle of the brain of drug naive mice and morphine-tolerant mice, both peptides inhibited PKA activity in the cytosolic, but not the particulate fraction of LSC. In addition, cytosolic and particulate PKA activities were inhibited by both peptides in thalamus. These results demonstrate that the inhibition of PKA reverses morphine tolerance. Moreover, the inhibition of PKA activity in specific brain regions and LSC from morphine-tolerant mice by PKI analogs administered i.c.v. is evidence that PKA plays a role in morphine tolerance.

Rheb Inhibits Protein Synthesis by Activating the PERK-eIF2α Signaling Cascade

Directory of Open Access Journals (Sweden)

Richa Tyagi

2015-02-01

Full Text Available Rheb, a ubiquitous small GTPase, is well known to bind and activate mTOR, which augments protein synthesis. Inhibition of protein synthesis is also physiologically regulated. Thus, with cell stress, the unfolded protein response system leads to phosphorylation of the initiation factor eIF2α and arrest of protein synthesis. We now demonstrate a major role for Rheb in inhibiting protein synthesis by enhancing the phosphorylation of eIF2α by protein kinase-like ER kinase (PERK. Interplay between the stimulatory and inhibitory roles of Rheb may enable cells to modulate protein synthesis in response to varying environmental stresses.

Raf kinase inhibitory protein: a signal transduction modulator and metastasis suppressor.

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Granovsky, Alexey E; Rosner, Marsha Rich

2008-04-01

Cells have a multitude of controls to maintain their integrity and prevent random switching from one biological state to another. Raf Kinase Inhibitory Protein (RKIP), a member of the phosphatidylethanolamine binding protein (PEBP) family, is representative of a new class of modulators of signaling cascades that function to maintain the "yin yang" or balance of biological systems. RKIP inhibits MAP kinase (Raf-MEK-ERK), G protein-coupled receptor (GPCR) kinase and NFkappaB signaling cascades. Because RKIP targets different kinases dependent upon its state of phosphorylation, RKIP also acts to integrate crosstalk initiated by multiple environmental stimuli. Loss or depletion of RKIP results in disruption of the normal cellular stasis and can lead to chromosomal abnormalities and disease states such as cancer. Since RKIP and the PEBP family have been reviewed previously, the goal of this analysis is to provide an update and highlight some of the unique features of RKIP that make it a critical player in the regulation of cellular signaling processes.

Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

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Pascal, John M; Armen, Roger S

2012-01-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain.

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Huber, Roland G; Fan, Hao; Bond, Peter J

2015-10-01

ZAP-70 (Zeta-chain-associated protein kinase 70) is a tyrosine kinase that interacts directly with the activated T-cell receptor to transduce downstream signals, and is hence a major player in the regulation of the adaptive immune response. Dysfunction of ZAP-70 causes selective T cell deficiency that in turn results in persistent infections. ZAP-70 is activated by a variety of signals including phosphorylation of the kinase domain (KD), and binding of its regulatory tandem Src homology 2 (SH2) domains to the T cell receptor. The present study investigates molecular mechanisms of activation and inhibition of ZAP-70 via atomically detailed molecular dynamics simulation approaches. We report microsecond timescale simulations of five distinct states of the ZAP-70 KD, comprising apo, inhibited and three phosphorylated variants. Extensive analysis of local flexibility and correlated motions reveal crucial transitions between the states, thus elucidating crucial steps in the activation mechanism of the ZAP-70 KD. Furthermore, we rationalize previously observed staurosporine-bound crystal structures, suggesting that whilst the KD superficially resembles an "active-like" conformation, the inhibitor modulates the underlying protein dynamics and restricts it in a compact, rigid state inaccessible to ligands or cofactors. Finally, our analysis reveals a novel, potentially druggable pocket in close proximity to the activation loop of the kinase, and we subsequently use its structure in fragment-based virtual screening to develop a pharmacophore model. The pocket is distinct from classical type I or type II kinase pockets, and its discovery offers promise in future design of specific kinase inhibitors, whilst mutations in residues associated with this pocket are implicated in immunodeficiency in humans.

The Structural Basis for Activation and Inhibition of ZAP-70 Kinase Domain.

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Roland G Huber

2015-10-01

Full Text Available ZAP-70 (Zeta-chain-associated protein kinase 70 is a tyrosine kinase that interacts directly with the activated T-cell receptor to transduce downstream signals, and is hence a major player in the regulation of the adaptive immune response. Dysfunction of ZAP-70 causes selective T cell deficiency that in turn results in persistent infections. ZAP-70 is activated by a variety of signals including phosphorylation of the kinase domain (KD, and binding of its regulatory tandem Src homology 2 (SH2 domains to the T cell receptor. The present study investigates molecular mechanisms of activation and inhibition of ZAP-70 via atomically detailed molecular dynamics simulation approaches. We report microsecond timescale simulations of five distinct states of the ZAP-70 KD, comprising apo, inhibited and three phosphorylated variants. Extensive analysis of local flexibility and correlated motions reveal crucial transitions between the states, thus elucidating crucial steps in the activation mechanism of the ZAP-70 KD. Furthermore, we rationalize previously observed staurosporine-bound crystal structures, suggesting that whilst the KD superficially resembles an "active-like" conformation, the inhibitor modulates the underlying protein dynamics and restricts it in a compact, rigid state inaccessible to ligands or cofactors. Finally, our analysis reveals a novel, potentially druggable pocket in close proximity to the activation loop of the kinase, and we subsequently use its structure in fragment-based virtual screening to develop a pharmacophore model. The pocket is distinct from classical type I or type II kinase pockets, and its discovery offers promise in future design of specific kinase inhibitors, whilst mutations in residues associated with this pocket are implicated in immunodeficiency in humans.

Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.

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Scarlatti, Francesca; Bauvy, Chantal; Ventruti, Annamaria; Sala, Giusy; Cluzeaud, Françoise; Vandewalle, Alain; Ghidoni, Riccardo; Codogno, Patrice

2004-04-30

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

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Neil Arvin Bretaña

Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

Science.gov (United States)

Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

2012-01-01

Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

Dual inhibition of γ-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase A-dependent mechanism.

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Jun, Hee-jin; Lee, Ji Hae; Cho, Bo-Ram; Seo, Woo-Duck; Kang, Hang-Won; Kim, Dong-Woo; Cho, Kang-Jin; Lee, Sung-Joon

2012-10-26

The in vitro effects on melanogenesis of γ-oryzanol (1), a rice bran-derived phytosterol, were investigated. The melanin content in B16F1 cells was significantly and dose-dependently reduced (-13% and -28% at 3 and 30 μM, respectively). Tyrosinase enzyme activity was inhibited by 1 both in a cell-free assay and when analyzed based on the measurement of cellular tyrosinase activity. Transcriptome analysis was performed to investigate the biological pathways altered by 1, and it was found that gene expression involving protein kinase A (PKA) signaling was markedly altered. Subsequent analyses revealed that 1 stimulation in B16 cells reduced cytosolic cAMP concentrations, PKA activity (-13% for cAMP levels and -40% for PKA activity), and phosphorylation of the cAMP-response element binding protein (-57%), which, in turn, downregulated the expression of microphthalmia-associated transcription factor (MITF; -59% for mRNA and -64% for protein), a key melanogenic gene transcription factor. Accordingly, tyrosinase-related protein 1 (TRP-1; -69% for mRNA and -82% for protein) and dopachrome tautomerase (-51% for mRNA and -92% for protein) in 1-stimulated B16F1 cells were also downregulated. These results suggest that 1 has dual inhibitory activities for cellular melanogenesis by inhibiting tyrosinase enzyme activity and reducing MITF and target genes in the PKA-dependent pathway.

Growth Inhibition by Bupivacaine Is Associated with Inactivation of Ribosomal Protein S6 Kinase 1

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Beigh, Mushtaq Ahmad; Showkat, Mehvish; Bashir, Basharat; Bashir, Asma; Hussain, Mahboob ul; Andrabi, Khurshid Iqbal

2014-01-01

Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation. PMID:24605337

Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis

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Kimon C. Kanelakis

2009-01-01

Full Text Available The heme-regulated inhibitor (HRI negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

KAUST Repository

Diaz Galicia, Miriam Escarlet

2018-05-01

Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

The Phosphoinositide 3-Kinaseα Selective Inhibitor, BYL719, Enhances the Effect of the Protein Kinase C Inhibitor, AEB071, in GNAQ/GNA11 Mutant Uveal Melanoma Cells

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Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K.

2014-01-01

G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy. PMID:24563540

Kinase inhibition by the Jamaican ball moss, Tillandsia recurvata L.

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Lowe, Henry I C; Watson, Charah T; Badal, Simone; Toyang, Ngeh J; Bryant, Joseph

2012-10-01

This research was undertaken in order to investigate the inhibitory potential of the Jamaican ball moss, Tillandsia recurvata against several kinases. The inhibition of these kinases has emerged as a potential solution to restoring the tight regulation of normal cellular growth, the loss of which leads to cancer cell formation. Kinase inhibition was investigated using competition binding (to the ATP sites) assays, which have been previously established and authenticated. Four hundred and fifty one kinases were tested against the Jamaican ball moss extract and a dose-response was tested on 40 kinases, which were inhibited by more than 35% compared to the control. Out of the 40 kinases, the Jamaican ball moss selectively inhibited 5 (CSNK2A2, MEK5, GAK, FLT and DRAK1) and obtained Kd(50)s were below 20 μg/ml. Since MEK5 and GAK kinases have been associated with aggressive prostate cancer, the inhibitory properties of the ball moss against them, coupled with its previously found bioactivity towards the PC-3 cell line, makes it promising in the arena of drug discovery towards prostate cancer.

Fibronectin phosphorylation by ecto-protein kinase

International Nuclear Information System (INIS)

Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru

1988-01-01

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 [γ- 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 [γ- 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

Protein kinase activity associated with the corticosteroid binder IB

International Nuclear Information System (INIS)

Vujicic, M.; Djordjevic-Markovic, R.; Radic, O.; Krstic, M.; Kanazir, D.

1997-01-01

The physiological effects elicited by glucocorticoids are mediated via glucocorticoid receptors (GR). Analysis of specific glucocorticoid binding to radioactively labelled [ 3 H] triamcinolone acetonide in rat liver cytosol and analysis by ion exchange chromatography have revealed the presence of two distinct molecular species. The major form, designated as binder II appears to correspond to the well characterized glucocorticoid receptor by virtue of its size, charge, steroid binding characteristics and ability to bind to DNA.The second form, designated as corticosteroid binder IB, is a minor binding component in the liver. The binder IB differs from the binder II receptor by virtue of its lower molecular weight and its elution in the pre gradient of DEAE-Sephadex A-50 column which retains the un activated binder II receptor complexes. We examined the kinase activity of partially purified corticosteroid binder IB. Using (γ 3 2 P) ATP we detected kinase activity associated with the IB fraction from the rat liver. This kinase phosphorylate mixed histones and and dose not phosphorylate IB protein in vitro. The kinase activity is completely inhibited by the addition of Mg 2 + ions and is partially inhibited by the addition of Ca 2 +ions. (author)

DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

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Adam J L Cook

2007-04-01

Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

DEFF Research Database (Denmark)

Massip, L; Garand, C; Labbé, A

2010-01-01

show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1......), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease...... activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast...

Insulin-induced decrease in protein phosphorylation in rat adipocytes not explained by decreased A-kinase activity

International Nuclear Information System (INIS)

Egan, J.J.; Greenberg, A.S.; Chang, M.K.; Londos, C.

1987-01-01

In isolated rat adipocytes, insulin inhibits lipolysis to a greater extent than would be predicted by the decrease in (-/+)cAMP activity ratio of cAMP-dependent protein kinase [A-kinase], from which it was speculated that insulin promotes the dephosphorylation of hormone-sensitive lipase. They have examined the phosphorylation state of cellular proteins under conditions of varying A-kinase activities in the presence and absence of insulin. Protein phosphorylation was determined by SDS-PAGE electrophoresis of extracts from 32 P-loaded cells; glycerol and A-kinase activity ratios were measured in the cytosolic extracts from control, non-radioactive cells. Increased protein phosphorylation in general occurred over the same range of A-kinase activity ratios, 0.1-0.3, associated with increased glycerol release. The insulin-induced decrease in lipolysis was associated with a decrease in the 32 P content of several proteins, an effect not explained by the modest reduction in A-kinase activity by insulin. This effect of insulin on protein phosphorylation was lost as the A-kinase activity ratios exceeded 0.5. The results suggest that insulin promotes the dephosphorylation of those adipocyte proteins which are subject to phosphorylation by A-kinase

A systematic evaluation of protein kinase a-a-kinase anchoring protein interaction motifs

NARCIS (Netherlands)

Burgers, Pepijn P|info:eu-repo/dai/nl/341566551; van der Heyden, Marcel A G; Kok, Bart; Heck, Albert J R|info:eu-repo/dai/nl/105189332; Scholten, Arjen|info:eu-repo/dai/nl/313939780

2015-01-01

Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs

NARCIS (Netherlands)

Burgers, Pepijn P; van der Heyden, MAG; Kok, Bart; Heck, Albert J R; Scholten, Arjen

2015-01-01

Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present

Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions.

Science.gov (United States)

Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

2016-01-01

Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+), or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+) but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+) than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+). Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+) entry in sperm through the Ca(2+)/CaM/CaMKKs/CaMKI pathway. The Ca(2+)/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2+) entry

Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs Effects on AMP-Activated Protein Kinase (AMPK Regulation of Chicken Sperm Functions.

Directory of Open Access Journals (Sweden)

Thi Mong Diep Nguyen

Full Text Available Sperm require high levels of energy to ensure motility and acrosome reaction (AR accomplishment. The AMP-activated protein kinase (AMPK has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+/calmodulin-dependent protein kinase kinases (CaMKKs mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+, or of CaMKKs inhibitor (STO-609. Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β, CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+. Our results show for the first time the presence of CaMKKs (α and β and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+ entry in sperm through the Ca(2+/CaM/CaMKKs/CaMKI pathway. The Ca(2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2

Prostate Cancer Cell Growth: Stimulatory Role of Neurotensin and Mechanism of Inhibition by Flavonoids as Related to Protein Kinase C

Science.gov (United States)

2010-01-01

cell lines (NCI-N417, NCI-H345, NCI-N592) were found to convert exogenous NT into the fragments NT1 –8 and NT9–13, reflecting the presence of...secrete NT. However, exogenous NT was degraded primarily to NT1 –11, consistent with the presence of neutral endopeptidase 3.4.24.11 in these cells . This...TITLE: Prostate Cancer Cell Growth: Stimulatory Role of Neurotensin and Mechanism of Inhibition by Flavonoids as Related to Protein Kinase C

Chitin and stress induced protein kinase activation

DEFF Research Database (Denmark)

Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

2017-01-01

The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

Receptor-interacting protein (RIP) kinase family

OpenAIRE

Zhang, Duanwu; Lin, Juan; Han, Jiahuai

2010-01-01

Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, incl...

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

International Nuclear Information System (INIS)

Crowe, David L; Ohannessian, Arthur

2004-01-01

Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines. Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

Directory of Open Access Journals (Sweden)

Ohannessian Arthur

2004-05-01

Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

Science.gov (United States)

Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

1999-03-05

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

A rice kinase-protein interaction map.

Science.gov (United States)

Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E; Ronald, Pamela C; Song, Wen-Yuan

2009-03-01

Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

Radioimmunoassay of bovine heart protein kinase

International Nuclear Information System (INIS)

Fleischer, N.; Rosen, O.M.; Reichlin, M.

1976-01-01

Immunization of guinea pigs with bovine cardiac cAMP-dependent protein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) resulted in the development of precipitating antibodies to the cAMP-binding subunit of the enzyme. Both the phosphorylated and nonphosphorylated cAMP-binding protein of the protein kinase reacted with the antiserum. A radioimmunoassay was developed that detects 10 ng of holoenzyme and permits measurement of enzyme concentrations in bovine cardiac muscle. Bovine liver, kidney, brain, and skeletal muscle contain protein kinases which are immunologically identical to those found in bovine cardiac muscle. However, the proportion of immunoreactive enzyme activity differed for each tissue. All of the immunologically nonreactive enzyme in skeletal muscle and heart was separable from immunoreactive enzyme by chromatography on DEAE-cellulose. Rat tissues and pig heart contained protein kinase activity that cross reacted immunologically in a nonparallel fashion with bovine cardiac enzyme. These results indicate that cAMP-dependent protein kinases within and between species are immunologically heterogeneous

Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

Science.gov (United States)

Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

2009-02-01

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD) or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR)-mediated eukaryotic initiation factor (eIF)2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.

Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

International Nuclear Information System (INIS)

Witters, L.A.; Bacon, G.W.

1985-01-01

The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32 P-ACC phosphorylated by the casein kinases was identified

HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients

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Zhaobo Li

2017-06-01

Full Text Available Inhibition of the ATPase cycle of the HSP90 chaperone promotes ubiquitylation and proteasomal degradation of its client proteins, which include many oncogenic protein kinases. This provides the rationale for HSP90 inhibitors as cancer therapeutics. However, the mechanism by which HSP90 ATPase inhibition triggers ubiquitylation is not understood, and the E3 ubiquitin ligases involved are largely unknown. Using a siRNA screen, we have identified components of two independent degradation pathways for the HSP90 client kinase CRAF. The first requires CUL5, Elongin B, and Elongin C, while the second requires the E3 ligase HECTD3, which is also involved in the degradation of MASTL and LKB1. HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain, which is mutationally disrupted in tumor cells with activated MAP kinase signaling. Our data implicate HECTD3 as a tumor suppressor modulating the activity of this important oncogenic signaling pathway.

dependent/calmodulin- stimulated protein kinase from moss

Indian Academy of Sciences (India)

Unknown

stimulated protein kinase; CDPK, calmodulin domain-like protein kinase; KM14, 14 amino acid synthetic peptide; .... used were obtained from Sigma Chemical Company, USA, ..... Plant chimeric Ca2+/Calmodulin-dependent protein kinase.

Targeting protein kinases to reverse multidrug resistance in sarcoma.

Science.gov (United States)

Chen, Hua; Shen, Jacson; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

2016-02-01

Sarcomas are a group of cancers that arise from transformed cells of mesenchymal origin. They can be classified into over 50 subtypes, accounting for approximately 1% of adult and 15% of pediatric cancers. Wide surgical resection, radiotherapy, and chemotherapy are the most common treatments for the majority of sarcomas. Among these therapies, chemotherapy can palliate symptoms and prolong life for some sarcoma patients. However, sarcoma cells can have intrinsic or acquired resistance after treatment with chemotherapeutics drugs, leading to the development of multidrug resistance (MDR). MDR attenuates the efficacy of anticancer drugs and results in treatment failure for sarcomas. Therefore, overcoming MDR is an unmet need for sarcoma therapy. Certain protein kinases demonstrate aberrant expression and/or activity in sarcoma cells, which have been found to be involved in the regulation of sarcoma cell progression, such as cell cycle, apoptosis, and survival. Inhibiting these protein kinases may not only decrease the proliferation and growth of sarcoma cells, but also reverse their resistance to chemotherapeutic drugs to subsequently reduce the doses of anticancer drugs and decrease drug side-effects. The discovery of novel strategies targeting protein kinases opens a door to a new area of sarcoma research and provides insight into the mechanisms of MDR in chemotherapy. This review will focus on the recent studies in targeting protein kinase to reverse chemotherapeutic drug resistance in sarcoma. Copyright © 2015 Elsevier Ltd. All rights reserved.

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

Science.gov (United States)

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

Science.gov (United States)

Catarsi, S; Drapeau, P

1997-08-01

Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

Uncaria rhynchophylla inhibits the production of nitric oxide and interleukin-1β through blocking nuclear factor κB, Akt, and mitogen-activated protein kinase activation in macrophages.

Science.gov (United States)

Kim, Ji-Hee; Bae, Chang Hwan; Park, Sun Young; Lee, Sang Joon; Kim, YoungHee

2010-10-01

The stems with hook of Uncaria rhynchophylla have been used in traditional medicine as an antipyretic, antihypertensive, and anticonvulsant in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of U. rhynchophylla in RAW 264.7 macrophages. The aqueous extract of U. rhynchophylla inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and interleukin (IL)-1β secretion as well as inducible NO synthase (iNOS) expression, without affecting cell viability. Furthermore, U. rhynchophylla suppressed LPS-induced nuclear factor κB (NF-κB) activation, phosphorylation, and degradation of inhibitory protein IκB (IκB)-α, phosphorylation of Akt, extracellular signal-regulated kinase 1/2, p38 kinase, and c-Jun N-terminal kinase. These results suggest that U. rhynchophylla has the inhibitory effects on LPS-induced NO and IL-1β production in macrophages through blockade in the phosphorylation of Akt and mitogen-activated protein kinases, following IκB-α degradation and NF-κB activation.

Toscana virus NSs protein promotes degradation of double-stranded RNA-dependent protein kinase.

Science.gov (United States)

Kalveram, Birte; Ikegami, Tetsuro

2013-04-01

Toscana virus (TOSV), which is transmitted by Phlebotomus spp. sandflies, is a major etiologic agent of aseptic meningitis and encephalitis in the Mediterranean. Like other members of the genus Phlebovirus of the family Bunyaviridae, TOSV encodes a nonstructural protein (NSs) in its small RNA segment. Although the NSs of Rift Valley fever virus (RVFV) has been identified as an important virulence factor, which suppresses host general transcription, inhibits transcription from the beta interferon promoter, and promotes the proteasomal degradation of double-stranded RNA-dependent protein kinase (PKR), little is known about the functions of NSs proteins encoded by less-pathogenic members of this genus. In this study we report that TOSV is able to downregulate PKR with similar efficiency as RVFV, while infection with the other phleboviruses-i.e., Punta Toro virus, sandfly fever Sicilian virus, or Frijoles virus-has no effect on cellular PKR levels. In contrast to RVFV, however, cellular transcription remains unaffected during TOSV infection. TOSV NSs protein promotes the proteasome-dependent downregulation of PKR and is able to interact with kinase-inactive PKR in infected cells.

Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

International Nuclear Information System (INIS)

Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

1986-01-01

A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn 2+ and [γ- 32 P]ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes

Opportunities to Target Specific Contractile Abnormalities with Smooth Muscle Protein Kinase Inhibitors

Directory of Open Access Journals (Sweden)

Annegret Ulke-Lemée

2010-05-01

Full Text Available Smooth muscle is a major component of most hollow organ systems (e.g., airways, vasculature, bladder and gut/gastrointestine; therefore, the coordinated regulation of contraction is a key property of smooth muscle. When smooth muscle functions normally, it contributes to general health and wellness, but its dysfunction is associated with morbidity and mortality. Rho-associated protein kinase (ROCK is central to calcium-independent, actomyosin-mediated contractile force generation in the vasculature, thereby playing a role in smooth muscle contraction, cell motility and adhesion. Recent evidence supports an important role for ROCK in the increased vasoconstriction and remodeling observed in various models of hypertension. This review will provide a commentary on the development of specific ROCK inhibitors and their clinical application. Fasudil will be discussed as an example of bench-to-bedside development of a clinical therapeutic that is used to treat conditions of vascular hypercontractility. Due to the wide spectrum of biological processes regulated by ROCK, many additional clinical indications might also benefit from ROCK inhibition. Apart from the importance of ROCK in smooth muscle contraction, a variety of other protein kinases are known to play similar roles in regulating contractile force. The zipper-interacting protein kinase (ZIPK and integrin-linked kinase (ILK are two well-described regulators of contraction. The relative contribution of each kinase to contraction depends on the muscle bed as well as hormonal and neuronal stimulation. Unfortunately, specific inhibitors for ZIPK and ILK are still in the development phase, but the success of fasudil suggests that inhibitors for these other kinases may also have valuable clinical applications. Notably, the directed inhibition of ZIPK with a pseudosubstrate molecule shows unexpected effects on the contractility of gastrointestinal smooth muscle.

Protein kinase D stabilizes aldosterone-induced ERK1/2 MAP kinase activation in M1 renal cortical collecting duct cells to promote cell proliferation.

LENUS (Irish Health Repository)

McEneaney, Victoria

2010-01-01

Aldosterone elicits transcriptional responses in target tissues and also rapidly stimulates the activation of protein kinase signalling cascades independently of de novo protein synthesis. Here we investigated aldosterone-induced cell proliferation and extra-cellular regulated kinase 1 and 2 (ERK1\\/2) mitogen activated protein (MAP) kinase signalling in the M1 cortical collecting duct cell line (M1-CCD). Aldosterone promoted the proliferative growth of M1-CCD cells, an effect that was protein kinase D1 (PKD1), PKCdelta and ERK1\\/2-dependent. Aldosterone induced the rapid activation of ERK1\\/2 with peaks of activation at 2 and 10 to 30 min after hormone treatment followed by sustained activation lasting beyond 120 min. M1-CCD cells suppressed in PKD1 expression exhibited only the early, transient peaks in ERK1\\/2 activation without the sustained phase. Aldosterone stimulated the physical association of PKD1 with ERK1\\/2 within 2 min of treatment. The mineralocorticoid receptor (MR) antagonist RU28318 inhibited the early and late phases of aldosterone-induced ERK1\\/2 activation, and also aldosterone-induced proliferative cell growth. Aldosterone induced the sub-cellular redistribution of ERK1\\/2 to the nuclei at 2 min and to cytoplasmic sites, proximal to the nuclei after 30 min. This sub-cellular distribution of ERK1\\/2 was inhibited in cells suppressed in the expression of PKD1.

Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

Directory of Open Access Journals (Sweden)

Tetsuro Ikegami

2009-02-01

Full Text Available Rift Valley fever virus (RVFV (genus Phlebovirus, family Bunyaviridae is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR-mediated eukaryotic initiation factor (eIF2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.

Inhibition of PKA anchoring to A-kinase anchoring proteins impairs consolidation and facilitates extinction of contextual fear memories

NARCIS (Netherlands)

Nijholt, Ingrid M.; Ostroveanu, Anghelus; Scheper, Wouter A.; Penke, Botond; Luiten, Paul G. M.; Van der Zee, Eddy A.; Eisel, Ulrich L. M.

Both genetic and pharmacological studies demonstrated that contextual fear conditioning is critically regulated by cyclic AMP-dependent protein kinase (PKA). Since PKA is a broad range protein kinase, a mechanism for confining its activity is required. It has been shown that intracellular spatial

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

Science.gov (United States)

Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

2016-07-29

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

Science.gov (United States)

Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

1997-09-01

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

Protein kinase and phosphatase activities of thylakoid membranes

International Nuclear Information System (INIS)

Michel, H.; Shaw, E.K.; Bennett, J.

1987-01-01

Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg 2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg 2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

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Mutsuki Amano

Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.

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Dikic, I; Tokiwa, G; Lev, S; Courtneidge, S A; Schlessinger, J

1996-10-10

The mechanisms by which mitogenic G-protein-coupled receptors activate the MAP kinase signalling pathway are poorly understood. Candidate protein tyrosine kinases that link G-protein-coupled receptors with MAP kinase include Src family kinases, the epidermal growth factor receptor, Lyn and Syk. Here we show that lysophosphatidic acid (LPA) and bradykinin induce tyrosine phosphorylation of Pyk2 and complex formation between Pyk2 and activated Src. Moreover, tyrosine phosphorylation of Pyk2 leads to binding of the SH2 domain of Src to tyrosine 402 of Pyk2 and activation of Src. Transient overexpression of a dominant interfering mutant of Pyk2 or the protein tyrosine kinase Csk reduces LPA- or bradykinin-induced activation of MAP kinase. LPA- or bradykinin-induced MAP kinase activation was also inhibited by overexpression of dominant interfering mutants of Grb2 and Sos. We propose that Pyk2 acts with Src to link Gi- and Gq-coupled receptors with Grb2 and Sos to activate the MAP kinase signalling pathway in PC12 cells.

Receptor-interacting protein (RIP) kinase family

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Zhang, Duanwu; Lin, Juan; Han, Jiahuai

2010-01-01

Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research. PMID:20383176

Indomethacin promotes apoptosis in gastric cancer cells through concomitant degradation of Survivin and Aurora B kinase proteins.

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Chiou, Shiun-Kwei; Hoa, Neil; Hodges, Amy; Ge, Lishen; Jadus, Martin R

2014-09-01

Regular usage of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with reduced incidence of a variety of cancers. The molecular mechanisms underlying these chemopreventive effects remain poorly understood. This current investigation showed that in gastric cancer cells: (1) Indomethacin treatment enhanced the degradation of chromosomal passenger proteins, Survivin and Aurora B kinase; (2) Indomethacin treatment down-regulated Aurora B kinase activity in a cell cycle-independent fashion; (3) siRNA knockdown of Survivin level promoted Aurora B kinase protein degradation, and vice versa; (4) ectopic overexpression of Survivin blocked reduction of Aurora B kinase level and activity by indomethacin treatment, and vice versa; (5) siRNA knockdown of Aurora B kinase level and AZD1152 inhibition of its activity induced apoptosis, and overexpression of Aurora B kinase inhibited indomethacin-induced apoptosis; (6) indomethacin treatment reduced Aurora B kinase level, coinciding with reduction of Survivin level and induction of apoptosis, in KATO III and HT-29 cells, and in mouse gastric mucosa. A role for Aurora B kinase function in NSAID-induced apoptosis was not previously explored. Thus this report provides better understanding of the molecular mechanisms underlying the anti-cancer effect of NSAIDs by elucidating a significant role for Aurora B kinase in indomethacin-induced apoptosis.

A framework for classification of prokaryotic protein kinases.

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Nidhi Tyagi

Full Text Available BACKGROUND: Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. METHODOLOGY/PRINCIPAL FINDINGS: We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. CONCLUSION/SIGNIFICANCE: Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular

Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

Energy Technology Data Exchange (ETDEWEB)

Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond (Toronto); (WU-MED)

2010-09-21

Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

International Nuclear Information System (INIS)

Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

2008-01-01

We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK)ζ, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGKζ siRNA transfection decreased H 2 O 2 -induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGKζ also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGKζ rapidly translocated to the cytoplasm following H 2 O 2 treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGKζ, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

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Inger Lindin

2014-03-01

Full Text Available The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD simulations of: (1 MK5 alone; (2 MK5 in complex with an inhibitor; and (3 MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

The Kinase STK3 Interacts with the Viral Structural Protein VP1 and Inhibits Foot-and-Mouth Disease Virus Replication

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Xue, Qiao

2017-01-01

Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects domestic and wild cloven-hoofed animals. The structural protein VP1 plays an important role in FMDV pathogenesis. However, the interacting partners of VP1 in host cells and the effects of these interactions in FMDV replication remain incompletely elucidated. Here, we identified a porcine cell protein, serine/threonine kinase 3 (STK3), which interacts with FMDV VP1 using the yeast two-hybrid system. The VP1-STK3 interaction was further confirmed by coimmunoprecipitation experiments in human embryonic kidney 293T and porcine kidney 15 (PK-15) cells. The carboxyl-terminal region (amino acids 180–214) of VP1 was essential for its interaction with STK3. The effects of overexpression and underexpressing of STK3 in PK-15 cells were assessed, and the results indicated that STK3 significantly inhibited FMDV replication. Our data expand the role of STK3 during viral infection, provide new information regarding the host cell kinases that are involved in viral replication, and identify potential targets for future antiviral strategies. PMID:29226127

TRESK background K(+ channel is inhibited by PAR-1/MARK microtubule affinity-regulating kinases in Xenopus oocytes.

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Gabriella Braun

Full Text Available TRESK (TWIK-related spinal cord K(+ channel, KCNK18 is a major background K(+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K(+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K(+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279 in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel.

Identification and Characterization of Amlexanox as a G Protein-Coupled Receptor Kinase 5 Inhibitor

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Kristoff T. Homan

2014-10-01

Full Text Available G protein-coupled receptor kinases (GRKs have been implicated in human diseases ranging from heart failure to diabetes. Previous studies have identified several compounds that selectively inhibit GRK2, such as paroxetine and balanol. Far fewer selective inhibitors have been reported for GRK5, a target for the treatment of cardiac hypertrophy, and the mechanism of action of reported compounds is unknown. To identify novel scaffolds that selectively inhibit GRK5, a differential scanning fluorometry screen was used to probe a library of 4480 compounds. The best hit was amlexanox, an FDA-approved anti-inflammatory, anti-allergic immunomodulator. The crystal structure of amlexanox in complex with GRK1 demonstrates that its tricyclic aromatic ring system forms ATP-like interactions with the hinge of the kinase domain, which is likely similar to how this drug binds to IκB kinase ε (IKKε, another kinase known to be inhibited by this compound. Amlexanox was also able to inhibit myocyte enhancer factor 2 transcriptional activity in neonatal rat ventricular myocytes in a manner consistent with GRK5 inhibition. The GRK1 amlexanox structure thus serves as a springboard for the rational design of inhibitors with improved potency and selectivity for GRK5 and IKKε.

Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

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Matej Zábrady

Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

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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

2009-03-01

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.

Involvement of protein kinase Mζ in the maintenance of long-term memory for taste aversion learning in young chicks.

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Tiunova, A A; Bezryadnov, D V; Anokhin, K V

2015-03-01

The effects of an inhibitor of protein kinase Mζ on long-term memory were studied using the model of taste aversion in newborn chicks. Memory was impaired by intracerebral injection of 10 or 20 nmol of ζ-inhibiting peptide 24 h after training. Memory impairment was found 2 h after peptide administration, and repeated examination 24 h after treatment showed no recovery. Memory impairment was not observed 24 h after inhibitor administration if the testing 2 h after treatment was not performed. The results indicate the contribution of protein kinase Mζ in the maintenance of long-term memory in the avian brain. These data confirm the hypothesis of several authors that inhibition of protein kinase Mζ does not abolish memory, but rather interacts with processes of memory retrieval and/or reconsolidation.

Insulin receptors mediate growth effects in cultured fetal neurons. II. Activation of a protein kinase that phosphorylates ribosomal protein S6

International Nuclear Information System (INIS)

Heidenreich, K.A.; Toledo, S.P.

1989-01-01

As an initial attempt to identify early steps in insulin action that may be involved in the growth responses of neurons to insulin, we investigated whether insulin receptor activation increases the phosphorylation of ribosomal protein S6 in cultured fetal neurons and whether activation of a protein kinase is involved in this process. When neurons were incubated for 2 h with 32Pi, the addition of insulin (100 ng/ml) for the final 30 min increased the incorporation of 32Pi into a 32K microsomal protein. The incorporation of 32Pi into the majority of other neuronal proteins was unaltered by the 30-min exposure to insulin. Cytosolic extracts from insulin-treated neurons incubated in the presence of exogenous rat liver 40S ribosomes and [gamma-32P]ATP displayed a 3- to 8-fold increase in the phosphorylation of ribosomal protein S6 compared to extracts from untreated cells. Inclusion of cycloheximide during exposure of the neurons to insulin did not inhibit the increased cytosolic kinase activity. Activation of S6 kinase activity by insulin was dose dependent (seen at insulin concentration as low as 0.1 ng/ml) and reached a maximum after 20 min of incubation. Addition of phosphatidylserine, diolein, and Ca2+ to the in vitro kinase reaction had no effect on the phosphorylation of ribosomal protein S6. Likewise, treatment of neurons with (Bu)2cAMP did not alter the phosphorylation of ribosomal protein S6 by neuronal cytosolic extracts. We conclude that insulin activates a cytosolic protein kinase that phosphorylates ribosomal S6 in neurons and is distinct from protein kinase-C and cAMP-dependent protein kinase. Stimulation of this kinase may play a role in insulin signal transduction in neurons

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones.

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Crossthwaite, Andrew J; Valli, Haseeb; Williams, Robert J

2004-03-01

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.

Inhibition of protein kinase A activity interferes with long-term, but not short-term, memory of conditioned taste aversions.

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Koh, Ming Teng; Thiele, Todd E; Bernstein, Ilene L

2002-12-01

The present experiments examined whether inhibition of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activity interferes with conditioned taste aversion (CTA) memories. Rats were centrally infused with the selective PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) before conditioning. Direct infusions of Rp-cAMPS into the amygdala showed no interference with short-term memory but did show significant attenuation of long-term memory and more rapid extinction. Results suggest that PKA activity is involved in the consolidation of long-term memory of CTAs, and that the amygdala may be 1 site that is important for this activity.

A phosphoinositide 3-kinase (PI3K)-serum- and glucocorticoid-inducible kinase 1 (SGK1) pathway promotes Kv7.1 channel surface expression by inhibiting Nedd4-2 protein

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Andersen, Martin Nybo; Krzystanek, Katarzyna; Petersen, Frederic

2013-01-01

Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium...... channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results...... as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant...

Insulin resistance enhances the mitogen-activated protein kinase signaling pathway in ovarian granulosa cells.

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Linghui Kong

Full Text Available The ovary is the main regulator of female fertility. Granulosa cell dysfunction may be involved in various reproductive endocrine disorders. Here we investigated the effect of insulin resistance on the metabolism and function of ovarian granulosa cells, and dissected the functional status of the mitogen-activated protein kinase signaling pathway in these cells. Our data showed that dexamethasone-induced insulin resistance in mouse granulosa cells reduced insulin sensitivity, accompanied with an increase in phosphorylation of p44/42 mitogen-activated protein kinase. Furthermore, up-regulation of cytochrome P450 subfamily 17 and testosterone and down-regulation of progesterone were observed in insulin-resistant mouse granulosa cells. Inhibition of p44/42 mitogen-activated protein kinase after induction of insulin resistance in mouse granulosa cells decreased phosphorylation of p44/42 mitogen-activated protein kinase, downregulated cytochrome P450 subfamily 17 and lowered progesterone production. This insulin resistance cell model can successfully demonstrate certain mechanisms such as hyperandrogenism, which may inspire a new strategy for treating reproductive endocrine disorders by regulating cell signaling pathways.

Redundant role of protein kinase C delta and epsilon during mouse embryonic development.

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Sergio Carracedo

Full Text Available Protein Kinase C delta and epsilon are mediators of important cellular events, such as cell proliferation, migration or apoptosis. The formation of blood vessels, i.e., vasculo- and angiogenesis, is a process where these isoforms have also been shown to participate. However, mice deficient in either Protein Kinase C delta or epsilon are viable and therefore their individual contribution to the formation of the vasculature appeared so far dispensable. In this study, we show that double null mutation of Protein Kinase C delta and epsilon causes embryonic lethality at approximately E9.5. At this stage, whole mount staining of the endothelial marker CD31 in double null embryos revealed defective blood vessel formation. Moreover, culture of double deficient mouse allantois showed impaired endothelial cell organization, and analyses of double deficient embryo sections showed dilated vessels, decreased endothelial-specific adherent junctions, and decreased contact of endothelial cells with mural cells. Protein kinase C delta and epsilon also appeared essential for vascular smooth muscle cell differentiation, since α-smooth muscle actin, a classical marker for vascular smooth muscle cells, was almost undetectable in double deficient embryonic aorta at E9.5. Subsequent qPCR analyses showed decreased VE-cadherin, Vegfr2, Cd31, Cdh2, Ets1, and Fli-1, among other angiogenesis related transcripts in double deficient embryos. Taken together, these data suggest for the first time an in vivo redundant role between members of the novel Protein Kinase C subfamily that allows for mutual compensation during mouse embryonic development, with vasculogenesis/angiogenesis as an obvious common function of these two Protein Kinase Cs. Protein Kinase C delta and epsilon might therefore be useful targets for inhibiting vasculo- and/or angiogenesis.

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

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Daniel Thomas

Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

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Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

2016-12-01

Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

Targeting the SH2-kinase interface in Bcr-Abl inhibits leukemogenesis.

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Grebien, Florian; Hantschel, Oliver; Wojcik, John; Kaupe, Ines; Kovacic, Boris; Wyrzucki, Arkadiusz M; Gish, Gerald D; Cerny-Reiterer, Sabine; Koide, Akiko; Beug, Hartmut; Pawson, Tony; Valent, Peter; Koide, Shohei; Superti-Furga, Giulio

2011-10-14

Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention. Copyright © 2011 Elsevier Inc. All rights reserved.

Partial purification and characterization of a Ca(2+)-dependent protein kinase from pea nuclei

Science.gov (United States)

Li, H.; Dauwalder, M.; Roux, S. J.

1991-01-01

Almost all the Ca(2+)-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca2+ and reaches half-maximal activation at about 3 x 10(-7) molar free Ca2+, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca(2+)-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca(2+)-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.

Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

DEFF Research Database (Denmark)

Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

2003-01-01

and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...

Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

Science.gov (United States)

Li, H.; Roux, S. J.

1992-01-01

A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

Inhibition of Cholesterol Synthesis in HepG2 Cells by GINST-Decreasing HMG-CoA Reductase Expression Via AMP-Activated Protein Kinase.

Science.gov (United States)

Han, Joon-Seung; Sung, Jong Hwan; Lee, Seung Kwon

2017-11-01

GINST, a hydrolyzed ginseng extract, has been reported to have antidiabetic effects and to reduce hyperglycemia and hyperlipidemia. Hypercholesterolemia is caused by diet or genetic factors and can lead to atherosclerosis and coronary heart disease. Thus, the purpose of this study is to determine whether GINST and the ginsenoside metabolite, IH-901 (compound K), reduce cholesterol synthesis in HepG2 cells and the signal transduction pathways involved. Concentrations of cholesterol were measured by using an enzymatic method. Expression levels of sterol regulatory element-binding protein 2 (SREBP2), HMG-CoA reductase (HMGCR), peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins α (C/EBPα), GAPDH, and phosphorylation of AMP-activated protein kinase α (AMPKα), protein kinase B (PKB, also known as Akt), and mechanistic target of rapamycin complex 1 (mTORC1) were measured using western blot. Total cholesterol concentration decreased after GINST treatment for 24 and 48 h. Expression of HMGCR decreased more with GINST than with the inhibitors, U18666A and atorvastatin, after 48 h in a dose-dependent manner. Phosphorylation of AMPKα increased 2.5x by GINST after 360 min of treatment, and phosphorylation of Akt decreased after 120 and 360 min. We separated compound K from GINST extracts flash chromatography. Compound K decreased cholesterol synthesis in HepG2 cells at 24 and 48 h. Therefore, we conclude that GINST inhibits cholesterol synthesis in HepG2 cells by decreasing HMGCR expression via AMPKα activation. GINST, a hydrolyzed ginseng extract, can inhibit cholesterol synthesis in liver cells via activation of AMPKα. IH-901 (compound K), which is the main component with bioactivity in GINST, also has anticholesterol effects. Thus, we suggest that GINST can be used to reduce hypercholesterolemia. © 2017 Institute of Food Technologists®.

Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A.

Science.gov (United States)

Canela, Núria; Orzáez, Mar; Fucho, Raquel; Mateo, Francesca; Gutierrez, Ricardo; Pineda-Lucena, Antonio; Bachs, Oriol; Pérez-Payá, Enrique

2006-11-24

The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

Resveratrol Inhibits Porcine Intestinal Glucose and Alanine Transport: Potential Roles of Na+/K+-ATPase Activity, Protein Kinase A, AMP-Activated Protein Kinase and the Association of Selected Nutrient Transport Proteins with Detergent Resistant Membranes

Directory of Open Access Journals (Sweden)

Stefanie Klinger

2018-03-01

Full Text Available Background: Beneficial effects of Resveratrol (RSV have been demonstrated, including effects on transporters and channels. However, little is known about how RSV influences intestinal transport. The aim of this study was to further characterize the effects of RSV on intestinal transport and the respective mechanisms. Methods: Porcine jejunum and ileum were incubated with RSV (300 µM, 30 min in Ussing chambers (functional studies and tissue bathes (detection of protein expression, phosphorylation, association with detergent resistant membranes (DRMs. Results: RSV reduced alanine and glucose-induced short circuit currents (ΔIsc and influenced forskolin-induced ΔIsc. The phosphorylation of sodium–glucose-linked transporter 1 (SGLT1, AMP-activated protein kinase (AMPK, protein kinase A substrates (PKA-S and liver kinase B1 (LKB1 increased but a causative relation to the inhibitory effects could not directly be established. The DRM association of SGLT1, peptide transporter 1 (PEPT1 and (phosphorylated Na+/H+-exchanger 3 (NHE3 did not change. Conclusion: RSV influences the intestinal transport of glucose, alanine and chloride and is likely to affect other transport processes. As the effects of protein kinase activation vary between the intestinal localizations, it would appear that increasing cyclic adenosine monophosphate (cAMP levels are part of the mechanism. Nonetheless, the physiological responses depend on cell type-specific structures.

Electrochemiluminescence resonance energy transfer between graphene quantum dots and graphene oxide for sensitive protein kinase activity and inhibitor sensing

Energy Technology Data Exchange (ETDEWEB)

Liang, Ru-Ping; Qiu, Wei-Bin; Zhao, Hui-Fang; Xiang, Cai-Yun; Qiu, Jian-Ding, E-mail: jdqiu@ncu.edu.cn

2016-01-21

Herein, a novel electrochemiluminescence resonance energy transfer (ECL-RET) biosensor using graphene quantum dots (GQDs) as donor and graphene oxide (GO) as acceptor for monitoring the activity of protein kinase was presented for the first time. Anti-phosphoserine antibody conjugated graphene oxide (Ab-GO) nonocomposite could be captured onto the phosphorylated peptide/GQDs modified electrode surface through antibody–antigen interaction in the presence of casein kinase II (CK2) and adenosine 5′-triphosphate (ATP), resulting in ECL from the GQDs quenching by closely contacting GO. This ECL quenching degree was positively correlated with CK2 activity. Therefore, on the basis of ECL-RET between GQDs and GO, the activity of protein kinase can be detected sensitively. This biosensor can also be used for quantitative analysis CK2 activity in serum samples and qualitative screening kinase inhibition, indicating the potential application of the developed method in biochemical fundamental research and clinical diagnosis. - Highlights: • We reported a novel ECL-RET biosensor for sensitive analysis of casein kinase II activity. • The successful ECL-RET between GQDs and GO could be established. • GQDs was employed for casein kinase II activity monitoring and inhibition assay. • Highly sensitive detection of CK2 activity and inhibition was achieved.

Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †

Science.gov (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

2009-01-01

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. PMID:19103740

Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases

Energy Technology Data Exchange (ETDEWEB)

Chen, Ying-Nan P.; LaMarche, Matthew J.; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G.; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G.; Dobson, Jason R.; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R.; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D.; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J.; Sellers, William R.; Stams, Travis; Fortin , Pascal D. (Novartis)

2016-06-29

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1, 2, 3, 4, 5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2, 3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6, 7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8, 9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Protein kinase CK2 in human diseases

DEFF Research Database (Denmark)

Guerra, Barbara; Issinger, Olaf-Georg

2008-01-01

Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. There are two paralog catalytic subunits, i.e. alpha (A1) and alpha' (A2). The alpha and alpha' subunits are linked to two beta subunits to produce a heterotetrameric structure...

Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer’s disease

Directory of Open Access Journals (Sweden)

Dhawan Gunjan

2012-07-01

Full Text Available Abstract Background Microglial activation is an important histologic characteristic of the pathology of Alzheimer’s disease (AD. One hypothesis is that amyloid beta (Aβ peptide serves as a specific stimulus for tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to disease. Therefore, inhibiting Aβ stimulation of microglia may prove to be an important therapeutic strategy for AD. Methods Primary murine microglia cultures and the murine microglia cell line, BV2, were used for stimulation with fibrillar Aβ1-42. The non-receptor tyrosine kinase inhibitor, dasatinib, was used to treat the cells to determine whether Src family kinase activity was required for the Aβ stimulated signaling response and subsequent increase in TNFα secretion using Western blot analysis and enzyme-linked immunosorbent assay (ELISA, respectively. A histologic longitudinal analysis was performed using an AD transgenic mouse model, APP/PS1, to determine an age at which microglial protein tyrosine kinase levels increased in order to administer dasatinib via mini osmotic pump diffusion. Effects of dasatinib administration on microglial and astroglial activation, protein phosphotyrosine levels, active Src kinase levels, Aβ plaque deposition, and spatial working memory were assessed via immunohistochemistry, Western blot, and T maze analysis. Results Aβ fibrils stimulated primary murine microglia via a tyrosine kinase pathway involving Src kinase that was attenuated by dasatinib. Dasatinib administration to APP/PS1 mice decreased protein phosphotyrosine, active Src, reactive microglia, and TNFα levels in the hippocampus and temporal cortex. The drug had no effect on GFAP levels, Aβ plaque load, or the related tyrosine kinase, Lyn. These anti-inflammatory changes correlated with improved performance on the T maze test in dasatinib infused animals compared to control animals. Conclusions These data suggest that amyloid

Early events elicited by Bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding

International Nuclear Information System (INIS)

Zachary, I.; Sinnett-Smith, J.W.; Rozengurt, E.

1986-01-01

Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an M/sub r/ 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent. The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125 I-labeled epidermal growth factor ( 125 I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca 2+ concentration does not mediate the bombesin inhibition of 125 I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125 I-EGF to its cellular receptor

An antisense oligodeoxynucleotide targeted against the type IIβ regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects

International Nuclear Information System (INIS)

Tortora, G.; Clair, T.; Cho-Chung, Y.S.

1990-01-01

The type II β regulatory subunit of cAMP-dependent protein kinase (RII β ) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII β antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII β antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII β protein. Exposure to RII β sense, RI α and RII α antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII β regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells

RGS16 inhibits breast cancer cell growth by mitigating phosphatidylinositol 3-kinase signaling.

Science.gov (United States)

Liang, Genqing; Bansal, Geetanjali; Xie, Zhihui; Druey, Kirk M

2009-08-07

Aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway supports growth of many tumors including those of breast, lung, and prostate. Resistance of breast cancer cells to targeted chemotherapies including tyrosine kinase inhibitors (TKI) has been linked to persistent PI3K activity, which may in part be due to increased membrane expression of epidermal growth factor (EGF) receptors (HER2 and HER3). Recently we found that proteins of the RGS (regulator of G protein signaling) family suppress PI3K activity downstream of the receptor by sequestering its p85alpha subunit from signaling complexes. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. RGS16 overexpression in MCF7 breast cancer cells inhibited EGF-induced proliferation and Akt phosphorylation, whereas shRNA-mediated extinction of RGS16 augmented cell growth and resistance to TKI treatment. Exposure to TKI also reduced RGS16 expression in MCF7 and BT474 cell lines. RGS16 bound the amino-terminal SH2 and inter-SH2 domains of p85alpha and inhibited its interaction with the EGF receptor-associated adapter protein Gab1. These results suggest that the loss of RGS16 in some breast tumors enhances PI3K signaling elicited by growth factors and thereby promotes proliferation and TKI evasion downstream of HER activation.

Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

KAUST Repository

Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inê s CR; Willige, Bjö rn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

2014-01-01

The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

KAUST Repository

Zourelidou, Melina

2014-06-19

The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

Simultaneous inhibition of aberrant cancer kinome using rationally designed polymer-protein core-shell nanomedicine.

Science.gov (United States)

Chandran, Parwathy; Gupta, Neha; Retnakumari, Archana Payickattu; Malarvizhi, Giridharan Loghanathan; Keechilat, Pavithran; Nair, Shantikumar; Koyakutty, Manzoor

2013-11-01

Simultaneous inhibition of deregulated cancer kinome using rationally designed nanomedicine is an advanced therapeutic approach. Herein, we have developed a polymer-protein core-shell nanomedicine to inhibit critically aberrant pro-survival kinases (mTOR, MAPK and STAT5) in primitive (CD34(+)/CD38(-)) Acute Myeloid Leukemia (AML) cells. The nanomedicine consists of poly-lactide-co-glycolide core (~250 nm) loaded with mTOR inhibitor, everolimus, and albumin shell (~25 nm thick) loaded with MAPK/STAT5 inhibitor, sorafenib and the whole construct was surface conjugated with monoclonal antibody against CD33 receptor overexpressed in AML. Electron microscopy confirmed formation of core-shell nanostructure (~290 nm) and flow cytometry and confocal studies showed enhanced cellular uptake of targeted nanomedicine. Simultaneous inhibition of critical kinases causing synergistic lethality against leukemic cells, without affecting healthy blood cells, was demonstrated using immunoblotting, cytotoxicity and apoptosis assays. This cell receptor plus multi-kinase targeted core-shell nanomedicine was found better specific and tolerable compared to current clinical regime of cytarabine and daunorubicin. These authors demonstrate simultaneous inhibition of critical kinases causing synergistic lethality against leukemic cells, without affecting healthy blood cells by using rationally designed polymer-protein core-shell nanomedicine, provoding an advanced method to eliminate cancer cells, with the hope of future therapeutic use. Copyright © 2013 Elsevier Inc. All rights reserved.

DNA-dependent protein kinase participates in the radiation activation of NF-kB

International Nuclear Information System (INIS)

Rosenzweig, Kenneth E.; Youmell, Matthew B.; Price, Brendan D.

1997-01-01

The NF-kB transcription factor is maintained in an inactive state by binding to the lkBa inhibitory protein. Activation requires phosphorylation and degradation of lkBa, releasing active NF-kB. NF-kB can be activated by cytokines, antigens, free radicals and X-ray irradiation. The protein kinase responsible for phosphorylation of lkBa in vivo has not been fully characterized. Here, we have examined the role of the DNA-dependent protein kinases (DNA-PK) in the radiation-activation of NF-kB. Wortmannin is an inhibitor of DNA-PK and related kinases. Exposure of SW480 cells to wortmannin inhibited the radioactivation of NF-kB DNA-binding. Analysis of lkBa levels by western blotting indicated that wortmannin blocked the radiation induced degradation of lkBa. In in vitro experiments, purified DNA-PK was able to efficiently phosphorylate lkBa, and this phosphorylation was inhibited by wortmannin. In contrast, the induction of NF-kB activity by TNFa was unaffected by wortmannin. The results suggest that DNA-PK may phosphorylate lkBa following irradiation, leading to degradation of lkBa and the release of active NF-kB. The inability of wortmannin to block TNFa activation of NF-kB indicates there may be more than one pathway for the activation of NF-kB

The multi-targeted kinase inhibitor sorafenib inhibits enterovirus 71 replication by regulating IRES-dependent translation of viral proteins.

Science.gov (United States)

Gao, Meng; Duan, Hao; Liu, Jing; Zhang, Hao; Wang, Xin; Zhu, Meng; Guo, Jitao; Zhao, Zhenlong; Meng, Lirong; Peng, Yihong

2014-06-01

The activation of ERK and p38 signal cascade in host cells has been demonstrated to be essential for picornavirus enterovirus 71 (EV71) replication and up-regulation of virus-induced cyclooxygenase-2 (COX-2)/prostaglandins E2 (PGE2) expression. The aim of this study was to examine the effects of sorafenib, a clinically approved anti-cancer multi-targeted kinase inhibitor, on the propagation and pathogenesis of EV71, with a view to its possible mechanism and potential use in the design of therapy regimes for Hand foot and mouth disease (HFMD) patients with life threatening neurological complications. In this study, non-toxic concentrations of sorafenib were shown to inhibit the yield of infectious progeny EV71 (clinical BC08 strain) by about 90% in three different cell types. A similar inhibitory effect of sorafenib was observed on the synthesis of both viral genomic RNA and the VP1 protein. Interestingly, sorafenib exerted obvious inhibition of the EV71 internal ribosomal entry site (IRES)-mediated translation, the first step in picornavirus replication, by linking it to a firefly luciferase reporter gene. Sorafenib was also able to prevent both EV71-induced CPE and the activation of ERK and p38, which contributes to up-regulation COX-2/PGE2 expression induced by the virus. Overall, this study shows that sorafenib strongly inhibits EV71 replication at least in part by regulating viral IRES-dependent translation of viral proteins, indicating a novel potential strategy for the treatment of HFMD patients with severe neurological complications. To our knowledge, this is the first report that investigates the mechanism by which sorafenib inhibits EV71 replication. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein kinase C regulates human pluripotent stem cell self-renewal.

Directory of Open Access Journals (Sweden)

Masaki Kinehara

Full Text Available The self-renewal of human pluripotent stem (hPS cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2 appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells.In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC, GF109203X (GFX, increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β, suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2 synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells.Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK, PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though h

Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal

Science.gov (United States)

Kinehara, Masaki; Kawamura, Suguru; Tateyama, Daiki; Suga, Mika; Matsumura, Hiroko; Mimura, Sumiyo; Hirayama, Noriko; Hirata, Mitsuhi; Uchio-Yamada, Kozue; Kohara, Arihiro; Yanagihara, Kana; Furue, Miho K.

2013-01-01

Background The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. Methodology/Principal Findings In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells. Conclusions/Significance Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long

Supplementation of chitosan alleviates high-fat diet-enhanced lipogenesis in rats via adenosine monophosphate (AMP)-activated protein kinase activation and inhibition of lipogenesis-associated genes.

Science.gov (United States)

Chiu, Chen-Yuan; Chan, Im-Lam; Yang, Tsung-Han; Liu, Shing-Hwa; Chiang, Meng-Tsan

2015-03-25

This study investigated the role of chitosan in lipogenesis in high-fat diet-induced obese rats. The lipogenesis-associated genes and their upstream regulatory proteins were explored. Diet supplementation of chitosan efficiently decreased the increased weights in body, livers, and adipose tissues in high-fat diet-fed rats. Chitosan supplementation significantly raised the lipolysis rate; attenuated the adipocyte hypertrophy, triglyceride accumulation, and lipoprotein lipase activity in epididymal adipose tissues; and decreased hepatic enzyme activities of lipid biosynthesis. Chitosan supplementation significantly activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and attenuated high-fat diet-induced protein expressions of lipogenic transcription factors (PPAR-γ and SREBP1c) in livers and adipose tissues. Moreover, chitosan supplementation significantly inhibited the expressions of downstream lipogenic genes (FAS, HMGCR, FATP1, and FABP4) in livers and adipose tissues of high-fat diet-fed rats. These results demonstrate for the first time that chitosan supplementation alleviates high-fat diet-enhanced lipogenesis in rats via AMPK activation and lipogenesis-associated gene inhibition.

Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

Science.gov (United States)

Morris, Dale L; O'Neil, Shawn P; Devraj, Rajesh V; Portanova, Joseph P; Gilles, Richard W; Gross, Cindy J; Curtiss, Sandra W; Komocsar, Wendy J; Garner, Debra S; Happa, Fernando A; Kraus, Lori J; Nikula, Kristen J; Monahan, Joseph B; Selness, Shaun R; Galluppi, Gerald R; Shevlin, Kimberly M; Kramer, Jeffrey A; Walker, John K; Messing, Dean M; Anderson, David R; Mourey, Robert J; Whiteley, Laurence O; Daniels, John S; Yang, Jerry Z; Rowlands, Philip C; Alden, Carl L; Davis, John W; Sagartz, John E

2010-06-01

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

International Nuclear Information System (INIS)

Chakraborty, Goutam; Saito, Mitsuo; Mao, Rui-Fen; Wang, Ray; Vadasz, Csaba; Saito, Mariko

2008-01-01

Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3β (GSK-3β), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3β, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3β, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways

Oryza sativa (Rice) Hull Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages by Suppressing Extracellular Signal-regulated Kinase, c-Jun N-terminal Kinase, and Nuclear Factor-κB Activation.

Science.gov (United States)

Ha, Sang Keun; Sung, Jeehye; Choi, Inwook; Kim, Yoonsook

2016-01-01

Rice ( Oryza sativa ) is a major cereal crop in many Asian countries and an important staple food source. Rice hulls have been reported to possess antioxidant activities. In this study, we evaluated the antiinflammatory effects of rice hull extract and associated signal transduction mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that rice hull extract inhibited nitric oxide (NO) and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively. The release of interleukin-1β and tumor necrosis factor-α was also reduced in a dose-dependent manner. Furthermore, rice hull extract attenuated the activation of nuclear factor-kappa B (NF-κB), as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in LPS-stimulated RAW264.7 cells. This suggests that rice hull extract decreases the production of inflammatory mediators by downregulating ERK and JNK and the NF-κB signal pathway in RAW 264.7 cells. Rice hull extract inhibits the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages.Rice hull extract inhibited nitric oxide and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively.Rice hull extract exerted anti-inflammatory effect through inhibition of nuclear factor-kappa B, extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways.Rice hull extract may provide a potential therapeutic approach for inflammatory diseases. Abbreviations used: COX-2: cyclooxygenase-2, ERK: extracellular signal-regulated kinase, IκB: inhibitory kappa B, IL-1β: interleukin-1β, iNOS: inducible NO synthase, JNK: c-Jun N-terminal kinase, LPS: lipopolysaccharide, MAPKs: mitogen-activated protein kinases, NF-κB: nuclear factor-κB, NO: nitric oxide, PGE2: prostaglandin E2, RHE: rice hull extract, ROS: reactive oxygen species

Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

Science.gov (United States)

Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

2016-12-21

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

5' adenosine monophosphate-activated protein kinase, metabolism and exercise.

Science.gov (United States)

Aschenbach, William G; Sakamoto, Kei; Goodyear, Laurie J

2004-01-01

The 5' adenosine monophosphate-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that functions as a metabolic 'fuel gauge' in skeletal muscle. AMPK is a ubiquitous heterotrimeric protein, consisting of an alpha catalytic, and beta and gamma regulatory subunits that exist in multiple isoforms and are all required for full enzymatic activity. During exercise, AMPK becomes activated in skeletal muscle in response to changes in cellular energy status (e.g. increased adenosine monophosphate [AMP]/adenosine triphosphate [ATP] and creatine/phosphocreatine ratios) in an intensity-dependent manner, and serves to inhibit ATP-consuming pathways, and activate pathways involved in carbohydrate and fatty-acid metabolism to restore ATP levels. Recent evidence shows that although AMPK plays this key metabolic role during acute bouts of exercise, it is also an important component of the adaptive response of skeletal muscles to endurance exercise training because of its ability to alter muscle fuel reserves and expression of several exercise-responsive genes. This review discusses the putative roles of AMPK in acute and chronic exercise responses, and suggests avenues for future AMPK research in exercise physiology and biochemistry.

Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

Energy Technology Data Exchange (ETDEWEB)

Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G. [Michigan; (Oxford)

2015-02-13

Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

Protein kinase CK2 in health and disease: Protein kinase CK2: from structures to insights

DEFF Research Database (Denmark)

Niefind, K; Raaf, J; Issinger, Olaf-Georg

2009-01-01

the critical region of CK2alpha recruitment is pre-formed in the unbound state. In CK2alpha the activation segment - a key element of protein kinase regulation - adapts invariably the typical conformation of the active enzymes. Recent structures of human CK2alpha revealed a surprising plasticity in the ATP......Within the last decade, 40 crystal structures corresponding to protein kinase CK2 (former name 'casein kinase 2'), to its catalytic subunit CK2alpha and to its regulatory subunit CK2beta were published. Together they provide a valuable, yet by far not complete basis to rationalize the biochemical...

The consequences of long-term glycogen synthase kinase-3 inhibition on normal and insulin resistant rat hearts.

Science.gov (United States)

Flepisi, T B; Lochner, Amanda; Huisamen, Barbara

2013-10-01

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase, discovered as a regulator of glycogen synthase. GSK-3 may regulate the expression of SERCA-2a potentially affecting myocardial contractility. It is known to phosphorylate and inhibit IRS-1, thus disrupting insulin signalling. This study aimed to determine whether myocardial GSK-3 protein and its substrate proteins are dysregulated in obesity and insulin resistance, and whether chronic GSK-3 inhibition can prevent or reverse this. Weight matched male Wistar rats were rendered obese by hyperphagia using a special diet (DIO) for 16 weeks and compared to chow fed controls. Half of each group was treated with the GSK-3 inhibitor CHIR118637 (30 mg/kg/day) from week 12 to16 of the diet period. Biometric and biochemical parameters were measured and protein expression determined by Western blotting and specific antibodies. Ca(2+)ATPase activity was determined spectrophotometrically. Cardiomyocytes were prepared by collagenase perfusion and insulin stimulated 2-deoxy-glucose uptake determined. DIO rats were significantly heavier than controls, associated with increased intra-peritoneal fat and insulin resistance. GSK-3 inhibition did not affect weight but improved insulin resistance, also on cellular level. It had no effect on GSK-3 expression but elevated its phospho/total ratio and elevated IRS-2 expression. Obesity lowered SERCA-2a expression and activity while GSK-3 inhibition alleviated this. The phospho/total ratio of phospholamban underscored inhibition of SERCA-2a in obesity. In addition, signs of myocardial hypertrophy were observed in treated control rats. GSK-3 inhibition could not reverse all the detrimental effects of obesity but may be harmful in normal rat hearts. It regulates IRS-2, SERCA-2a and phospholamban expression but not IRS-1.

Proteínas quinases: características estruturais e inibidores químicos Kinase protein: structural features and chemical inhibitors

Directory of Open Access Journals (Sweden)

Bárbara V. Silva

2009-01-01

Full Text Available Protein kinases are one of the largest protein families and they are responsible for regulation of a great number of signal transduction pathways in cells, through the phosphorylation of serine, threonine, or tyrosine residues. Deregulation of these enzymes is associated with several diseases including cancer, diabetes and inflammation. For this reason, specific inhibition of tyrosine or serine/threonine kinases may represent an interesting therapeutic approach. The most important types of protein kinases, their structural features and chemical inhibitors are discussed in this paper. Emphasis is given to the small-molecule drugs that target the ATP-binding sites of these enzymes.

Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation.

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Zhou, Haoming; Wang, Han; Ni, Ming; Yue, Shi; Xia, Yongxiang; Busuttil, Ronald W; Kupiec-Weglinski, Jerzy W; Lu, Ling; Wang, Xuehao; Zhai, Yuan

2018-02-13

Glycogen synthase kinase 3β (Gsk3β [Gsk3b]) is a ubiquitously expressed kinase with distinctive functions in different types of cells. Although its roles in regulating innate immune activation and ischaemia and reperfusion injuries (IRIs) have been well documented, the underlying mechanisms remain ambiguous, in part because of the lack of cell-specific tools in vivo. We created a myeloid-specific Gsk3b knockout (KO) strain to study the function of Gsk3β in macrophages in a murine liver partial warm ischaemia model. Compared with controls, myeloid Gsk3b KO mice were protected from IRI, with diminished proinflammatory but enhanced anti-inflammatory immune responses in livers. In bone marrow-derived macrophages, Gsk3β deficiency resulted in an early reduction of Tnf gene transcription but sustained increase of Il10 gene transcription on Toll-like receptor 4 stimulation in vitro. These effects were associated with enhanced AMP-activated protein kinase (AMPK) activation, which led to an accelerated and higher level of induction of the novel innate immune negative regulator small heterodimer partner (SHP [Nr0b2]). The regulatory function of Gsk3β on AMPK activation and SHP induction was confirmed in wild-type bone marrow-derived macrophages with a Gsk3 inhibitor. Furthermore, we found that this immune regulatory mechanism was independent of Gsk3β Ser9 phosphorylation and the phosphoinositide 3-kinase-Akt signalling pathway. In vivo, myeloid Gsk3β deficiency facilitated SHP upregulation by ischaemia-reperfusion in liver macrophages. Treatment of Gsk3b KO mice with either AMPK inhibitor or SHP small interfering RNA before the onset of liver ischaemia restored liver proinflammatory immune activation and IRI in these otherwise protected hosts. Additionally, pharmacological activation of AMPK protected wild-type mice from liver IRI, with reduced proinflammatory immune activation. Inhibition of the AMPK-SHP pathway by liver ischaemia was demonstrated in tumour resection

Complete inhibition of creatine kinase in isolated perfused rat hearts

International Nuclear Information System (INIS)

Fossel, E.T.; Hoefeler, H.

1987-01-01

Transient exposure of an isolated isovolumic perfused rat heart to low concentrations (0.5 mM) of perfusate-born iodoacetamide resulted in complete inhibition of creatine kinase and partial inhibition of glyceraldehyde-3-phosphate dehydrogenase in the heart. At low levels of developed pressure, hearts maintained mechanical function, ATP, and creatine phosphate levels at control values. However, iodoacetamide-inhibited hearts were unable to maintain control values of end diastolic pressure or peak systolic pressure as work load increased. Global ischemia resulted in loss of all ATP without loss of creatine phosphate, indicating lack of active creatine kinase. These results indicate that isovolumic perfused rat hearts are able to maintain normal function and normal levels of high-energy phosphates without active creatine kinase at low levels of developed pressure. 31 P-NMR of the heart was carried out

Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases.

