cGMP-dependent protein kinase type I is implicated in the regulation of the timing and quality of sleep and wakefulness.

Directory of Open Access Journals (Sweden)

Sonja Langmesser

Full Text Available Many effects of nitric oxide (NO are mediated by the activation of guanylyl cyclases and subsequent production of the second messenger cyclic guanosine-3',5'-monophosphate (cGMP. cGMP activates cGMP-dependent protein kinases (PRKGs, which can therefore be considered downstream effectors of NO signaling. Since NO is thought to be involved in the regulation of both sleep and circadian rhythms, we analyzed these two processes in mice deficient for cGMP-dependent protein kinase type I (PRKG1 in the brain. Prkg1 mutant mice showed a strikingly altered distribution of sleep and wakefulness over the 24 hours of a day as well as reductions in rapid-eye-movement sleep (REMS duration and in non-REM sleep (NREMS consolidation, and their ability to sustain waking episodes was compromised. Furthermore, they displayed a drastic decrease in electroencephalogram (EEG power in the delta frequency range (1-4 Hz under baseline conditions, which could be normalized after sleep deprivation. In line with the re-distribution of sleep and wakefulness, the analysis of wheel-running and drinking activity revealed more rest bouts during the activity phase and a higher percentage of daytime activity in mutant animals. No changes were observed in internal period length and phase-shifting properties of the circadian clock while chi-squared periodogram amplitude was significantly reduced, hinting at a less robust oscillator. These results indicate that PRKG1 might be involved in the stabilization and output strength of the circadian oscillator in mice. Moreover, PRKG1 deficiency results in an aberrant pattern, and consequently a reduced quality, of sleep and wakefulness, possibly due to a decreased wake-promoting output of the circadian system impinging upon sleep.

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

DEFF Research Database (Denmark)

Issinger, O G; Beier, H; Speichermann, N

1980-01-01

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were...... by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins...... from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates...

cGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland

Science.gov (United States)

De Jonge, Hugo R.; Tilly, Ben C.; Hogema, Boris M.; Pfau, Daniel J.; Kelley, Catherine A.; Kelley, Megan H.; Melita, August M.; Morris, Montana T.; Viola, Ryan M.

2013-01-01

The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG. PMID:24259420

Enhanced expression of a calcium-dependent protein kinase

Indian Academy of Sciences (India)

Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss ...

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

Directory of Open Access Journals (Sweden)

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

Regulation of the Na(+)-K(+)-2Cl(-) cotransporter by cGMP/cGMP-dependent protein kinase I after furosemide administration.

Science.gov (United States)

Limmer, Franziska; Schinner, Elisabeth; Castrop, Hayo; Vitzthum, Helga; Hofmann, Franz; Schlossmann, Jens

2015-10-01

Sodium chloride reabsorption in the thick ascending limb of the loop of Henle is mediated by the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). The loop diuretic furosemide is a potent inhibitor of NKCC2. However, less is known about the mechanism regulating the electrolyte transporter. Considering the well-established effects of nitric oxide on NKCC2 activity, cGMP is likely involved in this regulation. cGMP-dependent protein kinase I (cGKI; PKGI) is a cGMP target protein that phosphorylates different substrates after activation through cGMP. We investigated the potential correlation between the cGMP/cGKI pathway and NKCC2 regulation. We treated wild-type (wt) and cGKIα-rescue mice with furosemide. cGKIα-rescue mice expressed cGKIα only under the control of the smooth muscle-specific transgelin (SM22) promoter in a cGKI deficient background. Furosemide treatment increased the urine excretion of sodium and chloride in cGKIα-rescue mice compared to that in wt mice. We analyzed the phosphorylation of NKCC2 by western blotting and immunostaining using the phosphospecific antibody R5. The administration of furosemide significantly increased the phosphorylated NKCC2 signal in wt but not in cGKIα-rescue mice. NKCC2 activation led to its phosphorylation and membrane translocation. To examine whether cGKI was involved in this process, we analyzed vasodilator-stimulated phosphoprotein, which is phosphorylated by cGKI. Furosemide injection resulted in increased vasodilator-stimulated phosphoprotein phosphorylation in wt mice. We hypothesize that furosemide administration activated cGKI, leading to NKCC2 phosphorylation and membrane translocation. This cGKI-mediated pathway could be a mechanism to compensate for the inhibitory effect of furosemide on NKCC2. © 2015 FEBS.

Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase.