Science.gov (United States)

Yoshizumi, Masanori; Abe, Jun-Ichi; Tsuchiya, Koichiro; Berk, Bradford C; Tamaki, Toshiaki

2003-03-01

Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent findings suggest a steady laminar blood flow decreases EC apoptosis and inhibits TNF-mediated EC activation. EC apoptosis or activation is suggested to be involved in plaque erosion, which may lead to platelet aggregation. TNF-alpha regulates gene expression in ECs, in part, by stimulating mitogen-activated protein (MAP) kinases, which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAP kinases in ECs. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm(2)) on TNF-alpha-stimulated activity of three MAP kinases in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. TNF-alpha activated ERK1/2, JNK, and p38 maximally at 15 min in HUVEC. Pre-exposing HUVEC for 10 min to flow inhibited TNF-alpha activation of JNK, but showed no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, a specific ERK1/2 inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Transfection studies with dominant-negative constructs of the protein kinase MEK5 suggested an important role for big mitogen-activated protein kinase 1 (BMK1) in flow-mediated regulation of EC activation by TNF-alpha. Understanding the mechanisms by which steady laminar flow regulates JNK activation by cytokines may provide insight into the atheroprotective mechanisms induced by laminar blood flow.

Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

International Nuclear Information System (INIS)

Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

2011-01-01

The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-α-stimulated monocytes to endothelial cells and suppressed the TNF-α induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-α-induced nuclear factor-κB activation, which was attenuated by pretreatment with N G -nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: ► Puerarin induced the phosphorylation of eNOS and the production of NO. ► Puerarin activated eNOS through ER-dependent PI3-kinase and Ca 2+ -dependent AMPK. ► Puerarin-induced NO was involved in the inhibition of NF-kB activation. ► Puerarin may help for prevention of vascular dysfunction and diabetes.

In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.

Science.gov (United States)

Matsushita, Y; Hanazawa, K; Yoshioka, K; Oguchi, T; Kawakami, S; Watanabe, Y; Nishiguchi, M; Nyunoya, H

2000-08-01

The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.

Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

International Nuclear Information System (INIS)

Ikeda, Kikuko; Nakayama, Yuji; Togashi, Yuuki; Obata, Yuuki; Kuga, Takahisa; Kasahara, Kousuke; Fukumoto, Yasunori; Yamaguchi, Naoto

2008-01-01

Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification

The Link between Protein Kinase CK2 and Atypical Kinase Rio1

Directory of Open Access Journals (Sweden)

Konrad Kubiński

2017-02-01

Full Text Available The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors.

Role of adiponectin/phosphatidylinositol 3-kinase/protein kinase B ...

African Journals Online (AJOL)

The adiponectin/phosphatidylinositol 3-kinase/protein kinase B (ADP/PI3k/Akt) signal transduction pathway has an important role in promoting cell survival. This study was designed to determine if the ADP/PI3K/Akt signaling pathway has a role in the mechanism of ischemia–reperfusion injury in vivo. Sprague–Dawley rats ...

Protein Kinases in Shaping Plant Architecture.

Science.gov (United States)

Wu, Juan; Wang, Bo; Xin, Xiaoyun; Ren, Dongtao

2018-02-13

Plant architecture, the three-dimensional organization of the plant body, includes the branching pattern and the size, shape, and position of organs. Plant architecture is genetically controlled and is influenced by environmental conditions. The regulations occur at most of the stages from the first division of the fertilized eggs to the final establishment of plant architecture. Among the various endogenous regulators, protein kinases and their associated signaling pathways have been shown to play important roles in regulating the process of plant architecture establishment. In this review, we summarize recent progress in the understanding of the mechanisms by which plant architecture formation is regulated by protein kinases, especially mitogen-activated protein kinase (MAPK). Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Novel adenosine 3',5'-cyclic monophosphate dependent protein kinases in a marine diatom

International Nuclear Information System (INIS)

Lin, P.P.C.; Volcani, B.E.

1989-01-01

Two novel adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg 2+ and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser( 32 P)-Ser-Asn-Ala-Arg and have an apparent M r of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M r of about 78,000 is photolabeled with 8-azido[ 32 P]cAMP and is also phosphorylated with [γ- 32 P]ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids

GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

Science.gov (United States)

Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

2016-06-01

Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Aurora A kinase RNAi and small molecule inhibition of Aurora kinases with VE-465 induce apoptotic death in multiple myeloma cells.

Science.gov (United States)

Evans, Robert; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Keats, Jonathan; Maxwell, Christopher; Perry, Troy; Chau, Heidi; Belch, Andrew; Pilarski, Linda; Reiman, Tony

2008-03-01

The expression of RHAMM and other centrosome-associated genes are known to correlate with the extent of centrosome amplification in multiple myeloma, and with poor prognosis. RHAMM has a significant interaction with TPX2, a protein which regulates the localization and action of Aurora A kinase (AURKA) at the spindle poles. AURKA is known to be a central determinant of centrosome and spindle function and is a target for cancer therapy. Given these observations, we investigated the role of Aurora kinases as therapeutic targets in myeloma. Here we report that AURKA is expressed ubiquitously in myeloma, to varying degrees, in both cell lines and patients' bone marrow plasma cells. siRNA targeting AURKA induces apoptotic cell death in myeloma cell lines. The Aurora kinase inhibitor VE-465 also induces apoptosis and death in myeloma cell lines and primary myeloma plasma cells. The combination of VE-465 and dexamethasone improves cell killing compared with the use of either agent alone, even in cells resistant to the single agents. The phenotype of myeloma cells treated with VE-465 is consistent with published reports on the effects of Aurora kinase inhibition. Aurora kinase inhibitors should be pursued as potential treatments for myeloma.

The ROCO Kinase QkgA Is Necessary for Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum▿

OpenAIRE

Phillips, Jonathan E.; Gomer, Richard H.

2010-01-01

AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in Dictyostelium discoideum. Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that qkgA− cells accumulate normal levels of ex...

Taurine Inhibits K+-Cl− Cotransporter KCC2 to Regulate Embryonic Cl− Homeostasis via With-no-lysine (WNK) Protein Kinase Signaling Pathway*

Science.gov (United States)

Inoue, Koichi; Furukawa, Tomonori; Kumada, Tatsuro; Yamada, Junko; Wang, Tianying; Inoue, Rieko; Fukuda, Atsuo

2012-01-01

GABA inhibits mature neurons and conversely excites immature neurons due to lower K+-Cl− cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl− homeostasis, which is required for normal brain development. PMID:22544747

Investigating small molecules to inhibit germinal center kinase-like kinase (GLK/MAP4K3) upstream of PKCθ phosphorylation: Potential therapy to modulate T cell dependent immunity.

Science.gov (United States)

May-Dracka, Tricia L; Arduini, Robert; Bertolotti-Ciarlet, Andrea; Bhisetti, Govinda; Brickelmaier, Margot; Cahir-McFarland, Ellen; Enyedy, Istvan; Fontenot, Jason D; Hesson, Thomas; Little, Kevin; Lyssikatos, Joe; Marcotte, Douglas; McKee, Timothy; Murugan, Paramasivam; Patterson, Thomas; Peng, Hairuo; Rushe, Mia; Silvian, Laura; Spilker, Kerri; Wu, Ping; Xin, Zhili; Burkly, Linda C

2018-06-01

Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines. Copyright © 2018 Elsevier Ltd. All rights reserved.

Tyrosine and aurora kinase inhibitors diminish transport function of multidrug resistance-associated protein (MRP 4 and breast cancer resistance protein (BCRP

Directory of Open Access Journals (Sweden)

Rhiannon N. Hardwick

2016-12-01

Full Text Available Tyrosine and aurora kinases are important effectors in signal transduction pathways that are often involved in aberrant cancer cell growth. Tyrosine (TKI and aurora (AKI kinase inhibitors are anti-cancer agents specifically designed to target such signaling pathways through TKI/AKI binding to the ATP-binding pocket of kinases thereby leading to diminished kinase activity. Some TKIs have been identified as inhibitors of ATP-binding cassette (ABC transporters such as P-glycoprotein and breast cancer resistance protein (BCRP, which are commonly upregulated in malignant cells. TKI/AKIs have been investigated as ABC transporter inhibitors in order to facilitate the accumulation of concomitantly administered chemo-therapeutics within cancer cells. However, ABC transporters are prominently expressed in the liver and other eliminating organs, and their inhibition has been linked to intracellular accumulation of drugs, altered disposition, and toxicity. The potential for TKIs/AKIs to inhibit other important hepatic efflux transporters, particularly multidrug resistance-associated proteins (MRPs, remains unknown. The aim of the current study was to compare the inhibitory potency of 20 selected TKI/AKIs against MRP4 and BCRP through the use of inverted membrane vesicle assays. Relative IC50 values were estimated by determining TKI/AKI inhibition of MRP4-mediated [3H]-dehydroepiandrosterone sulfate uptake and BCRP-mediated [3H]-estrone sulfate uptake. To provide insight to the clinical relevance of TKI/AKI inhibition of ABC efflux transporters, the ratio of the steady-state maximum total plasma concentration (Css to the IC50 for each compound was calculated with Css/IC50 ratio >0.1 deemed potentially clinically relevant. Such analysis identified several potentially clinically relevant inhibitors of MRP4: alisertib, danusertib, erlotinib, lapatinib, neratinib, nilotinib, pazopanib, sorafenib, and tozasertib. The potentially clinically relevant inhibition of

Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

Science.gov (United States)

Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

2017-08-15

The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Activation of AMP-activated protein kinase by kainic acid mediates brain-derived neurotrophic factor expression through a NF-kappaB dependent mechanism in C6 glioma cells

International Nuclear Information System (INIS)

Yoon, Hana; Oh, Young Taek; Lee, Jung Yeon; Choi, Ji Hyun; Lee, Ju Hie; Baik, Hyung Hwan; Kim, Sung Soo; Choe, Wonchae; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug

2008-01-01

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. Kainic acid (KA), a prototype excitotoxin is known to induce brain-derived neurotrophic factor (BDNF) in brain. In this study, we examined the role of AMPK in KA-induced BDNF expression in C6 glioma cells. We showed that KA and KA receptor agonist induced activation of AMPK and KA-induced AMPK activation was blocked by inhibition of Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) β. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPKα1 blocked KA-induced BDNF mRNA and protein expression. Inhibition of AMPK blocked KA-induced phosphorylation of CaMKII and I kappaB kinase (IKK) in C6 cells. Finally, we showed that inhibition of AMPK reduced DNA binding and transcriptional activation of nuclear factor-kappaB (NF-κB) in KA-treated cells. These results suggest that AMPK mediates KA-induced BDNF expression by regulating NF-κB activation

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

Science.gov (United States)

Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Jenkins, Jermaine L; Heimiller, Chelsea; Maines, Mahin D

2016-08-01

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner

Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

Science.gov (United States)

Shabani Nashtaei, M; Amidi, F; Sedighi Gilani, M A; Aleyasin, A; Bakhshalizadeh, Sh; Naji, M; Nekoonam, S

2017-03-01

Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 μm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and

The Protein Kinase RSK Family - Roles in Prostate Cancer

National Research Council Canada - National Science Library

Lannigan, Deborah

2006-01-01

The Ser/Thr protein kinase p90-kDa ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase but its roles in prostate cancer have not been previously examined...

A Stabilized Demethoxyviridin Derivative Inhibits PI3 kinase

Science.gov (United States)

Yuan, Hushan; Pupo, Monica T.; Blois, Joe; Smith, Adam; Weissleder, Ralph; Clardy, Jon; Josephson, Lee

2009-01-01

The viridins like demethoxyviridin (Dmv) and wortmannin (Wm) are nanomolar inhibitors of the PI3 kinases, a family of enzymes that play key roles in a host of regulatory processes. Central to the use of these compounds to investigate the role of PI3 kinase in biological systems, or as scaffolds for drug development, are the interrelated issues of stability, chemical reactivity, and bioactivity as inhibitors of PI3 kinase. We found that Dmv was an even more potent inhibitor of PI3 kinase than Wm. However, Dmv was notably less stable than Wm in PBS, with a half-life of 26 min vs Wm’s half-life of 3470 min. Dmv, like Wm, disappeared in culture media with a half-life of less than 1 min. To overcome Dmv’s instability, it was esterified at the C1 position, and then reacted with glycine at the C20 position. The resulting Dmv derivative, termed SA-DmvC20-Gly had a half-life of 218 min in PBS and 64 min in culture media. SA-DmvC20-Gly underwent an exchange reaction at the C20 position with N-acetyl lysine in a manner similar to a WmC20 derivative, WmC20-Proline. SA-DmvC20-Gly inhibited PI3 kinase with an IC50 of 44 nM, compared to Wm’s IC50 of 12 nM. These results indicate that the stability of Dmv can be manipulated by reactions at the C1 and C20 positions, while substantially maintaining its ability to inhibit PI3 kinase. Our results indicate it may be possible to obtain stabilized Dmv derivatives for use as PI3 kinase inhibitors in biological systems. PMID:19523825

Cajaninstilbene acid relaxes rat renal arteries: roles of Ca2+ antagonism and protein kinase C-dependent mechanism.

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Dong-Mei Zhang

Full Text Available Cajaninstilbene acid (CSA is a major active component present in the leaves of Cajanus cajan (L. Millsp. The present study explores the underlying cellular mechanisms for CSA-induced relaxation in rat renal arteries. Vascular reactivity was examined in arterial rings that were suspended in a Multi Myograph System and the expression of signaling proteins was assessed by Western blotting method. CSA (0.1-10 µM produced relaxations in rings pre-contracted by phenylephrine, serotonin, 9, 11-dideoxy-9α, 11α-epoxymethanoprostaglandin F(2α (U46619, and 60 mM KCl. CSA-induced relaxations did not show difference between genders and were unaffected by endothelium denudation, nor by treatment with N(G-nitro-L-arginine methyl ester, indomethacin, ICI-182780, tetraethylammonium ion, BaCl(2, glibenclamide, 4-aminopyridine or propranolol. CSA reduced contraction induced by CaCl(2 (0.01-5 mM in Ca(2+-free 60 mM KCl solution and by 30 nM (--Bay K8644 in 15 mM KCl solution. CSA inhibited 60 mM KCl-induced Ca(2+ influx in smooth muscle of renal arteries. In addition, CSA inhibited contraction evoked by phorbol 12-myristate 13-acetate (PMA, protein kinase C agonist in Ca(2+-free Krebs solution. Moreover, CSA reduced the U46619- and PMA-induced phosphorylation of myosin light chain (MLC at Ser19 and myosin phosphatase target subunit 1 (MYPT1 at Thr853 which was associated with vasoconstriction. CSA also lowered the phosphorylation of protein kinase C (PKCδ at Thr505. In summary, the present results suggest that CSA relaxes renal arteries in vitro via multiple cellular mechanisms involving partial inhibition of calcium entry via nifedipine-sensitive calcium channels, protein kinase C and Rho kinase.

Structural Requirements for Yersinia YopJ Inhibition of MAP Kinase Pathways

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Burdette, Dara; Mukherjee, Sohini; Keitany, Gladys; Goldsmith, Elizabeth; Orth, Kim

2008-01-01

MAPK signaling cascades are evolutionally conserved. The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK) and prevent activation by phosphorylation. YopJ is also able to block yeast MAPK signaling pathways using this mechanism. Based on these observations, we performed a genetic screen to isolate mutants in the yeast MKK, Pbs2, that suppress YopJ inhibition. One suppressor contains a mutation in a conserved tyrosine residue and bypasses YopJ inhibition by increasing the basal activity of Pbs2. Mutations on the hydrophobic face of the conserved G α-helix in the kinase domain prevent both binding and acetylation by YopJ. Corresponding mutants in human MKKs showed that they are conserved not only structurally, but also functionally. These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition. PMID:18167536

Structural requirements for Yersinia YopJ inhibition of MAP kinase pathways.

Directory of Open Access Journals (Sweden)

Yi-Heng Hao

2008-01-01

Full Text Available MAPK signaling cascades are evolutionally conserved. The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK and prevent activation by phosphorylation. YopJ is also able to block yeast MAPK signaling pathways using this mechanism. Based on these observations, we performed a genetic screen to isolate mutants in the yeast MKK, Pbs2, that suppress YopJ inhibition. One suppressor contains a mutation in a conserved tyrosine residue and bypasses YopJ inhibition by increasing the basal activity of Pbs2. Mutations on the hydrophobic face of the conserved G alpha-helix in the kinase domain prevent both binding and acetylation by YopJ. Corresponding mutants in human MKKs showed that they are conserved not only structurally, but also functionally. These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition.

Protein kinase C and α 2-adrenoceptor-mediated inhibition of noradrenaline release from the rat tail artery

International Nuclear Information System (INIS)

Bucher, B.; Neuburger, J.; Illes, P.

1991-01-01

In isolated rat tail arteries preincubated with [3H]noradrenaline, electrical field stimulation evoked the overflow of tritium. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activating phorbol ester, time-dependently increased the overflow at 1 mumol/L but not at 0.1 mumol/L. In contrast, the overflow was not altered by phorbol 13-acetate (PA, 1 mumol/L), which does not influence the activity of PKC. Polymyxin B (70 mumol/L), an inhibitor of PKC, depressed the overflow when given alone and, in addition, attenuated the effect of PMA, 1 mumol/L. The selective alpha 2-adrenoceptor agonist B-HT 933 depressed the overflow; PMA, 1 mumol/L, did not interfere with the effect of B-HT 933, 10 mumol/L. The results provide evidence for the participation of prejunctionally located PKC in the release of noradrenaline. However, PKC does not seem to be involved in the alpha 2-adrenoceptor-agonist-mediated inhibition of noradrenaline release

Protein kinase C β inhibits autophagy and sensitizes cervical cancer Hela cells to cisplatin.

Science.gov (United States)

Li, Na; Zhang, Wei

2017-04-28

Recently, autophagy has been indicated to play an essential role in various biological events, such as the response of cervical cancer cells to chemotherapy. However, the exact signalling mechanism that regulates autophagy during chemotherapy remains unclear. In the present study, we investigated the regulation by cisplatin on protein kinase C β (PKC β), on B-cell lymphoma 2 (Bcl-2) and on apoptosis in cervical cancer Hela cells. And then we examined the regulation by cisplatin on autophagy and the role of autophagy on the chemotherapy in Hela cells. In addition, the regulation of the PKC β on the autophagy was also investigated. Our results indicated that cisplatin promoted PKC β in Hela cells. The PKC β inhibitor reduced the cisplatin-induced apoptosis, whereas increased the cisplatin-induced autophagy in Hela cells. On the other side, the PKC β overexpression aggravated the cisplatin-induced apoptosis, whereas down-regulated the cisplatin-induced autophagy. Taken together, our study firstly recognized the involvement of PKC β in the cytotoxicity of cisplatin via inhibiting autophagy in cervical cancer cells. We propose that PKC β would sensitize cervical cancer cells to chemotherapy via reducing the chemotherapy induced autophagy in cancer cells. © 2017 The Author(s).

Protein kinase D1 (PKD1) influences androgen receptor (AR) function in prostate cancer cells

International Nuclear Information System (INIS)

Mak, Paul; Jaggi, Meena; Syed, Viqar; Chauhan, Subhash C.; Hassan, Sazzad; Biswas, Helal; Balaji, K.C.

2008-01-01

Protein kinase D1 (PKD1), founding member of PKD protein family, is down-regulated in advanced prostate cancer (PCa). We demonstrate that PKD1 and androgen receptor (AR) are present as a protein complex in PCa cells. PKD1 is associated with a transcriptional complex which contains AR and promoter sequence of the Prostate Specific Antigen (PSA) gene. Ectopic expression of wild type PKD1 and the kinase dead mutant PKD1 (K628W) attenuated the ligand-dependent transcriptional activation of AR in prostate cancer cells and yeast cells indicating that PKD1 can affect AR transcription activity, whereas knocking down PKD1 enhanced the ligand-dependent transcriptional activation of AR. Co-expression of kinase dead mutant with AR significantly inhibited androgen-mediated cell proliferation in both LNCaP and DU145 PC cells. Our data demonstrate for the first time that PKD1 can influence AR function in PCa cells

3-Phosphoinositide-dependent PDK1 negatively regulates transforming growth factor-beta-induced signaling in a kinase-dependent manner through physical interaction with Smad proteins.

Science.gov (United States)

Seong, Hyun-A; Jung, Haiyoung; Kim, Kyong-Tai; Ha, Hyunjung

2007-04-20

We have reported previously that PDK1 physically interacts with STRAP, a transforming growth factor-beta (TGF-beta) receptor-interacting protein, and enhances STRAP-induced inhibition of TGF-beta signaling. In this study we show that PDK1 coimmunoprecipitates with Smad proteins, including Smad2, Smad3, Smad4, and Smad7, and that this association is mediated by the pleckstrin homology domain of PDK1. The association between PDK1 and Smad proteins is increased by insulin treatment but decreased by TGF-beta treatment. Analysis of the interacting proteins shows that Smad proteins enhance PDK1 kinase activity by removing 14-3-3, a negative regulator of PDK1, from the PDK1-14-3-3 complex. Knockdown of endogenous Smad proteins, including Smad3 and Smad7, by transfection with small interfering RNA produced the opposite trend and decreased PDK1 activity, protein kinase B/Akt phosphorylation, and Bad phosphorylation. Moreover, coexpression of Smad proteins and wild-type PDK1 inhibits TGF-beta-induced transcription, as well as TGF-beta-mediated biological functions, such as apoptosis and cell growth arrest. Inhibition was dose-dependent on PDK1, but no inhibition was observed in the presence of an inactive kinase-dead PDK1 mutant. In addition, confocal microscopy showed that wild-type PDK1 prevents translocation of Smad3 and Smad4 from the cytoplasm to the nucleus, as well as the redistribution of Smad7 from the nucleus to the cytoplasm in response to TGF-beta. Taken together, our results suggest that PDK1 negatively regulates TGF-beta-mediated signaling in a PDK1 kinase-dependent manner via a direct physical interaction with Smad proteins and that Smad proteins can act as potential positive regulators of PDK1.

Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

Energy Technology Data Exchange (ETDEWEB)

Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

2011-11-15

The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

Protein kinase Cepsilon is important for migration of neuroblastoma cells

International Nuclear Information System (INIS)

Stensman, Helena; Larsson, Christer

2008-01-01

Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration

Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

Science.gov (United States)

Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

Sibiriline, a new small chemical inhibitor of receptor-interacting protein kinase 1, prevents immune-dependent hepatitis.

Science.gov (United States)

Le Cann, Fabienne; Delehouzé, Claire; Leverrier-Penna, Sabrina; Filliol, Aveline; Comte, Arnaud; Delalande, Olivier; Desban, Nathalie; Baratte, Blandine; Gallais, Isabelle; Piquet-Pellorce, Claire; Faurez, Florence; Bonnet, Marion; Mettey, Yvette; Goekjian, Peter; Samson, Michel; Vandenabeele, Peter; Bach, Stéphane; Dimanche-Boitrel, Marie-Thérèse

2017-09-01

Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis. © 2017 Federation of European Biochemical Societies.

Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

DEFF Research Database (Denmark)

Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

2008-01-01

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

Science.gov (United States)

Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

2016-11-01

To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Resveratrol alleviates diabetes-induced testicular dysfunction by inhibiting oxidative stress and c-Jun N-terminal kinase signaling in rats

Energy Technology Data Exchange (ETDEWEB)

Faid, Iman; Al-Hussaini, Heba; Kilarkaje, Narayana, E-mail: knarayana@hsc.edu.kw

2015-12-15

Diabetes adversely affects reproductive functions in humans and animals. The present study investigated the effects of Resveratrol on diabetes-induced alterations in oxidative stress, c-Jun N-terminal kinase (JNK) signaling and apoptosis in the testis. Adult male Wistar rats (13–15 weeks; n = 6/group) were segregated into 1) normal control, 2) Resveratrol-treated (5 mg/kg; ip; given during last 3 weeks), 3) Streptozotocin-induced diabetic and, 4) Resveratrol-treated diabetic groups, and euthanized on day 42 after the confirmation of diabetes. Resveratrol did not normalize blood glucose levels in diabetic rats. Resveratrol supplementation recovered diabetes-induced decreases in reproductive organ weights, sperm count and motility, intra-testicular levels of superoxide dismutase, catalase, and glutathione peroxidase and an increase in 4-hydroxynonenal activities (P < 0.05). Resveratrol also recovered diabetes-induced increases in JNK signaling pathway proteins, namely, ASK1 (apoptosis signal-regulating kinase 1), JNKs (46 and 54 kDa isoforms) and p-JNK to normal control levels (P < 0.05). Interestingly, the expression of a down-stream target of ASK1, MKK4 (mitogen-activated protein kinase kinase 4) and its phosphorylated form (p-MKK4) did not change in experimental groups. Resveratrol inhibited diabetes-induced increases in AP-1 (activator protein-1) components, c-Jun and ATF2 (activating transcription factor 2), but not their phosphorylated forms, to normal control levels (P < 0.05). Further, Resveratrol inhibited diabetes-induced increase in cleaved-caspase-3 to normal control levels. In conclusion, Resveratrol alleviates diabetes-induced apoptosis in testis by modulating oxidative stress, JNK signaling pathway and caspase-3 activities, but not by inhibiting hyperglycemia, in rats. These results suggest that Resveratrol supplementation may be a useful strategy to treat diabetes-induced testicular dysfunction. - Highlights: • Resveratrol up-regulates glutathione

Resveratrol alleviates diabetes-induced testicular dysfunction by inhibiting oxidative stress and c-Jun N-terminal kinase signaling in rats

International Nuclear Information System (INIS)

Faid, Iman; Al-Hussaini, Heba; Kilarkaje, Narayana

2015-01-01

Diabetes adversely affects reproductive functions in humans and animals. The present study investigated the effects of Resveratrol on diabetes-induced alterations in oxidative stress, c-Jun N-terminal kinase (JNK) signaling and apoptosis in the testis. Adult male Wistar rats (13–15 weeks; n = 6/group) were segregated into 1) normal control, 2) Resveratrol-treated (5 mg/kg; ip; given during last 3 weeks), 3) Streptozotocin-induced diabetic and, 4) Resveratrol-treated diabetic groups, and euthanized on day 42 after the confirmation of diabetes. Resveratrol did not normalize blood glucose levels in diabetic rats. Resveratrol supplementation recovered diabetes-induced decreases in reproductive organ weights, sperm count and motility, intra-testicular levels of superoxide dismutase, catalase, and glutathione peroxidase and an increase in 4-hydroxynonenal activities (P < 0.05). Resveratrol also recovered diabetes-induced increases in JNK signaling pathway proteins, namely, ASK1 (apoptosis signal-regulating kinase 1), JNKs (46 and 54 kDa isoforms) and p-JNK to normal control levels (P < 0.05). Interestingly, the expression of a down-stream target of ASK1, MKK4 (mitogen-activated protein kinase kinase 4) and its phosphorylated form (p-MKK4) did not change in experimental groups. Resveratrol inhibited diabetes-induced increases in AP-1 (activator protein-1) components, c-Jun and ATF2 (activating transcription factor 2), but not their phosphorylated forms, to normal control levels (P < 0.05). Further, Resveratrol inhibited diabetes-induced increase in cleaved-caspase-3 to normal control levels. In conclusion, Resveratrol alleviates diabetes-induced apoptosis in testis by modulating oxidative stress, JNK signaling pathway and caspase-3 activities, but not by inhibiting hyperglycemia, in rats. These results suggest that Resveratrol supplementation may be a useful strategy to treat diabetes-induced testicular dysfunction. - Highlights: • Resveratrol up-regulates glutathione

Fisetin Ameliorated Photodamage by Suppressing the Mitogen-Activated Protein Kinase/Matrix Metalloproteinase Pathway and Nuclear Factor-κB Pathways.

Science.gov (United States)

Chiang, Hsiu-Mei; Chan, Shih-Yun; Chu, Yin; Wen, Kuo-Ching

2015-05-13

Ultraviolet (UV) irradiation is one of the most important extrinsic factors contributing to skin photodamage. After UV irradiation, a series of signal transductions in the skin will be activated, leading to inflammatory response and photoaged skin. In this study, fisetin, a flavonol that exists in fruits and vegetables, was investigated for its photoprotective effects. The results revealed that 5-25 μM fisetin inhibits cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP)-1, MMP-3, MMP-9 expression induced by ultraviolet B (UVB) irradiation in human skin fibroblasts. In addition, fisetin suppressed UVB-induced collagen degradation. With regard to its effect on upper-stream signal transduction, we found that fisetin reduced the expression of ultraviolet (UV)-induced ERK, JNK, and p38 phosphorylation in the mitogen-activated protein kinase (MAP kinase) pathway. Furthermore, fisetin reduced inhibitor κB (IκB) degradation and increased the amount of p65, which is a major subunit of nuclear factor-κB (NF-κB), in cytoplasm. It also suppressed NF-κB translocated to the nucleus and inhibited cAMP response element-binding protein (CREB) Ser-133 phosphorylation level in the phosphoinositide 3-kinase/protein kinase B/CREB (PI3K/AKT/CREB) pathway. Finally, fisetin inhibited UV-induced intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), and nitric oxide (NO) generation. The mentioned effects and mechanisms suggest that fisetin can be used in the development of photoprotective agents.

Protein kinase that phosphorylates light-harvesting complex is autophosphorylated and is associated with photosystem II

International Nuclear Information System (INIS)

Coughlan, S.J.; Hind, G.

1987-01-01

Thylakoid membranes were phosphorylated with [γ- 32 P]ATP and extracted with octyl glucoside and cholate. Among the radiolabeled phosphoproteins in the extract was a previously characterized protein kinase of 64-kDa apparent mass. The ability of this enzyme to undergo autophosphorylation in situ was used to monitor its distribution in the membrane. Fractionation studies showed that the kinase is confined to granal regions of the thylakoid, where it appears to be associated with the light-harvesting chlorophyll-protein complex of photosystem II. The kinetics of kinase autophosphorylation were investigated both in situ and in extracted, purified enzyme. In the membrane, autophosphorylation saturated within 20-30 min and was reversed with a half-time of 7-8 min upon removal of ATP or oxidative inactivation of the kinase; the accompanying dephosphorylation of light-harvesting complex was slower and kinetically complex. Fluoride (10 mM) inhibited these dephosphorylations. Autophosphorylation of the isolated kinase was independent of enzyme concentration, indicative of an intramolecular mechanism. A maximum of one serine residue per mole of kinase was esterified. Autophosphorylation was more rapid in the presence of histone IIIs, an exogenous substrate. Dephosphorylation of the isolated enzyme was not observed

Purification and characterization of a thylakoid protein kinase

International Nuclear Information System (INIS)

Coughlan, S.J.; Hind, G.

1986-01-01

Control of state transitions in the thylakoid by reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHC-II) is modulated by a kinase. The kinase catalyzing this phosphorylation is associated with the thylakoid membrane, and is regulated by the redox state of the plastoquinone pool. The isolation and partial purification from spinach thylakoids of two protein kinases (CPK1, CPK2) of apparent molecular masses 25 kDa and 38 kDa has been reported. Neither enzyme utilizes isolated LHC-II as a substrate. The partial purification of a third protein kinase (LHCK) which can utilize both lysine-rich histones (IIIs and Vs) and isolated LHC-II as substrate has now been purified to homogeneity and characterized by SDS-polyacrylamide gel electrophoresis as a 64 kDa peptide. From a comparison of the two isolation procedures we have concluded that CPK1 is indeed a protein kinase, but has a lower specific activity than that of LHCK. 8 refs., 4 figs

KCl cotransport regulation and protein kinase G in cultured vascular smooth muscle cells.

Science.gov (United States)

Adragna, N C; Zhang, J; Di Fulvio, M; Lincoln, T M; Lauf, P K

2002-05-15

K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p <0.01). At 0.5 mM, NEM stimulated K-Cl cotransport by 5-fold in PKG- but not in PKG+ cells. However, NEM was more potent although less effective to activate K-Cl cotransport in normal (passage 1-3) and PKG+ than in PKG- cells. In PKG- cells, [(dihydroindenyl) oxy] alkanoic acid (300 mM) but not furosemide (1 mM) inhibited K-Cl cotransport. Furthermore, no difference in K-Cl cotransport mRNA expression was observed between these cells. In conclusion, this study shows that manipulation of PKG expression in vascular smooth muscle cells affects K-Cl cotransport activity and its activation by NEM.

Phosphotyrosine phosphatase and tyrosine kinase inhibition modulate airway pressure-induced lung injury.

Science.gov (United States)

Parker, J C; Ivey, C L; Tucker, A

1998-11-01

We determined whether drugs which modulate the state of protein tyrosine phosphorylation could alter the threshold for high airway pressure-induced microvascular injury in isolated perfused rat lungs. Lungs were ventilated for successive 30-min periods with peak inflation pressures (PIP) of 7, 20, 30, and 35 cmH2O followed by measurement of the capillary filtration coefficient (Kfc), a sensitive index of hydraulic conductance. In untreated control lungs, Kfc increased by 1.3- and 3.3-fold relative to baseline (7 cmH2O PIP) after ventilation with 30 and 35 cmH2O PIP. However, in lungs treated with 100 microM phenylarsine oxide (a phosphotyrosine phosphatase inhibitor), Kfc increased by 4.7- and 16.4-fold relative to baseline at these PIP values. In lungs treated with 50 microM genistein (a tyrosine kinase inhibitor), Kfc increased significantly only at 35 cmH2O PIP, and the three groups were significantly different from each other. Thus phosphotyrosine phosphatase inhibition increased the susceptibility of rat lungs to high-PIP injury, and tyrosine kinase inhibition attenuated the injury relative to the high-PIP control lungs.

Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells.

Science.gov (United States)

Caligiuri, Alessandra; Bertolani, Cristiana; Guerra, Cristina Tosti; Aleffi, Sara; Galastri, Sara; Trappoliere, Marco; Vizzutti, Francesco; Gelmini, Stefania; Laffi, Giacomo; Pinzani, Massimo; Marra, Fabio

2008-02-01

Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. Activation of AMPK negatively modulates the activated phenotype of HSCs.

Diversity, classification and function of the plant protein kinase superfamily

OpenAIRE

Lehti-Shiu, Melissa D.; Shiu, Shin-Han

2012-01-01

Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase r...

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

Science.gov (United States)

Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

CIKS, a connection to IκB kinase and stress-activated protein kinase

Science.gov (United States)

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

Inhibition of phosphatidylinositol-3-kinase causes increased sensitivity to radiation through a PKB-dependent mechanism

International Nuclear Information System (INIS)

Gottschalk, Alexander R.; Doan, Albert; Nakamura, Jean L.; Stokoe, David; Haas-Kogan, Daphne A.

2005-01-01

Purpose: To identify whether inhibition of phosphatidylinositol-3-kinase (PI3K) causes increased radiosensitivity through inhibition of protein kinase B (PKB), implicating PKB as an important therapeutic target in prostate cancer. Methods and Materials: The prostate cancer cell line LNCaP was treated with the PI3K inhibitor LY294002, radiation, and combinations of the two therapies. Apoptosis and survival were measured by cell cycle analysis, Western blot analysis for cleaved poly (ADP-ribose) polymerase, and clonogenic survival. To test the hypothesis that inhibition of PKB is responsible for LY294002-induced radiosensitivity, LNCaP cells expressing a constitutively active form of PKB were used. Results: The combination of PI3K inhibition and radiation caused an increase in apoptosis and a decrease in clonogenic survival when compared to either modality alone. The expression of constitutively activated PKB blocked apoptosis induced by combination of PI3K inhibition and radiation and prevented radiosensitization by LY294002. Conclusion: These data indicate that PI3K inhibition increases sensitivity of prostate cancer cell lines to ionizing radiation through inactivation of PKB. Therefore, PTEN mutations, which lead to PKB activation, may play an important role in the resistance of prostate cancer to radiation therapy. Targeted therapy against PKB could be beneficial in the management of prostate cancer patients

Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy

Science.gov (United States)

Sarcocystis neurona is the most frequent cause of Equine Protozoal Myeloencephalitis (EPM), a debilitating neurologic disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma...

AMP N1-Oxide, a Unique Compound of Royal Jelly, Induces Neurite Outgrowth from PC12 Vells via Signaling by Protein Kinase A Independent of that by Mitogen-Activated Protein Kinase

Directory of Open Access Journals (Sweden)

Noriko Hattori

2010-01-01

Full Text Available Earlier we identified adenosine monophosphate (AMP N1-oxide as a unique compound of royal jelly (RJ that induces neurite outgrowth (neuritegenesis from cultured rat pheochromocytoma PC12 cells via the adenosine A2A receptor. Now, we found that AMP N1-oxide stimulated the phosphorylation of not only mitogen-activated protein kinase (MAPK but also that of cAMP/calcium-response element-binding protein (CREB in a dose-dependent manner. Inhibition of MAPK activation by a MEK inhibitor, PD98059, did not influence the AMP N1-oxide-induced neuritegenesis, whereas that of protein kinase A (PKA by a selective inhibitor, KT5720, significantly reduced neurite outgrowth. AMP N1-oxide also had the activity of suppressing the growth of PC12 cells, which correlated well with the neurite outgrowth-promoting activity. KT5720 restored the growth of AMP N1-oxide-treated PC12 cells. It is well known that nerve growth factor suppresses proliferation of PC12 cells before causing stimulation of neuronal differentiation. Thus, AMP N1-oxide elicited neuronal differentiation of PC12 cells, as evidenced by generation of neurites, and inhibited cell growth through adenosine A2A receptor-mediated PKA signaling, which may be responsible for characteristic actions of RJ.

Crystal structure of human protein kinase CK2

DEFF Research Database (Denmark)

Niefind, K; Guerra, B; Ermakowa, I

2001-01-01

The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalyt...... as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure.......The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic...... subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit...

Selective blockade of protein kinase B protects the rat and human myocardium against ischaemic injury

Science.gov (United States)

Linares-Palomino, José; Husainy, Muhammad A; Lai, Vien K; Dickenson, John M; Galiñanes, Manuel

2010-01-01

Protein kinase B (PKB/Akt) plays a critical role in cell survival but the investigation of its involvement has been limited by the lack of specific pharmacological agents. In this study, using novel PKB inhibitors (VIII and XI), we investigated the role of PKB in cardioprotection of the rat and human myocardium, the location of PKB in relation to mitoKATP channels and p38 mitogen-activated protein kinase (p38 MAPK), and whether the manipulation of PKB can overcome the unresponsiveness to protection of the diabetic myocardium. Myocardial slices from rat left ventricle and from the right atrial appendage of patients undergoing elective cardiac surgery were subjected to 90 min ischaemia/120 min reoxygenation at 37°C. Tissue injury was assessed by creatine kinase (CK) released and determination of cell necrosis and apoptosis. The results showed that blockade of PKB activity caused significant reduction of CK release and cell death, a benefit that was as potent as ischaemic preconditioning and could be reproduced by blockade of phosphatidylinositol 3-kinase (PI-3K) with wortmannin and LY 294002. The protection was time dependent with maximal benefit seen when PKB and PI-3K were inhibited before ischaemia or during both ischaemia and reoxygenation. In addition, it was revealed that PKB is located downstream of mitoKATP channels but upstream of p38 MAPK. PKB inhibition induced a similar degree of protection in the human and rat myocardium and, importantly, it reversed the unresponsiveness to protection of the diabetic myocardium. In conclusion, inhibition of PKB plays a critical role in protection of the mammalian myocardium and may represent a clinical target for the reduction of ischaemic injury. PMID:20403980

Development of a phosphorylated Momordica charantia protein system for inhibiting susceptible dose-dependent C. albicans to available antimycotics: An allosteric regulation of protein.

Science.gov (United States)

Qiao, Yuanbiao; Song, Li; Zhu, Chenchen; Wang, Qian; Guo, Tianyan; Yan, Yanhua; Li, Qingshan

2017-11-15

A regulatory Momordica charantia protein system was constructed allosterically by in vitro protein phosphorylation, in an attempt to evaluate antimycological pluripotency against dose-dependent susceptibilities in C. albicans. Fungal strain lineages susceptible to ketoconazole, econazole, miconazole, 5-flucytosine, nystatin and amphotericin B were prepared in laboratory, followed by identification via antifungal susceptibility testing. Protein phosphorylation was carried out in reactions with 5'-adenylic, guanidylic, cytidylic and uridylic acids and cyclic adenosine triphosphate, through catalysis of cyclin-dependent kinase 1, protein kinase A and protein kinase C respectively. Biochemical analysis of enzymatic reactions indicated the apparent Michaelis-Menten constants and maximal velocity values of 16.57-91.97mM and 55.56-208.33μM·min -1 , together with an approximate 1:1 reactant stoichiometric ratio. Three major protein phosphorylation sites were theoretically predicted at Thr255, Thr102 and Thr24 by a KinasePhos tool. Additionally, circular dichroism spectroscopy demonstrated that upon phosphorylation, protein folding structures were decreased in random coil, β6-sheet and α1-helix partial regions. McFarland equivalence standard testing yielded the concentration-dependent inhibition patterns, while fungus was grown in Sabouraud's dextrose agar. The minimal inhibitory concentrations of 0.16-0.51μM (at 50% response) were obtained for free protein and phosphorylated counterparts. With respect to the 3-cycling susceptibility testing regimen, individuals of total protein forms were administrated in-turn at 0.14μM/cycle. Relative inhibition ratios were retained to 66.13-81.04% of initial ones regarding the ketoconazole-susceptible C. albicans growth. An inhibitory protein system, with an advantage of decreasing antifungal susceptibilities to diverse antimycotics, was proposed because of regulatory pluripotency whereas little contribution to susceptibility in

Protein kinase A regulatory subunit distribution in medulloblastoma

International Nuclear Information System (INIS)

Mucignat-Caretta, Carla; Denaro, Luca; Redaelli, Marco; D'Avella, Domenico; Caretta, Antonio

2010-01-01

Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

AR-v7 protein expression is regulated by protein kinase and phosphatase

Science.gov (United States)

Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

2015-01-01

Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

Phosphorylation of protein kinase A (PKA) regulatory subunit RIα by protein kinase G (PKG) primes PKA for catalytic activity in cells.

Science.gov (United States)

Haushalter, Kristofer J; Casteel, Darren E; Raffeiner, Andrea; Stefan, Eduard; Patel, Hemal H; Taylor, Susan S

2018-03-23

cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro , whereupon phosphorylated RIα no longer inhibits PKAc at normal (1:1) stoichiometric ratios. However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant "desensitization" of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser 101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a "sensitized intermediate" state that is in effect primed to trigger PKAc activity.

Regulation of Ca2+ influx by a protein kinase C activator in chromaffin cells: differential role of P/Q- and L-type Ca2+ channels.

Science.gov (United States)

Sena, C M; Santos, R M; Boarder, M R; Rosário, L M

1999-02-05

Phorbol esters reduce depolarization-evoked Ca2+ influx in adrenal chromaffin cells, suggesting that voltage-sensitive Ca2+ channels (VSCCs) are inhibited by protein kinase C-mediated phosphorylation. We now address the possibility that L- and P/Q-type Ca2+ channel subtypes might be differentially involved in phorbol ester action. In bovine chromaffin cells, short-term (10 min) incubations with phorbol 12-myristate 13-acetate (PMA) inhibited early high K+-evoked rises in cytosolic free Ca2+ concentration ([Ca2+]i) and the early component of the depolarization-evoked Mn2+ quenching of fura-2 fluorescence in a dose-dependent manner (IC50: 18 and 7 nM; maximal inhibitions: 45 and 48%, respectively). The protein kinase C inhibitor staurosporine (100 nM) reverted the inhibitory action of PMA. PMA (0.1-1 microM) inhibited the early and late phases of the ionomycin (2 microM)-evoked [Ca2+]i transients by 14-23%. Omega-agatoxin IVA, a blocker of P/Q-type Ca2+ channels, inhibited high K+-evoked [Ca2+]i rises in a dose-dependent fashion (IC50 = 50 nM). In contrast, 0.1 microM omega-conotoxin GVIA, a blocker of N-type channels, was without effect. A sizeable (< 45%) component of early Ca2+ influx persisted in the combined presence of omega-agatoxin IVA (100 nM) and nitrendipine (1 microM). Simultaneous exposure to omega-agatoxin IVA and PMA inhibited both the early [Ca2+]i transients and Mn2+ quenching to a much greater extent than each drug separately. Inhibition of the [Ca2+]i transients by nitrendipine and PMA did not significantly exceed that produced by PMA alone. It is concluded that phorbol ester-mediated activation of protein kinase C inhibits preferentially L-type VSCCs over P/Q type channels in adrenal chromaffin cells. However, the possibility cannot be ruled out that dihydropyridine-resistant, non-P/Q type channels might also be negatively regulated by protein kinase C. This may represent an important pathway for the specific control of VSCCs by protein kinase C

Gabapentin Inhibits Protein Kinase C Epsilon Translocation in Cultured Sensory Neurons with Additive Effects When Coapplied with Paracetamol (Acetaminophen).