Directory of Open Access Journals (Sweden)

Christine S Hopp

Full Text Available Signalling by 3'-5'-cyclic guanosine monophosphate (cGMP exists in virtually all eukaryotes. In the apicomplexan parasite Plasmodium, the cGMP-dependent protein kinase (PKG has previously been reported to play a critical role in four key stages of the life cycle. The Plasmodium falciparum isoform (PfPKG is essential for the initiation of gametogenesis and for blood stage schizont rupture and work on the orthologue from the rodent malaria parasite P. berghei (PbPKG has shown additional roles in ookinete differentiation and motility as well as liver stage schizont development. In the present study, PfPKG expression and subcellular location in asexual blood stages was investigated using transgenic epitope-tagged PfPKG-expressing P. falciparum parasites. In Western blotting experiments and immunofluorescence analysis (IFA, maximal PfPKG expression was detected at the late schizont stage. While IFA suggested a cytosolic location, a degree of overlap with markers of the endoplasmic reticulum (ER was found and subcellular fractionation showed some association with the peripheral membrane fraction. This broad localisation is consistent with the notion that PfPKG, as with the mammalian orthologue, has numerous cellular substrates. This idea is further supported by the global protein phosphorylation pattern of schizonts which was substantially changed following PfPKG inhibition, suggesting a complex role for PfPKG during schizogony.

cGMP-dependent protein kinase I, the circadian clock, sleep and learning.

Science.gov (United States)

Feil, Robert; Hölter, Sabine M; Weindl, Karin; Wurst, Wolfgang; Langmesser, Sonja; Gerling, Andrea; Feil, Susanne; Albrecht, Urs

2009-07-01

The second messenger cGMP controls cardiovascular and gastrointestinal homeostasis in mammals. However, its physiological relevance in the nervous system is poorly understood.1 Now, we have reported that the cGMP-dependent protein kinase type I (PRKG1) is implicated in the regulation of the timing and quality of sleep and wakefulness.2Prkg1 mutant mice showed altered distribution of sleep and wakefulness as well as reduction in rapid-eye-movement sleep (REMS) duration and in non-REMS consolidation. Furthermore, the ability to sustain waking episodes was compromised. These observations were also reflected in wheel-running and drinking activity. A decrease in electroencephalogram power in the delta frequency range (1-4 Hz) under baseline conditions was observed, which was normalized after sleep deprivation. Together with the finding that circadian clock amplitude is reduced in Prkg1 mutants these results indicate a decrease of the wake-promoting output of the circadian system affecting sleep. Because quality of sleep might affect learning we tested Prkg1 mutants in several learning tasks and find normal spatial learning but impaired object recognition memory in these animals. Our findings indicate that Prkg1 impinges on circadian rhythms, sleep and distinct aspects of learning.

Aging has the opposite effect on cAMP and cGMP circadian variations in rat Leydig cells.

Science.gov (United States)

Baburski, Aleksandar Z; Sokanovic, Srdjan J; Andric, Silvana A; Kostic, Tatjana S

2017-05-01

The Leydig cell physiology displays a circadian rhythm driven by a complex interaction of the reproductive axis hormones and circadian system. The final output of this regulatory process is circadian pattern of steroidogenic genes expression and testosterone production. Aging gradually decreases robustness of rhythmic testosterone secretion without change in pattern of LH secretion. Here, we analyzed effect of aging on circadian variation of cAMP and cGMP signaling in Leydig cells. Results showed opposite effect of aging on cAMP and cGMP daily variation. Reduced amplitude of cAMP circadian oscillation was probably associated with changed expression of genes involved in cAMP production (increased circadian pattern of Adcy7, Adcy9, Adcy10 and decreased Adcy3); cAMP degradation (increased Pde4a, decreased Pde8b, canceled rhythm of Pde4d, completely reversed circadian pattern of Pde7b and Pde8a); and circadian expression of protein kinase A subunits (Prkac/PRKAC and Prkar2a). Aging stimulates expression of genes responsible for cGMP production (Nos2, Gucy1a3 and Gucy1b3/GUCYB3) and degradation (Pde5a, Pde6a and Pde6h) but the overall net effect is elevation of cGMP circadian oscillations in Leydig cells. In addition, the expression of cGMP-dependent kinase, Prkg1/PRKG1 is up-regulated. It seems that aging potentiate cGMP- and reduce cAMP-signaling in Leydig cells. Since both signaling pathways affect testosterone production and clockwork in the cells, further insights into these signaling pathways will help to unravel disorders linked to the circadian timing system, aging and reproduction.

Nuclear cGMP-dependent kinase regulates gene expression via activity-dependent recruitment of a conserved histone deacetylase complex.