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Vellani, Vittorio; Giacomoni, Chiara

2017-01-01

Gabapentin is a well-established anticonvulsant drug which is also effective for the treatment of neuropathic pain. Although the exact mechanism leading to relief of allodynia and hyperalgesia caused by neuropathy is not known, the blocking effect of gabapentin on voltage-dependent calcium channels has been proposed to be involved. In order to further evaluate its analgesic mechanisms, we tested the efficacy of gabapentin on protein kinase C epsilon (PKC ε ) translocation in cultured peripheral neurons isolated from rat dorsal root ganglia (DRGs). We found that gabapentin significantly reduced PKC ε translocation induced by the pronociceptive peptides bradykinin and prokineticin 2, involved in both inflammatory and chronic pain. We recently showed that paracetamol (acetaminophen), a very commonly used analgesic drug, also produces inhibition of PKC ε . We tested the effect of the combined use of paracetamol and gabapentin, and we found that the inhibition of translocation adds up. Our study provides a novel mechanism of action for gabapentin in sensory neurons and suggests a mechanism of action for the combined use of paracetamol and gabapentin, which has recently been shown to be effective, with a cumulative behavior, in the control of postoperative pain in human patients.

Gabapentin Inhibits Protein Kinase C Epsilon Translocation in Cultured Sensory Neurons with Additive Effects When Coapplied with Paracetamol (Acetaminophen

Directory of Open Access Journals (Sweden)

Vittorio Vellani

2017-01-01

Full Text Available Gabapentin is a well-established anticonvulsant drug which is also effective for the treatment of neuropathic pain. Although the exact mechanism leading to relief of allodynia and hyperalgesia caused by neuropathy is not known, the blocking effect of gabapentin on voltage-dependent calcium channels has been proposed to be involved. In order to further evaluate its analgesic mechanisms, we tested the efficacy of gabapentin on protein kinase C epsilon (PKCε translocation in cultured peripheral neurons isolated from rat dorsal root ganglia (DRGs. We found that gabapentin significantly reduced PKCε translocation induced by the pronociceptive peptides bradykinin and prokineticin 2, involved in both inflammatory and chronic pain. We recently showed that paracetamol (acetaminophen, a very commonly used analgesic drug, also produces inhibition of PKCε. We tested the effect of the combined use of paracetamol and gabapentin, and we found that the inhibition of translocation adds up. Our study provides a novel mechanism of action for gabapentin in sensory neurons and suggests a mechanism of action for the combined use of paracetamol and gabapentin, which has recently been shown to be effective, with a cumulative behavior, in the control of postoperative pain in human patients.

Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic β-cells

Energy Technology Data Exchange (ETDEWEB)

Chen, Shan-Shan [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China); Jiang, Teng [Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing (China); Wang, Yi; Gu, Li-Ze [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China); Wu, Hui-Wen [Laboratory Center for Basic Medical Sciences, Nanjing Medical University, Nanjing (China); Tan, Lan [Department of Neurology, Qingdao Municipal Hospital, Nanjing Medical University, Nanjing (China); Guo, Jun, E-mail: Guoj@njmu.edu.cn [Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing Medical University, Nanjing (China); Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing (China)

2014-01-17

Highlights: •PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in β-cells. •Activated PKR inhibited β-cell proliferation by arresting cell cycle at G1 phase. •Activated PKR fully abrogated the pro-proliferative effects of IGF-I on β-cells. -- Abstract: Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM.

Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic β-cells

International Nuclear Information System (INIS)

Chen, Shan-Shan; Jiang, Teng; Wang, Yi; Gu, Li-Ze; Wu, Hui-Wen; Tan, Lan; Guo, Jun

2014-01-01

Highlights: •PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in β-cells. •Activated PKR inhibited β-cell proliferation by arresting cell cycle at G1 phase. •Activated PKR fully abrogated the pro-proliferative effects of IGF-I on β-cells. -- Abstract: Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM

Effect of triiodothyronine on rat liver chromatin protein kinase

International Nuclear Information System (INIS)

Kruh, J.; Tichonicky, L.

1976-01-01

1) Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin nonhistone proteins. A significant increase was found after two daily injections. A 4-fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p-hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3, 3', 5'-triiodothyronine have no effect on protein kinase. 2) Chromatin non-histone proteins isolated from rats injected with triiodothyronine incorporated more 32 P when incubated with [γ- 32 P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32 P incorporation. It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non-histone proteins. (orig.) [de

Maintaining glycogen synthase kinase-3 activity is critical for mTOR kinase inhibitors to inhibit cancer cell growth.

Science.gov (United States)

Koo, Junghui; Yue, Ping; Gal, Anthony A; Khuri, Fadlo R; Sun, Shi-Yong

2014-05-01

mTOR kinase inhibitors that target both mTORC1 and mTORC2 are being evaluated in cancer clinical trials. Here, we report that glycogen synthase kinase-3 (GSK3) is a critical determinant for the therapeutic response to this class of experimental drugs. Pharmacologic inhibition of GSK3 antagonized their suppressive effects on the growth of cancer cells similarly to genetic attenuation of GSK3. Conversely, expression of a constitutively activated form of GSK3β sensitized cancer cells to mTOR inhibition. Consistent with these findings, higher basal levels of GSK3 activity in a panel of human lung cancer cell lines correlated with more efficacious responses. Mechanistic investigations showed that mTOR kinase inhibitors reduced cyclin D1 levels in a GSK3β-dependent manner, independent of their effects on suppressing mTORC1 signaling and cap binding. Notably, selective inhibition of mTORC2 triggered proteasome-mediated cyclin D1 degradation, suggesting that mTORC2 blockade is responsible for GSK3-dependent reduction of cyclin D1. Silencing expression of the ubiquitin E3 ligase FBX4 rescued this reduction, implicating FBX4 in mediating this effect of mTOR inhibition. Together, our findings define a novel mechanism by which mTORC2 promotes cell growth, with potential implications for understanding the clinical action of mTOR kinase inhibitors. ©2014 AACR.

Significance of host cell kinases in herpes simplex virus type 1 egress and lamin-associated protein disassembly from the nuclear lamina

International Nuclear Information System (INIS)

Leach, Natalie R.; Roller, Richard J.

2010-01-01

The nuclear lamina is thought to be a steric barrier to the herpesvirus capsid. Disruption of the lamina accompanied by phosphorylation of lamina proteins is a conserved feature of herpesvirus infection. In HSV-1-infected cells, protein kinase C (PKC) alpha and delta isoforms are recruited to the nuclear membrane and PKC delta has been implicated in phosphorylation of emerin and lamin B. We tested two critical hypotheses about the mechanism and significance of lamina disruption. First, we show that chemical inhibition of all PKC isoforms reduced viral growth five-fold and inhibited capsid egress from the nucleus. However, specific inhibition of either conventional PKCs or PKC delta does not inhibit viral growth. Second, we show hyperphosphorylation of emerin by viral and cellular kinases is required for its disassociation from the lamina. These data support hypothesis that phosphorylation of lamina components mediates lamina disruption during HSV nuclear egress.

Inhibition of IGF-1 receptor kinase blocks the differentiation into cardiomyocyte-like cells of BMSCs induced by IGF-1.

Science.gov (United States)

Gong, Haibin; Wang, Xiuli; Wang, Lei; Liu, Ying; Wang, Jie; Lv, Qian; Pang, Hui; Zhang, Qinglin; Wang, Zhenquan

2017-07-01

Bone marrow mesenchymal stem cells (BMSCs) have the potential to transdifferentiate into cardiomyocyte‑like cells (CLCs) if an appropriate cardiac environment is provided. Insulin‑like growth factor‑1 (IGF‑1) plays an important role in the cell migration, survival and differentiation of BMSCs. However, the effect of IGF‑1 on the cellular differentiation remains unclear. In the present study, BMSCs were isolated from rat femurs and tibias and the cells were purified at passage 6 (P6). IGF‑1 and IGF‑1 receptor (IGF‑1R) kinase inhibitor I‑OMe AG538 were added to detect if IGF‑1 could induce BMSCs to transdifferentiate into CLCs and if I‑OMe AG538 could inhibit IGF‑1‑mediated receptor activation and downstream signaling. Immunostaining demonstrated that all P6 BMSCs express CD29 and CD44 but not CD45. BMSCs induced by 15 ng/ml IGF‑1 revealed positivity for cardiac troponin‑T and cardiac troponin‑I. The optimal induction time was 14 days but the expression of these proteins were incompletely inhibited by 300 nmol/l I‑OMe AG538 and completely inhibited by 10 µmol/l I‑OMe AG538. Western blotting showed that the level of IGF‑1R autophosphorylation and the expression of cTnT and cTnI were higher when BMSCs were induced for 14 days. I‑OMe AG538 selectively inhibited IGF‑1‑mediated growth and signal transduction and the inhibitory effect of I‑OMe AG538 were not reverted in the presence of exogenous IGF‑1. In addition, when a time course analysis of the effects of I‑OMe AG538 on mitogen‑activated protein kinase kinase and phosphatidylinositol 3‑kinase signaling were done, we observed a transient inhibitory effect on Erk1/2 and Akt phosphorylation, in keeping with the inhibitory effects on cell growth. Taken together, these data indicate that I‑OMe AG538 could inhibit IGF-1-induced CLCs in BMSCs and this effect is time- and concentration-dependent.

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

Science.gov (United States)

Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

2017-10-13

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

SOcK, MiSTs, MASK and STicKs: the GCKIII (germinal centre kinase III) kinases and their heterologous protein-protein interactions.

Science.gov (United States)

Sugden, Peter H; McGuffin, Liam J; Clerk, Angela

2013-08-15

The GCKIII (germinal centre kinase III) subfamily of the mammalian Ste20 (sterile 20)-like group of serine/threonine protein kinases comprises SOK1 (Ste20-like/oxidant-stress-response kinase 1), MST3 (mammalian Ste20-like kinase 3) and MST4. Initially, GCKIIIs were considered in the contexts of the regulation of mitogen-activated protein kinase cascades and apoptosis. More recently, their participation in multiprotein heterocomplexes has become apparent. In the present review, we discuss the structure and phosphorylation of GCKIIIs and then focus on their interactions with other proteins. GCKIIIs possess a highly-conserved, structured catalytic domain at the N-terminus and a less-well conserved C-terminal regulatory domain. GCKIIIs are activated by tonic autophosphorylation of a T-loop threonine residue and their phosphorylation is regulated primarily through protein serine/threonine phosphatases [especially PP2A (protein phosphatase 2A)]. The GCKIII regulatory domains are highly disorganized, but can interact with more structured proteins, particularly the CCM3 (cerebral cavernous malformation 3)/PDCD10 (programmed cell death 10) protein. We explore the role(s) of GCKIIIs (and CCM3/PDCD10) in STRIPAK (striatin-interacting phosphatase and kinase) complexes and their association with the cis-Golgi protein GOLGA2 (golgin A2; GM130). Recently, an interaction of GCKIIIs with MO25 has been identified. This exhibits similarities to the STRADα (STE20-related kinase adaptor α)-MO25 interaction (as in the LKB1-STRADα-MO25 heterotrimer) and, at least for MST3, the interaction may be enhanced by cis-autophosphorylation of its regulatory domain. In these various heterocomplexes, GCKIIIs associate with the Golgi apparatus, the centrosome and the nucleus, as well as with focal adhesions and cell junctions, and are probably involved in cell migration, polarity and proliferation. Finally, we consider the association of GCKIIIs with a number of human diseases, particularly

Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

DEFF Research Database (Denmark)

Sayed, M; Kim, S O; Salh, B S

2000-01-01

Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

A Global Protein Kinase and Phosphatase Interaction Network in Yeast

Science.gov (United States)

Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

2011-01-01

The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

Analysis of the complexity of protein kinases within the phloem sieve tube system. Characterization of Cucurbita maxima calmodulin-like domain protein kinase 1.

Science.gov (United States)

Yoo, Byung-Chun; Lee, Jung-Youn; Lucas, William J

2002-05-03

In angiosperms, functional, mature sieve elements lack nuclei, vacuoles, ribosomes, and most of the endomembrane network. In this study, the complexity, number, and nature of protein kinases within the phloem sap of Cucurbita maxima were investigated to test the hypothesis that the enucleate sieve tube system utilizes a simplified signal transduction network. Supporting evidence was obtained in that only five putative protein kinases (three calcium-independent and two calcium-dependent protein kinases) were detected within the phloem sap extracted from stem tissues. Biochemical methods were used to purify one such calcium-dependent protein kinase. The gene for this C. maxima calmodulin-like domain protein kinase 1 (CmCPK1), was cloned using peptide microsequences. A combination of mass spectrometry, peptide fingerprinting, and amino-terminal sequencing established that, in the phloem sap, CmCPK1 exists as an amino-terminally cleaved protein. A second highly homologous isoform, CmCPK2, was identified, but although transcripts could be detected in the companion cells, peptide fingerprint analysis suggested that CmCPK2 does not enter the phloem sap. Potential substrates for CmCPK1, within the phloem sap, were also detected using an on-membrane phosphorylation assay. Entry of CmCPK1 into sieve elements via plasmodesmata and the potential roles played by these phloem protein kinases are discussed.

Fasting potentiates the anticancer activity of tyrosine kinase inhibitors by strengthening MAPK signaling inhibition

Science.gov (United States)

Caffa, Irene; D'Agostino, Vito; Damonte, Patrizia; Soncini, Debora; Cea, Michele; Monacelli, Fiammetta; Odetti, Patrizio; Ballestrero, Alberto; Provenzani, Alessandro; Longo, Valter D.; Nencioni, Alessio

2015-01-01

Tyrosine kinase inhibitors (TKIs) are now the mainstay of treatment in many types of cancer. However, their benefit is frequently short-lived, mandating the search for safe potentiation strategies. Cycles of fasting enhance the activity of chemo-radiotherapy in preclinical cancer models and dietary approaches based on fasting are currently explored in clinical trials. Whether combining fasting with TKIs is going to be potentially beneficial remains unknown. Here we report that starvation conditions increase the ability of commonly administered TKIs, including erlotinib, gefitinib, lapatinib, crizotinib and regorafenib, to block cancer cell growth, to inhibit the mitogen-activated protein kinase (MAPK) signaling pathway and to strengthen E2F-dependent transcription inhibition. In cancer xenografts models, both TKIs and cycles of fasting slowed tumor growth, but, when combined, these interventions were significantly more effective than either type of treatment alone. In conclusion, cycles of fasting or of specifically designed fasting-mimicking diets should be evaluated in clinical studies as a means to potentiate the activity of TKIs in clinical use. PMID:25909220

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

International Nuclear Information System (INIS)

Sun Haipeng; Xu Beibei; Sheveleva, Elena; Chen, Qin M.

2008-01-01

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca 2+ concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes

Inhibition of protein kinase CK2 reduces CYP24A1 expression and enhances 1,25-dihydroxyvitamin D3 anti-tumor activity in human prostate cancer cells

Science.gov (United States)

Luo, Wei; Yu, Wei-Dong; Ma, Yingyu; Chernov, Mikhail; Trump, Donald L.; Johnson, Candace S.

2013-01-01

Vitamin D has broad range of physiological functions and anti-tumor effects. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme for degrading many forms of vitamin D including the most active form, 1,25D3. Inhibition of CYP24A1 enhances 1,25D3 anti-tumor activity. In order to isolate regulators of CYP24A1 expression in prostate cancer cells, we established a stable prostate cancer cell line PC3 with CYP24A1 promoter driving luciferase expression to screen a small molecular library for compounds that inhibit CYP24A1 promoter activity. From this screening, we identified, 4,5,6,7-tetrabromobenzimidazole (TBBz), a protein kinase CK2 selective inhibitor as a disruptor of CYP24A1 promoter activity. We show that TBBz inhibits CYP24A1 promoter activity induced by 1,25D3 in prostate cancer cells. In addition, TBBz downregulates endogenous CYP24A1 mRNA level in TBBz treated PC3 cells. Furthermore, siRNA-mediated CK2 knockdown reduces 1,25D3 induced CYP24A1 mRNA expression in PC3 cells. These results suggest that CK2 contributes to 1,25D3 mediated target gene expression. Lastly, inhibition of CK2 by TBBz or CK2 siRNA significantly enhanced 1,25D3 mediated anti-proliferative effect in vitro and in vivo in a xenograft model. In summary, our findings reveal that protein kinase CK2 is involved in the regulation of CYP24A1 expression by 1,25D3 and CK2 inhibitor enhances 1,25D3 mediated anti-tumor effect. PMID:23358686

A casein-kinase-2-related protein kinase is tightly associated with the large T antigen of simian virus 40

DEFF Research Database (Denmark)

Götz, C; Koenig, M G; Issinger, O G

1995-01-01

by the addition of protein kinase CK2 suggest that at least one of the T-antigen-associated protein kinases is CK2 or a protein-kinase-CK2-related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitro binding studies. Experiments with temperature-sensitive SV40-transformed......The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we...... show that immuno-purified T antigen from SV40-transformed cells and from baculovirus-infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation...

Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

International Nuclear Information System (INIS)

Honma, Yuichi; Harada, Masaru

2013-01-01

Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. -- Graphical abstract: Display Omitted -- Highlights: •We examined the cytotoxic mechanisms of sorafenib in hepatoma cells. •Sorafenib induces cell death via apoptotic and necrotic fashion. •Sorafenib inhibits protein ubiquitination and unfolded protein response. •Autophagy induced by sorafenib may affect its cytotoxicity. •Sorafenib inhibits keratin phosphorylation and cytoplasmic inclusion formation

Inhibition of gap-junctional intercellular communication and activation of mitogen-activated protein kinases by cyanobacterial extracts--indications of novel tumor-promoting cyanotoxins?

Science.gov (United States)

Bláha, Ludĕk; Babica, Pavel; Hilscherová, Klára; Upham, Brad L

2010-01-01

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) - ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5-5mgd.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.

Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

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Hou Ssu-Yu

2010-06-01

Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

MicroRNA-200b Suppresses Arsenic-transformed Cell Migration by Targeting Protein Kinase Cα and Wnt5b-Protein Kinase Cα Positive Feedback Loop and Inhibiting Rac1 Activation*

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Wang, Zhishan; Humphries, Brock; Xiao, Hua; Jiang, Yiguo; Yang, Chengfeng

2014-01-01

MicroRNA-200b (miR-200b) is a member of miR-200 family that has been found to inhibit cell migration and cancer metastasis; however, the underlying mechanism is not well understood. We previously reported that miR-200 expression is depleted in arsenic-transformed human bronchial epithelial cells with highly migratory and invasive characteristics, whereas stably re-expressing miR-200b strongly suppresses arsenic-transformed cell migration. This study was performed to investigate how miR-200b inhibits arsenic-transformed cell migration. We found that protein kinase Cα (PKCα) is significantly up-regulated in arsenic-transformed cells. Combining bioinformatics analysis with PKCα 3′-untranslated region vector luciferase reporter assays, we showed that PKCα is a direct target of miR-200b. Inhibiting PKCα activity or knocking down PKCα expression drastically reduced cell migration, phenocoping the inhibitory effect of overexpressing miR-200b. In contrast, forced expression of PKCα in miR-200b overexpressing cells impaired the inhibitory effect of miR-200b on cell migration. In addition, we also found a positive feedback loop between Wnt5b and PKCα in arsenic-transformed cells. Knocking down Wnt5b expression reduced phospho-PKC levels and cell migration; and knocking down PKCα expression decreased Wnt5b level and cell migration. Moreover, forced expression of PKCα increased Wnt5b and phospho-PKC levels and cell migration. Further mechanistic studies revealed that Rac1 is highly activated in arsenic-transformed cells and stably expressing miR-200b abolishes Rac1 activation changing actin cytoskeleton organization. Manipulating PKCα or Wnt5b expression levels significantly altered the level of active Rac1. Together, these findings indicate that miR-200b suppresses arsenic-transformed cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop and subsequently inhibiting Rac1 activation. PMID:24841200

MicroRNA-200b suppresses arsenic-transformed cell migration by targeting protein kinase Cα and Wnt5b-protein kinase Cα positive feedback loop and inhibiting Rac1 activation.

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Wang, Zhishan; Humphries, Brock; Xiao, Hua; Jiang, Yiguo; Yang, Chengfeng

2014-06-27

MicroRNA-200b (miR-200b) is a member of miR-200 family that has been found to inhibit cell migration and cancer metastasis; however, the underlying mechanism is not well understood. We previously reported that miR-200 expression is depleted in arsenic-transformed human bronchial epithelial cells with highly migratory and invasive characteristics, whereas stably re-expressing miR-200b strongly suppresses arsenic-transformed cell migration. This study was performed to investigate how miR-200b inhibits arsenic-transformed cell migration. We found that protein kinase Cα (PKCα) is significantly up-regulated in arsenic-transformed cells. Combining bioinformatics analysis with PKCα 3'-untranslated region vector luciferase reporter assays, we showed that PKCα is a direct target of miR-200b. Inhibiting PKCα activity or knocking down PKCα expression drastically reduced cell migration, phenocoping the inhibitory effect of overexpressing miR-200b. In contrast, forced expression of PKCα in miR-200b overexpressing cells impaired the inhibitory effect of miR-200b on cell migration. In addition, we also found a positive feedback loop between Wnt5b and PKCα in arsenic-transformed cells. Knocking down Wnt5b expression reduced phospho-PKC levels and cell migration; and knocking down PKCα expression decreased Wnt5b level and cell migration. Moreover, forced expression of PKCα increased Wnt5b and phospho-PKC levels and cell migration. Further mechanistic studies revealed that Rac1 is highly activated in arsenic-transformed cells and stably expressing miR-200b abolishes Rac1 activation changing actin cytoskeleton organization. Manipulating PKCα or Wnt5b expression levels significantly altered the level of active Rac1. Together, these findings indicate that miR-200b suppresses arsenic-transformed cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop and subsequently inhibiting Rac1 activation. © 2014 by The American Society for Biochemistry and Molecular

Inhibition of Glycogen Synthase Kinase or the Apoptotic Protein p53 Lowers the Threshold of Helium Cardioprotection In Vivo: The Role of Mitochondrial Permeability Transition

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Pagel, Paul S.; Krolikowski, John G.; Pratt, Phillip F.; Shim, Yon Hee; Amour, Julien; Warltier, David C.; Weihrauch, Dorothee

2008-01-01

BACKGROUND Prosurvival signaling kinases inhibit glycogen synthase kinase-3β (GSK-3β) activity and stimulate apoptotic protein p53 degradation. Helium produces cardioprotection by activating prosurvival kinases, but whether GSK and p53 inhibition mediate this process is unknown. We tested the hypothesis that inhibition of GSK or p53 lowers the threshold of helium cardioprotection via a mitochondrial permeability transition pore (mPTP)-dependent mechanism. METHODS Rabbits (n = 85) instrumented for hemodynamic measurement and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), or 1, 3, or 5 cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture (fraction of inspired oxygen concentration = 0.30) before LAD occlusion. Other rabbits received the GSK inhibitor SB 216763 (SB21; 0.2 or 0.6 mg/kg), the p53 inhibitor pifithrin-α (PIF; 1.5 or 3.0 mg/kg), or SB21 (0.2 mg/kg) or PIF (1.5 mg/kg) plus helium (1 cycle) before LAD occlusion in the presence or absence of the mPTP opener atractyloside (5 mg/kg). RESULTS Helium reduced (P < 0.05) myocardial infarct size (35 ± 6 [n = 7], 25 ± 4 [n = 7], and 20 ± 3% [n = 6] of area at risk, 1, 3, and 5 cycles, respectively) compared with control (44 ± 6% [n = 7]). SB21 (0.6 [n = 7] but not 0.2 mg/kg [n = 6]) and PIF (3.0 [n = 6] but not 1.5 mg/kg [n = 7]) also reduced necrosis. SB21 (0.2 mg/kg) or 1.5 mg/kg PIF (1.5 mg/kg) plus helium (1 cycle; n = 6 per group) decreased infarct size to an equivalent degree as three cycles of helium alone, and this cardioprotection was blocked by atractyloside (n = 7 per group). CONCLUSIONS Inhibition of GSK or p53 lowers the threshold of helium-induced preconditioning via a mPTP-dependent mechanism in vivo. PMID:18713881

Magnolol Alleviates Inflammatory Responses and Lipid Accumulation by AMP-Activated Protein Kinase-Dependent Peroxisome Proliferator-Activated Receptor α Activation

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Ye Tian

2018-02-01

Full Text Available Magnolol (MG is a kind of lignin isolated from Magnolia officinalis, which serves several different biological functions, such as antifungal, anticancer, antioxidant, and hepatoprotective functions. This study aimed to evaluate the protective effect of MG against oleic acid (OA-induced hepatic steatosis and inflammatory damage in HepG2 cells and in a tyloxapol (Ty-induced hyperlipidemia mouse model. Our findings indicated that MG can effectively inhibit OA-stimulated tumor necrosis factor α (TNF-α secretion, reactive oxygen species generation, and triglyceride (TG accumulation. Further study manifested that MG significantly suppressed OA-activated mitogen-activated protein kinase (MAPK and nuclear factor-kappa B (NF-κB signaling pathways and that these inflammatory responses can be negated by pretreatment with inhibitors of extracellular regulated protein kinase and c-Jun N-terminal kinase (U0126 and SP600125, respectively. In addition, MG dramatically upregulated peroxisome proliferator-activated receptor α (PPARα translocation and reduced sterol regulatory element-binding protein 1c (SREBP-1c protein synthesis and excretion, both of which are dependent upon the phosphorylation of adenosine monophosphate (AMP-activated protein kinase (AMPK, acetyl-CoA carboxylase, and AKT kinase (AKT. However, MG suspended the activation of PPARα expression and was thus blocked by pretreatment with LY294002 and compound c (specific inhibitors of AKT and AMPK. Furthermore, MG clearly alleviated serum TG and total cholesterol release; upregulated AKT, AMPK, and PPARα expression; suppressed SREBP-1c generation; and alleviated hepatic steatosis and dyslipidemia in Ty-induced hyperlipidemia mice. Taken together, these results suggest that MG exerts protective effects against steatosis, hyperlipidemia, and the underlying mechanism, which may be closely associated with AKT/AMPK/PPARα activation and MAPK/NF-κB/SREBP-1c inhibition.

Interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway in renal carcinogenesis of uninephrectomized rats.

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Yang, Ke-Ke; Sui, Yi; Zhou, Hui-Rong; Zhao, Hai-Lu

2017-05-01

Renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway both play important roles in carcinogenesis, but the interplay of renin-angiotensin system and adenosine monophosphate-activated protein kinase in carcinogenesis is not clear. In this study, we researched the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase in renal carcinogenesis of uninephrectomized rats. A total of 96 rats were stratified into four groups: sham, uninephrectomized, and uninephrectomized treated with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker. Renal adenosine monophosphate-activated protein kinase and its downstream molecule acetyl coenzyme A carboxylase were detected by immunohistochemistry and western blot at 10 months after uninephrectomy. Meanwhile, we examined renal carcinogenesis by histological transformation and expressions of Ki67 and mutant p53. During the study, fasting lipid profiles were detected dynamically at 3, 6, 8, and 10 months. The results indicated that adenosine monophosphate-activated protein kinase expression in uninephrectomized rats showed 36.8% reduction by immunohistochemistry and 89.73% reduction by western blot. Inversely, acetyl coenzyme A carboxylase expression increased 83.3% and 19.07% in parallel to hyperlipidemia at 6, 8, and 10 months. The histopathology of carcinogenesis in remnant kidneys was manifested by atypical proliferation and carcinoma in situ, as well as increased expressions of Ki67 and mutant p53. Intervention with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker significantly prevented the inhibition of adenosine monophosphate-activated protein kinase signaling pathway and renal carcinogenesis in uninephrectomized rats. In conclusion, the novel findings suggest that uninephrectomy-induced disturbance in adenosine monophosphate-activated protein kinase signaling pathway resulted in hyperlipidemia and

SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor.

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Bucholc, Maria; Ciesielski, Arkadiusz; Goch, Grażyna; Anielska-Mazur, Anna; Kulik, Anna; Krzywińska, Ewa; Dobrowolska, Grażyna

2011-02-04

SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb(3+) as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 10(5) M(-1). The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.

Characterization of pathogenic germline mutations in human Protein Kinases

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Orengo Christine A

2011-07-01

Full Text Available Abstract Background Protein Kinases are a superfamily of proteins involved in crucial cellular processes such as cell cycle regulation and signal transduction. Accordingly, they play an important role in cancer biology. To contribute to the study of the relation between kinases and disease we compared pathogenic mutations to neutral mutations as an extension to our previous analysis of cancer somatic mutations. First, we analyzed native and mutant proteins in terms of amino acid composition. Secondly, mutations were characterized according to their potential structural effects and finally, we assessed the location of the different classes of polymorphisms with respect to kinase-relevant positions in terms of subfamily specificity, conservation, accessibility and functional sites. Results Pathogenic Protein Kinase mutations perturb essential aspects of protein function, including disruption of substrate binding and/or effector recognition at family-specific positions. Interestingly these mutations in Protein Kinases display a tendency to avoid structurally relevant positions, what represents a significant difference with respect to the average distribution of pathogenic mutations in other protein families. Conclusions Disease-associated mutations display sound differences with respect to neutral mutations: several amino acids are specific of each mutation type, different structural properties characterize each class and the distribution of pathogenic mutations within the consensus structure of the Protein Kinase domain is substantially different to that for non-pathogenic mutations. This preferential distribution confirms previous observations about the functional and structural distribution of the controversial cancer driver and passenger somatic mutations and their use as a proxy for the study of the involvement of somatic mutations in cancer development.

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

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Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

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Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

Type II cGMP‑dependent protein kinase inhibits the migration, invasion and proliferation of several types of human cancer cells.

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Wu, Min; Wu, Yan; Qian, Hai; Tao, Yan; Pang, Ji; Wang, Ying; Chen, Yongchang

2017-10-01

Previous studies have indicated that type II cyclic guanosine monophosphate (cGMP)‑dependent protein kinase (PKG II) could inhibit the proliferation and migration of gastric cancer cells. However, the effects of PKG II on the biological functions of other types of cancer cells remain to be elucidated. Therefore, the aim of the present study was to investigate the effects of PKG II on cancer cells derived from various types of human tissues, including A549 lung, HepG2 hepatic, OS‑RC‑2 renal, SW480 colon cancer cells and U251 glioma cells. Cancer cells were infected with adenoviral constructs coding PKG II (Ad‑PKG II) to up‑regulate PKG II expression, and treated with 8‑(4‑chlorophenylthio) (8‑pCPT)‑cGMP to activate the kinase. A Cell Counting kit 8 assay was used to detect cell proliferation. Cell migration was measured using a Transwell assay, whereas a terminal deoxynucleotidyl transferase 2'‑deoxyuridine, 5'‑triphosphate nick‑end labeling assay was used to detect cell apoptosis. A pull‑down assay was used to investigate the activation of Ras‑related C3 botulinum toxin substrate (Rac) 1 and western blotting was used to detect the expression of proteins of interest. The present results demonstrated that EGF (100 ng/ml, 24 h) promoted the proliferation and migration of cancer cells, and it suppressed their apoptosis. In addition, treatment with EGF enhanced the activation of Rac1, and up‑regulated the protein expression of proliferating cell nuclear antigen, matrix metalloproteinase (MMP)2, MMP7 and B‑cell lymphoma (Bcl)‑2, whereas it down‑regulated the expression of Bcl‑2‑associated X protein. Transfection of cancer cells with Ad‑PKG II, and PKG II activation with 8‑pCPT‑cGMP, was identified to counteract the effects triggered by EGF. The present results suggested that PKG II may exert inhibitory effects on the proliferation and migration of various types of cancer cells.

In vitro, inhibition of mitogen-activated protein kinase pathways protects against bupivacaine- and ropivacaine-induced neurotoxicity

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Lirk, Philipp; Haller, Ingrid; Colvin, Hans Peter; Lang, Leopold; Tomaselli, Bettina; Klimaschewski, Lars; Gerner, Peter

2008-01-01

Animal models show us that specific activation of the p38 mitogen-activated protein kinase (MAPK) may be a pivotal step in lidocaine neurotoxicity, but this has not been investigated in the case of two very widely used local anesthetics, bupivacaine and ropivacaine. We investigated the hypotheses

Involvement of both protein kinase C and G proteins in superoxide production after IgE triggering in guinea pig eosinophils

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Toshiya Aizawa

1997-01-01

Full Text Available To study the function and mechanism of eosinophils via the low affinity IgE receptor (FceRII, we examined the production of 02 metabolites by measuring the luminol-dependent chemiluminescence (LDCL response and the generation of cysteinyl leukotrienes. Eosinophils obtained from guinea pig peritoneal fluid sensitized with horse serum were purified. Luminol-dependent chemiluminescence was induced by stimulation with monoclonal anti-CD23 antibody, but not by mouse serum (controls. The mean (±SEM value of LDCL was 20.6±1.3X103 c.p.m. This reaction consisted of an initial rapid phase and a propagation phase and ended within lOmin. Guinea pig eosinophils were histochemically stained with monoclonal anti-CD23 antibody. The major product generated in the LDCL response was superoxide, as determined by the measurement of superoxide by cytochrome c reduction and the complete inhibitory effect of superoxide dismutase on the LDCL response. Pretreatment with either pertussis toxin or cholera toxin inhibited the LDCL reaction. Depletion of bivalent ions by EDTA inhibited this response and the protein kinase C inhibitor D-sphingosin inhibited both 1-oleoyl-2-acetyl-glycerol-induced and FcϵRII-mediated LDCL. These findings suggest that the NADPH-protein kinase C pathway may be involved in the FceRII-mediated LDCL response in guinea pig eosinophils.

Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

KAUST Repository

Diaz Galicia, Miriam Escarlet

2018-01-01

is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain

Semiconductor technology in protein kinase research and drug discovery: sensing a revolution.

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Bhalla, Nikhil; Di Lorenzo, Mirella; Estrela, Pedro; Pula, Giordano

2017-02-01

Since the discovery of protein kinase activity in 1954, close to 600 kinases have been discovered that have crucial roles in cell physiology. In several pathological conditions, aberrant protein kinase activity leads to abnormal cell and tissue physiology. Therefore, protein kinase inhibitors are investigated as potential treatments for several diseases, including dementia, diabetes, cancer and autoimmune and cardiovascular disease. Modern semiconductor technology has recently been applied to accelerate the discovery of novel protein kinase inhibitors that could become the standard-of-care drugs of tomorrow. Here, we describe current techniques and novel applications of semiconductor technologies in protein kinase inhibitor drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase

International Nuclear Information System (INIS)

Nakanishi, S.; Yamada, K.; Kase, H.; Nakamura, S.; Nonomura, Y.

1988-01-01

Effects of K-252a, purified from the culture broth of Nocardiopsis sp., on the activity of myosin (light chain kinase were investigated. 1) K-252a affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca 2+ -dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca 2+ -independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10 -6 M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10 -4 M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lowere in the presence of 100 μM ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using [γ- 32 P]ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP. These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

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Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

2013-06-10

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc

Didymin Alleviates Hepatic Fibrosis Through Inhibiting ERK and PI3K/Akt Pathways via Regulation of Raf Kinase Inhibitor Protein

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Xing Lin

2016-12-01

Full Text Available Background: Didymin has been reported to have anti-cancer potential. However, the effect of didymin on liver fibrosis remains illdefined. Methods: Hepatic fibrosis was induced by CCl4 in rats. The effects of didymin on liver pathology and collagen accumulation were observed by hematoxylin-eosin and Masson's trichrome staining, respectively. Serum transaminases activities and collagen-related indicators levels were determined by commercially available kits. Moreover, the effects of didymin on hepatic stellate cell apoptosis and cell cycle were analyzed by flow cytometry. Mitochondrial membrane potential was detected by using rhodamine-123 dye. The expression of Raf kinase inhibitor protein (RKIP and the phosphorylation of the ERK/MAPK and PI3K/Akt pathways were assessed by Western blot. Results: Didymin significantly ameliorated chronic liver injury and collagen deposition. It strongly inhibited hepatic stellate cells proliferation, induced apoptosis and caused cell cycle arrest in G2/M phase. Moreover, didymin notably attenuated mitochondrial membrane potential, accompanied by release of cytochrome C. Didymin significantly inhibited the ERK/MAPK and PI3K/Akt pathways. The effects of didymin on the collagen accumulation in rats and on the biological behaviors of hepatic stellate cells were largely abolished by the specific RKIP inhibitor locostatin. Conclusion: Didymin alleviates hepatic fibrosis by inhibiting ERK/MAPK and PI3K/Akt pathways via regulation of RKIP expression.

cGMP-Dependent Protein Kinase Inhibitors in Health and Disease

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Jens Schlossmann

2013-02-01

Full Text Available cGMP-dependent protein kinases (PKG exhibit diverse physiological functions in the mammalian system e.g., in vascular and gastrointestinal smooth muscles, in platelets, in kidney, in bone growth, nociception and in the central nervous system. Furthermore, PKG were found in insects and in the malaria parasite Plasmodium falciparum. Two different genes of PKG exist: a the PKG-I gene that is expressed as cytosolic PKG-Iα or PKG-Iβ isoform, and b the PKG-II gene, which expresses the membrane associated PKG-II protein. The enzyme kinetics, the localization and the substrates of these PKG enzymes differ utilizing different physiological functions. Various inhibitors of PKG were developed directed against diverse functional regions of the kinase. These inhibitors of PKG have been used to analyse the specific functions of these enzymes. The review article will summarize these different inhibitors regarding their specificity and their present applications in vitro and in vivo. Furthermore, it will be discussed that the distinct inhibition of the PKG enzymes could be used as a valuable pharmacological target e.g., in the treatment of cardiovascular diseases, diarrhea, cancer or malaria.

A natural product from Cannabis sativa subsp. sativa inhibits homeodomain-interacting protein kinase 2 (HIPK2), attenuating MPP+-induced apoptosis in human neuroblastoma SH-SY5Y cells.

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Wang, Guan; Zhu, Lingjuan; Zhao, Yuqian; Gao, Suyu; Sun, Dejuan; Yuan, Jingquan; Huang, Yuxin; Zhang, Xue; Yao, Xinsheng

2017-06-01

Homeodomain-interacting protein kinase 2 (HIPK2) is a conserved serine/threonine kinase, which regulate transcription, cell differentiation, proliferation and apoptosis. Previous evidences indicated that HIPK2 could be involved in the pathogenesis of neurodegenerative diseases, suggesting as a novel target for Parkinson's disease (PD) therapeutic development. Herein, gene microarray analysis was performed to verify the key regulatory function of HIPK2 in PD. (Z)-methylp-hydroxycinnamate (ZMHC, 7) with other eighteen compounds were isolated from Cannabis sativa subsp. sativa, growing in Bama Yao Autonomous County, one of the five largest longevity regions of the world. Intriguingly, ZMHC was identified to bind HIPK2 with high affinity through molecular modeling and molecular dynamics (MD) simulations. Moreover, cell morphology, flow cytometry and western blot assay suggested that ZMHC inhibited HIPK2, which attenuated MPP + -induced apoptosis in SH-SY5Y cells. In conclusion, these findings discovered a natural product that inhibited HIPK2, and highlighted that ZMHC could be a potential precursor agent for future PD therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.

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Leonie Unterholzner

2011-09-01

Full Text Available Recognition of viruses by pattern recognition receptors (PRRs causes interferon-β (IFN-β induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1 and IκB kinase-ε (IKKε, which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.

The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

Science.gov (United States)

Aoubala, M; Holt, J; Clegg, R A; Rowlands, D J; Harris, M

2001-07-01

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission.

Science.gov (United States)

Baker, David A; Stewart, Lindsay B; Large, Jonathan M; Bowyer, Paul W; Ansell, Keith H; Jiménez-Díaz, María B; El Bakkouri, Majida; Birchall, Kristian; Dechering, Koen J; Bouloc, Nathalie S; Coombs, Peter J; Whalley, David; Harding, Denise J; Smiljanic-Hurley, Ela; Wheldon, Mary C; Walker, Eloise M; Dessens, Johannes T; Lafuente, María José; Sanz, Laura M; Gamo, Francisco-Javier; Ferrer, Santiago B; Hui, Raymond; Bousema, Teun; Angulo-Barturén, Iñigo; Merritt, Andy T; Croft, Simon L; Gutteridge, Winston E; Kettleborough, Catherine A; Osborne, Simon A

2017-09-05

To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG). The most potent compound (ML10) has an IC 50 of 160 pM in a PfPKG kinase assay and inhibits P. falciparum blood stage proliferation in vitro with an EC 50 of 2.1 nM. Oral dosing renders blood stage parasitaemia undetectable in vivo using a P. falciparum SCID mouse model. The series targets both merozoite egress and erythrocyte invasion, but crucially, also blocks transmission of mature P. falciparum gametocytes to Anopheles stephensi mosquitoes. A co-crystal structure of PvPKG bound to ML10, reveals intimate molecular contacts that explain the high levels of potency and selectivity we have measured. The properties of this series warrant consideration for further development to produce an antimalarial drug.Protein kinases are promising drug targets for treatment of malaria. Here, starting with a medicinal chemistry approach, Baker et al. generate an imidazopyridine that selectively targets Plasmodium falciparum PKG, inhibits blood stage parasite growth in vitro and in mice and blocks transmission to mosquitoes.

Identification of the protein kinase C phosphorylation site in neuromodulin

International Nuclear Information System (INIS)

Apel, E.D.; Byford, M.F.; Au, D.; Walsh, K.A.; Storm, D.R.

1990-01-01

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin and the authors have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar K m values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [γ- 32 P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32 P-labeled tryptic peptide was generated from phosphorylated neuromodulin. They conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmoculin to neuromodulin. The proximity of serine-41 to the calmodulin binding domain in neuromodulin very likely explains the effect of phosphorylation on the affinity of neuromodulin for calmodulin

Pre-ERCP infusion of semapimod, a mitogen-activated protein kinases inhibitor, lowers post-ERCP hyperamylasemia but not pancreatitis incidence

NARCIS (Netherlands)

van Westerloo, David J.; Rauws, Erik A.; Hommes, Daan; de Vos, Alex F.; van der Poll, Tom; Powers, Barbara L.; Fockens, Paul; Dijkgraaf, Marcel G. W.; Bruno, Marco J.

2008-01-01

BACKGROUND: Acute pancreatitis and hyperamylasemia are frequent complications of an ERCP. Semapimod is a synthetic guanylhydrazone that inhibits the mitogen-activated protein kinase (MAPK) pathway, macrophage activation, and the production of several inflammatory cytokines. OBJECTIVE: This study

SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-Type Protein Kinase, Is Important for Abscisic Acid Responses in Arabidopsis through Phosphorylation of ABSCISIC ACID-INSENSITIVE51[OPEN

Science.gov (United States)

Zhou, Xiaona; Hao, Hongmei; Zhang, Yuguo; Bai, Yili; Zhu, Wenbo; Qin, Yunxia; Yuan, Feifei; Zhao, Feiyi; Wang, Mengyao; Hu, Jingjiang; Xu, Hong; Guo, Aiguang; Zhao, Huixian; Zhao, Yang; Cao, Cuiling; Yang, Yongqing; Schumaker, Karen S.; Guo, Yan; Xie, Chang Gen

2015-01-01

Abscisic acid (ABA) plays an essential role in seed germination. In this study, we demonstrate that one SNF1-RELATED PROTEIN KINASE3-type protein kinase, SOS2-LIKE PROTEIN KINASE5 (PKS5), is involved in ABA signal transduction via the phosphorylation of an interacting protein, ABSCISIC ACID-INSENSITIVE5 (ABI5). We found that pks5-3 and pks5-4, two previously identified PKS5 superactive kinase mutants with point mutations in the PKS5 FISL/NAF (a conserved peptide that is necessary for interaction with SOS3 or SOS3-LIKE CALCIUM BINDING PROTEINs) motif and the kinase domain, respectively, are hypersensitive to ABA during seed germination. PKS5 was found to interact with ABI5 in yeast (Saccharomyces cerevisiae), and this interaction was further confirmed in planta using bimolecular fluorescence complementation. Genetic studies revealed that ABI5 is epistatic to PKS5. PKS5 phosphorylates a serine (Ser) residue at position 42 in ABI5 and regulates ABA-responsive gene expression. This phosphorylation was induced by ABA in vivo and transactivated ABI5. Expression of ABI5, in which Ser-42 was mutated to alanine, could not fully rescue the ABA-insensitive phenotypes of the abi5-8 and pks5-4abi5-8 mutants. In contrast, mutating Ser-42 to aspartate rescued the ABA insensitivity of these mutants. These data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for the ABA regulation of seed germination and gene expression in Arabidopsis (Arabidopsis thaliana). PMID:25858916

Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

International Nuclear Information System (INIS)

Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

2014-01-01

Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li 2 CO 3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li 2 CO 3 did not affect PI3K-mediated PI(3,4,5)P 3 production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li 2 CO 3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li 2 CO 3 significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li 2 CO 3 significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity

cAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner.

Science.gov (United States)

Nedvetsky, Pavel I; Zhao, Xiaocheng; Mathivet, Thomas; Aspalter, Irene M; Stanchi, Fabio; Metzger, Ross J; Mostov, Keith E; Gerhardt, Holger

2016-10-01

cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior. © 2016. Published by The Company of Biologists Ltd.

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

DEFF Research Database (Denmark)

John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette

2010-01-01

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified...... LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric...... kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co...

Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

Energy Technology Data Exchange (ETDEWEB)

Voss, Kelsey; Amaya, Moushimi [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Mueller, Claudius [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Roberts, Brian [Leidos Health Life Sciences, 5202 Presidents Court, Suite 110, Frederick, MD (United States); Kehn-Hall, Kylene; Bailey, Charles [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Petricoin, Emanuel [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Narayanan, Aarthi, E-mail: anaraya1@gmu.edu [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States)

2014-11-15

New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells.

Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

International Nuclear Information System (INIS)

Voss, Kelsey; Amaya, Moushimi; Mueller, Claudius; Roberts, Brian; Kehn-Hall, Kylene; Bailey, Charles; Petricoin, Emanuel; Narayanan, Aarthi

2014-01-01

New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells

Anti-glycophorin C induces mitochondrial membrane depolarization and a loss of extracellular regulated kinase 1/2 protein kinase activity that is prevented by pretreatment with cytochalasin D: implications for hemolytic disease of the fetus and newborn caused by anti-Ge3.

Science.gov (United States)

Micieli, Jonathan A; Wang, Duncheng; Denomme, Gregory A

2010-08-01

Anti-glycophorin C (GPC), blood group antibodies of which cause hemolytic disease of the fetus and newborn (HDFN), is a potent inhibitor of erythroid progenitor cell growth. The cellular mechanism for growth inhibition has not been characterized. K562 cells were incubated in the presence of either anti-GPC, an immunoglobulin G isotype control, an inhibitor of actin polymerization called cytochalasin D with anti-GPC, or cytochalasin D alone. The JC-1 cationic dye was used to detect mitochondrial depolarization and the activity of the mitogen-activated protein kinases was assessed by Western blotting. Anti-GPC inhibits the activity of extracellular regulated kinase (ERK)1/2 within 10 minutes but does not alter the activity of p38 or c-Jun N-terminal kinase. After 24 hours there was a significant loss of mitochondrial membrane potential compared to isotype control–treated cells. Both the ERK1/2 inhibition and the loss of mitochondrial potential were prevented by pretreatment with cytochalasin D. A cell surface antibody can cause anemia by altering the signaling pathways in erythroid cells by promoting depolarization of mitochondria via cytoskeletal rearrangement. The observation that neonates with anti-GPC HDFN are unresponsive to erythropoietin can be explained by the antibody inhibiting a protein kinase through which this hematopoietic growth factor achieves its effects.

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

Science.gov (United States)

Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

dsRNA-Dependent Protein Kinase PKR and its Role in Stress, Signaling and HCV Infection

Directory of Open Access Journals (Sweden)

Eliane F. Meurs

2012-10-01

Full Text Available The double-stranded RNA-dependent protein kinase PKR plays multiple roles in cells, in response to different stress situations. As a member of the interferon (IFN‑Stimulated Genes, PKR was initially recognized as an actor in the antiviral action of IFN, due to its ability to control translation, through phosphorylation, of the alpha subunit of eukaryotic initiation factor 2 (eIF2a. As such, PKR participates in the generation of stress granules, or autophagy and a number of viruses have designed strategies to inhibit its action. However, PKR deficient mice resist most viral infections, indicating that PKR may play other roles in the cell other than just acting as an antiviral agent. Indeed, PKR regulates several signaling pathways, either as an adapter protein and/or using its kinase activity. Here we review the role of PKR as an eIF2a kinase, its participation in the regulation of the NF-kB, p38MAPK and insulin pathways, and we focus on its role during infection with the hepatitis C virus (HCV. PKR binds the HCV IRES RNA, cooperates with some functions of the HCV core protein and may represent a target for NS5A or E2. Novel data points out for a role of PKR as a pro-HCV agent, both as an adapter protein and as an eIF2a-kinase, and in cooperation with the di-ubiquitin-like protein ISG15. Developing pharmaceutical inhibitors of PKR may help in resolving some viral infections as well as stress-related damages.

Alteration of sodium, potassium-adenosine triphosphatase activity in rabbit ciliary processes by cyclic adenosine monophosphate-dependent protein kinase

International Nuclear Information System (INIS)

Delamere, N.A.; Socci, R.R.; King, K.L.

1990-01-01

The response of sodium, potassium-adenosine triphosphatase (Na,K-ATPase) to cyclic adenosine monophosphate (cAMP)-dependent protein kinase was examined in membranes obtained from rabbit iris-ciliary body. In the presence of the protein kinase together with 10(-5) M cAMP, Na,K-ATPase activity was reduced. No change in Na,K-ATPase activity was detected in response to the protein kinase without added cAMP. Likewise cAMP alone did not alter Na,K-ATPase activity. Reduction of Na,K-ATPase activity was also observed in the presence of the cAMP-dependent protein kinase catalytic subunit. The response of the enzyme to the kinase catalytic subunit was also examined in membranes obtained from rabbit ciliary processes. In the presence of 8 micrograms/ml of the catalytic subunit, ciliary process Na,K-ATPase activity was reduced by more than 50%. To examine whether other ATPases were suppressed by the protein kinase, calcium-stimulated ATPase activity was examined; its activity was stimulated by the catalytic subunit. To test whether the response of the ciliary process Na,K-ATPase is unique, experiments were also performed using membrane preparations from rabbit lens epithelium or rabbit kidney; the catalytic subunit significantly reduced the activity of Na,K-ATPase from the kidney but not the lens. These Na,K-ATPase studies suggest that in the iris-ciliary body, cAMP may alter sodium pump activity. In parallel 86Rb uptake studies, we observed that ouabain-inhibitable potassium uptake by intact pieces of iris-ciliary body was reduced by exogenous dibutryl cAMP or by forskolin

Protein kinase A antagonist inhibits β-catenin nuclear translocation, c-Myc and COX-2 expression and tumor promotion in ApcMin/+ mice

Directory of Open Access Journals (Sweden)

Brudvik Kristoffer W

2011-12-01

Full Text Available Abstract Background The adenomatous polyposis coli (APC protein is part of the destruction complex controlling proteosomal degradation of β-catenin and limiting its nuclear translocation, which is thought to play a gate-keeping role in colorectal cancer. The destruction complex is inhibited by Wnt-Frz and prostaglandin E2 (PGE2 - PI-3 kinase pathways. Recent reports show that PGE2-induced phosphorylation of β-catenin by protein kinase A (PKA increases nuclear translocation indicating two mechanisms of action of PGE2 on β-catenin homeostasis. Findings Treatment of ApcMin/+ mice that spontaneously develop intestinal adenomas with a PKA antagonist (Rp-8-Br-cAMPS selectively targeting only the latter pathway reduced tumor load, but not the number of adenomas. Immunohistochemical characterization of intestines from treated and control animals revealed that expression of β-catenin, β-catenin nuclear translocation and expression of the β-catenin target genes c-Myc and COX-2 were significantly down-regulated upon Rp-8-Br-cAMPS treatment. Parallel experiments in a human colon cancer cell line (HCT116 revealed that Rp-8-Br-cAMPS blocked PGE2-induced β-catenin phosphorylation and c-Myc upregulation. Conclusion Based on our findings we suggest that PGE2 act through PKA to promote β-catenin nuclear translocation and tumor development in ApcMin/+ mice in vivo, indicating that the direct regulatory effect of PKA on β-catenin nuclear translocation is operative in intestinal cancer.

AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

Directory of Open Access Journals (Sweden)

Maayan Shaked

Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

Aurora B kinase inhibition in mitosis: strategies for optimising the use of aurora kinase inhibitors such as AT9283.

Science.gov (United States)

Curry, Jayne; Angove, Hayley; Fazal, Lynsey; Lyons, John; Reule, Matthias; Thompson, Neil; Wallis, Nicola

2009-06-15

Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.

A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

Science.gov (United States)

Hwang, I.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2000-01-01

The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

Phosphatidylinositol-3-kinase-dependent phosphorylation of SLP-76 by the lymphoma-associated ITK-SYK fusion-protein

International Nuclear Information System (INIS)

Hussain, Alamdar; Faryal, Rani; Nore, Beston F.; Mohamed, Abdalla J.; Smith, C.I. Edvard

2009-01-01

Recurrent chromosomal translocations have long been implicated in various types of lymphomas and other malignancies. Novel recurrent t(5;9)(q33;q22) has been recently discovered in un-specified peripheral T-cell lymphoma. To elucidate the role of this translocation, the corresponding fusion construct encoding the N-terminal portion of the ITK kinase and the C-terminal catalytic region of the SYK kinase was generated. We herein show that the ITK-SYK fusion-protein is constitutively active. Moreover, we demonstrate that ITK-SYK is phosphorylated on key tyrosine residues and is capable of potently phosphorylating the related adapter proteins BLNK and SLP-76. In transiently transfected cells, SYK was phosphorylated at Y352 but not detectably at the activation-loop tyrosines Y525/Y526. In contrast, ITK-SYK was phosphorylated both at Y212 and the activation-loop tyrosines Y385/Y386, corresponding to Y352 and Y525/Y526 in SYK, respectively. In resting primary lymphocytes, ITK-SYK predominantly localizes to the cell surface. In addition, we demonstrate that following stimulation, the ITK-SYK fusion-protein in cell lines translocates to the cell membrane and, moreover, that this phenomenon as well as SLP-76 phosphorylation are blocked upon phosphatidylinositol-3-kinase (PI3-kinase) inhibition.

Adenovirus DNA binding protein inhibits SrCap-activated CBP and CREB-mediated transcription

International Nuclear Information System (INIS)

Xu Xiequn; Tarakanova, Vera; Chrivia, John; Yaciuk, Peter

2003-01-01

The SNF2-related CBP activator protein (SrCap) is a potent activator of transcription mediated by CBP and CREB. We have previously demonstrated that the Adenovirus 2 DNA Binding Protein (DBP) binds to SrCap and inhibits the transcription mediated by the carboxyl-terminal region of SrCap (amino acids 1275-2971). We report here that DBP inhibits the ability of full-length SrCap (1-2971) to activate transcription mediated by Gal-CREB and Gal-CBP. In addition, DBP also inhibits the ability of SrCap to enhance Protein Kinase A (PKA) activated transcription of the enkaphalin promoter. DBP was found to dramatically inhibit transcription of a mammalian two-hybrid system that was dependent on the interaction of SrCap and CBP binding domains. We also found that DBP has no effect on transcription mediated by a transcriptional activator that is not related to SrCap, indicating that our reported transcriptional inhibition is specific for SrCap and not due to nonspecific effects of DBP's DNA binding activity on the CAT reporter plasmid. Taken together, these results suggest a model in which DBP inhibits cellular transcription mediated by the interaction between SrCap and CBP

Silibinin activates AMP-activated protein kinase to protect neuronal cells from oxygen and glucose deprivation-re-oxygenation.

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Xie, Zhi; Ding, Sheng-quan; Shen, Ya-fang

2014-11-14

In this study, we explored the cytoprotective potential of silibinin against oxygen-glucose deprivation (OGD)-induced neuronal cell damages, and studied underling mechanisms. In vitro model of ischemic stroke was created by keeping neuronal cells (SH-SY5Y cells and primary mouse cortical neurons) in an OGD condition followed by re-oxygenation. Pre-treatment of silibinin significantly inhibited OGD/re-oxygenation-induced necrosis and apoptosis of neuronal cells. OGD/re-oxygenation-induced reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) reduction were also inhibited by silibinin. At the molecular level, silibinin treatment in SH-SY5Y cells and primary cortical neurons led to significant AMP-activated protein kinase (AMPK) signaling activation, detected by phosphorylations of AMPKα1, its upstream kinase liver kinase B1 (LKB1) and the downstream target acetyl-CoA Carboxylase (ACC). Pharmacological inhibition or genetic depletion of AMPK alleviated the neuroprotective ability of silibinin against OGD/re-oxygenation. Further, ROS scavenging ability by silibinin was abolished with AMPK inhibition or silencing. While A-769662, the AMPK activator, mimicked silibinin actions and suppressed ROS production and neuronal cell death following OGD/re-oxygenation. Together, these results show that silibinin-mediated neuroprotection requires activation of AMPK signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

Torilin Inhibits Inflammation by Limiting TAK1-Mediated MAP Kinase and NF-κB Activation

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Mehari Endale

2017-01-01

Full Text Available Torilin, a sesquiterpene isolated from the fruits of Torilis japonica, has shown antimicrobial, anticancer, and anti-inflammatory properties. However, data on the mechanism of torilin action against inflammation is limited. This study aimed at determining the anti-inflammatory property of torilin in LPS-induced inflammation using in vitro model of inflammation. We examined torilin’s effect on expression levels of inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 macrophages. The involvement of NF-kB and AP-1, MAP kinases, and adaptor proteins were assessed. Torilin strongly inhibited LPS-induced NO release, iNOS, PGE2, COX-2, NF-α, IL-1β, IL-6, and GM-CSF gene and protein expressions. In addition, MAPKs were also suppressed by torilin pretreatment. Involvement of ERK1/2, P38MAPK, and JNK1/2 was further confirmed by PD98059, SB203580, and SP600125 mediated suppression of iNOS and COX-2 proteins. Furthermore, torilin attenuated NF-kB and AP-1 translocation, DNA binding, and reporter gene transcription. Interestingly, torilin inhibited TAK1 kinase activation with the subsequent suppression of MAPK-mediated JNK, p38, ERK1/2, and AP-1 (ATF-2 and c-jun activation and IKK-mediated I-κBα degradation, p65/p50 activation, and translocation. Together, the results revealed the suppression of NF-κB and AP-1 regulated inflammatory mediator and cytokine expressions, suggesting the test compound’s potential as a candidate anti-inflammatory agent.

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

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Elena Elizabeth Bagley

2014-06-01

Full Text Available Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1 currents in periaqueductal gray (PAG neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1

How protein kinases co-ordinate mitosis in animal cells.

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Ma, Hoi Tang; Poon, Randy Y C

2011-04-01

Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

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Ngo, HT; Pham, Long; Kim, JW

2013-01-01

Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway

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Joubert, Pierre-Emmanuel; Stapleford, Kenneth; Guivel-Benhassine, Florence; Vignuzzi, Marco; Schwartz, Olivier; Albert, Matthew L.

2015-01-01

Chikungunya virus (CHIKV), the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell’s cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K) and MAP kinase-activated protein kinase (MnKs) activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition. PMID:26317997

Inhibition of mTORC1 Enhances the Translation of Chikungunya Proteins via the Activation of the MnK/eIF4E Pathway.

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Pierre-Emmanuel Joubert

2015-08-01

Full Text Available Chikungunya virus (CHIKV, the causative agent of a major epidemic spanning five continents, is a positive stranded mRNA virus that replicates using the cell's cap-dependent translation machinery. Despite viral infection inhibiting mTOR, a metabolic sensor controls cap-dependent translation, viral proteins are efficiently translated. Rapalog treatment, silencing of mtor or raptor genes, but not rictor, further enhanced CHIKV infection in culture cells. Using biochemical assays and real time imaging, we demonstrate that this effect is independent of autophagy or type I interferon production. Providing in vivo evidence for the relevance of our findings, mice treated with mTORC1 inhibitors exhibited increased lethality and showed a higher sensitivity to CHIKV. A systematic evaluation of the viral life cycle indicated that inhibition of mTORC1 has a specific positive effect on viral proteins, enhancing viral replication by increasing the translation of both structural and nonstructural proteins. Molecular analysis defined a role for phosphatidylinositol-3 kinase (PI3K and MAP kinase-activated protein kinase (MnKs activation, leading to the hyper-phosphorylation of eIF4E. Finally, we demonstrated that in the context of CHIKV inhibition of mTORC1, viral replication is prioritized over host translation via a similar mechanism. Our study reveals an unexpected bypass pathway by which CHIKV protein translation overcomes viral induced mTORC1 inhibition.

Characterization of goat inner cell mass derived cells in double kinase inhibition condition

International Nuclear Information System (INIS)

Wei, Qiang; Xi, Qihui; Liu, Xiaokun; Meng, Kai; Zhao, Xiaoe; Ma, Baohua

2017-01-01

The identification of small molecular inhibitors, which were reported to promote the derivation of mouse and human embryonic stem cells (ESCs), provides a potential strategy for the derivation of domesticated ungulate ESCs. In present study, goat inner cell mass (ICM) derived cells in the double inhibition (2i) condition, in which, mitogen-activated protein kinase kinase (MAP2K) and glycogen synthase kinase 3 (GSK3) were inhibited by PD0325901 and BIO respectively, were characterized. The results showed that goat ICM derived cells in 2i medium adding leukaemia inhibitor factor (LIF) possessed a mouse ES-like morphology. But these cells had much compromised proliferation capacity, resulting in difficulty in expansion. In 2i alone medium, goat ICM derived cells possessed primate ES-like morphology. These cells expressed pluripotent markers and could differentiate into derivatives of three germ layers in vitro. However, these cells could not be proliferated in long-term (persisted for 15 passages) because of spontaneously neural differentiation. Additionally, goat ICM derived cells could be inducing differentiated into neural lineage in vitro. Although goat ESCs could not be established in PD0325901 and BIO alone medium, this derivation condition provides a useful research system to find signaling molecular those regulate early embryonic development and pluripotency in goat. - Highlights: • Goat inner cell mass derived cells possessed finite pluripotency in 2i condition. • These cells could not be proliferated in long-term in 2i condition. • These cells could spontaneously and inductively differentiate into neural lineage.

Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition

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Ciomperlik, Jessica J. [Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 (United States); Basta, Holly A. [Department of Biology, Rocky Mountain College, Billings, MT (United States); Palmenberg, Ann C., E-mail: acpalmen@wisc.edu [Institute for Molecular Virology and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706 (United States)

2016-01-15

Cardiovirus Leader proteins (L{sub X}) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus L{sub M} structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus L{sub E}, and also larger complexes with L{sub E}:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant L{sub S} and L{sub T} from Theiloviruses. When mutations were introduced to alter the L{sub E} zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on L{sub E} must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by L{sub E}.

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

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Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

2015-01-01

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

Salidroside pretreatment attenuates apoptosis and autophagy during hepatic ischemia–reperfusion injury by inhibiting the mitogen-activated protein kinase pathway in mice

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Feng J

2017-07-01

Full Text Available Jiao Feng,1,* Qinghui Zhang,2,* Wenhui Mo,3,* Liwei Wu,1 Sainan Li,1 Jingjing Li,1 Tong Liu,1 Shizan Xu,4 Xiaoming Fan,5 Chuanyong Guo1 1Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, 2Department of Clinical Laboratory, Kunshan First People’s Hospital Affiliated to Jiangsu University, Kunshan, JiangSu, 3Department of Gastroenterology, Minhang Hospital, Fudan University, Shanghai, 4Department of Gastroenterology, Shanghai Tenth People’s Hospital, School of Clinical Medicine of Nanjing Medical University, Shanghai, 5Department of Gastroenterology, Jinshan Hospital of Fudan University, Jinshan, Shanghai, China *These authors contributed equally to this work Abstract: Ischemia–reperfusion injury (IRI contributes to liver damage in many clinical situations, such as liver resection and liver transplantation. In the present study, we investigated the effects of the antioxidant, anti-inflammatory, and anticancer agent salidroside (Sal on hepatic IRI in mice. The mice were randomly divided into six groups: normal control, Sham, Sal (20 mg/kg, IRI, IRI + Sal (10 mg/kg, and IRI + Sal (20 mg/kg. We measured liver enzymes, proinflammatory cytokines, TNF-α and interleukin-6, and apoptosis- and autophagy-related marker proteins at 2, 8, and 24 hours after reperfusion. Components of mitogen-activated protein kinase (MAPK signaling, including P-38, jun N-terminal kinase (JNK, and extracellular signal-regulated kinase (ERK, were also measured using an MAPK activator anisomycin to deduce their roles in hepatic IRI. Our results show that Sal safely protects hepatocytes from IRI by reducing levels of liver enzymes in the serum. These findings were confirmed by histopathology. We concluded that Sal protects hepatocytes from IRI partly by inhibiting the activation of MAPK signaling, including the phosphorylation of P38, JNK, and ERK. This ameliorates inflammatory reactions, apoptosis, and

Involvement of stress-activated protein kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents.

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Saleem, A; Datta, R; Yuan, Z M; Kharbanda, S; Kufe, D

1995-12-01

The cellular response to 1-beta-D-arabinofuranosylcytosine (ara-C) includes activation of Jun/AP-1, induction of c-jun transcription, and programmed cell death. The stress-activated protein (SAP) kinases stimulate the transactivation function of c-jun by amino terminal phosphorylation. The present work demonstrates that ara-C activates p54 SAP kinase. The finding that SAP kinase is also activated by alkylating agents (mitomycin C and cisplatinum) and the topoisomerase I inhibitor 9-amino-camptothecin supports DNA damage as an initial signal in this cascade. The results demonstrate that ara-C also induces binding of SAP kinase to the SH2/SH3-containing adapter protein Grb2. SAP kinase binds to the SH3 domains of Grb2, while interaction of the p85 alpha-subunit of phosphatidylinositol 3-kinase complex. The results also demonstrate that ara-C treatment is associated with inhibition of lipid and serine kinase activities of PI 3-kinase. The potential significance of the ara-C-induced interaction between SAP kinase and PI 3-kinase is further supported by the demonstration that Wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. The finding that Wortmannin treatment is also associated with internucleosomal DNA fragmentation may support a potential link between PI 3-kinase and regulation of both SAP kinase and programmed cell death.

Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

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Annika Sommerfeld

2015-05-01

Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

Extract of Polygala tenuifolia Alleviates Stress-Exacerbated Atopy-Like Skin Dermatitis through the Modulation of Protein Kinase A and p38 Mitogen-Activated Protein Kinase Signaling Pathway

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Bongjun Sur

2017-01-01

Full Text Available Atopic dermatitis (AD and stress create a vicious cycle: stress exacerbates atopic symptoms, and atopic disease elicits stress and anxiety. Targeting multiple pathways including stress and allergic inflammation is, therefore, important for treating AD. In this study, we investigated the remedial value of Polygala tenuifolia Willd. (PTW for treating immobilization (IMO stress-exacerbated atopy-like skin dermatitis and its underlying mechanism. Trimellitic anhydride (TMA was applied to dorsal skin for sensitization and subsequently both ears for eliciting T-cell-dependent contact hypersensitivity in mice, which underwent 2 h-IMO stress and PTW administration for the latter 6 and 9 days in the ear exposure period of TMA, respectively. To elicit in vitro degranulation of human mast cell line-1 (HMC-1, 10 µM substance P (SP and 200 nM corticotrophin-releasing factor (CRF were sequentially added with 48 h-interval. PTW extract (500 µg/mL was added 30 min before CRF treatment. IMO stress exacerbated TMA-induced scratching behavior by 252%, and increased their blood corticosterone levels by two-fold. Treatment with 250 mg/kg PTW significantly restored IMO stress-exacerbated scratching behavior and other indicators such as skin inflammation and water content, lymph node weights, and serum histamine and immunoglobulin E (lgE levels. Furthermore, it also reversed TMA-stimulated expression of tumor necrosis factor (TNF-α and interleukin (IL-4 mRNAs in ear tissues. PTW significantly inhibited SP/CRF-stimulated degranulation of HMC-1 cells, subsequent tryptase secretion, and protein kinase A (PKA activity. PTW also selectively inhibited p38 mitogen-activated protein kinase (MAPK phosphorylation in SP/CRF-treated HMC-1 cells. PTW significantly inhibited HMC-1 cell degranulation and alleviated IMO stress-exacerbated atopic dermatitis symptoms by modulating the PKA/p38 MAPK signaling pathway.