Directory of Open Access Journals (Sweden)

Yan Hao

2011-05-01

Full Text Available Elevation of the second messenger cGMP by nitric oxide (NO activates the cGMP-dependent protein kinase PKG, which is key in regulating cardiovascular, intestinal, and neuronal functions in mammals. The NO-cGMP-PKG signaling pathway is also a major therapeutic target for cardiovascular and male reproductive diseases. Despite widespread effects of PKG activation, few molecular targets of PKG are known. We study how EGL-4, the Caenorhabditis elegans PKG ortholog, modulates foraging behavior and egg-laying and seeks the downstream effectors of EGL-4 activity. Using a combination of unbiased forward genetic screen and proteomic analysis, we have identified a conserved SAEG-1/SAEG-2/HDA-2 histone deacetylase complex that is specifically recruited by activated nuclear EGL-4. Gene expression profiling by microarrays revealed >40 genes that are sensitive to EGL-4 activity in a SAEG-1-dependent manner. We present evidence that EGL-4 controls egg laying via one of these genes, Y45F10C.2, which encodes a novel protein that is expressed exclusively in the uterine epithelium. Our results indicate that, in addition to cytoplasmic functions, active EGL-4/PKG acts in the nucleus via a conserved Class I histone deacetylase complex to regulate gene expression pertinent to behavioral and physiological responses to cGMP. We also identify transcriptional targets of EGL-4 that carry out discrete components of the physiological response.

The role of DNA dependent protein kinase in synapsis of DNA ends

NARCIS (Netherlands)

E.P.W.C. Weterings (Eric); N.S. Verkaik (Nicole); H.T. Brüggenwirth (Hennie); D.C. van Gent (Dik); J.H.J. Hoeijmakers (Jan)

2003-01-01

textabstractDNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks

The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

OpenAIRE

Jette, Nicholas; Lees-Miller, Susan P.

2014-01-01

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemi...

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.

Similar expression patterns of bestrophin-4 and cGMP dependent Ca2+-activated chloride channel activity in the vasculature

DEFF Research Database (Denmark)

Bouzinova, Elena V.; Larsen, Per; Matchkov, Vladimir

2008-01-01

(abstract by Matchkov et. al) that siRNA mediated downregulation of bestrophin-4 is associated with the disappearance of a recently demonstrated2 cGMP-dependent Ca2+-activated Cl- current in vascular smooth muscle cells (SMCs). Here we study the distribution of bestrophin-4-and cGMP dependent Cl- channel...... expressed epitope) Western blot detected a ~65 kDa band in cell lysates from rat mesenteric small arteries and aorta, which was not seen in pulmonary arteries and when preincubated with the immunizing peptide. The distribution of bestrophin-4 mRNA and protein has a pattern similar to the cGMP-dependent Cl......- current in SMCs of different origins. Immunohistochemistry identified bestrophin-4 both in endothelial and SMCs of the vascular tree in the brain, heart, kidney and mesentery, but not in the lungs. We suggest that bestrophin-4 is important for the cGMP dependent, Ca2+ activated Cl- conductance in many...

cGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

Directory of Open Access Journals (Sweden)

Steiner Jennifer A

2009-08-01

Full Text Available Abstract Background The Na+/Cl--dependent serotonin (5-hydroxytryptamine, 5-HT transporter (SERT is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear. Results In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting

Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

Science.gov (United States)

Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

2013-07-01

Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

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

Presenilin dependence of phospholipase C and protein kinase C signaling

DEFF Research Database (Denmark)

Dehvari, Nodi; Cedazo-Minguez, Angel; Isacsson, Ola

2007-01-01

-stimulated phospholipase C (PLC) activity which was gamma-secretase dependent. To further evaluate the dependence of PLC on PSs we measured PLC activity and the activation of variant protein kinase C (PKC) isoforms in mouse embryonic fibroblasts (MEFs) lacking either PS1, PS2, or both. PLC activity and PKCalpha...

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.

Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

KAUST Repository

Irving, Helen R.; Kwezi, Lusisizwe; Wheeler, Janet I.; Gehring, Christoph A

2012-01-01

Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

KAUST Repository

Irving, Helen R.

2012-02-01

Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

Activity of cAMP-dependent protein kinases and cAMP-binding proteins of rat kidney cytosol during dehydration

International Nuclear Information System (INIS)

Zelenina, M.N.; Solenov, E.I.; Ivanova, L.N.

1985-01-01

The activity of cAMP-dependent protein kinases, the binding of cAMP, and the spectrum of cAMP-binding proteins in the cytosol of the renal papilla was studied in intact rats and in rats after 24 h on a water-deprived diet. It was found that the activation of protein kinases by 10 -6 M cAMP is significantly higher in the experimental animals than in the intact animals. In chromatography on DEAE-cellulose, the positions of the peaks of specific reception of cAMP corresponded to the peaks of the regulatory subunits of cAMP-dependent protein kinases of types I and II. In this case, in intact animals more than 80% of the binding activity was detected in peaks II, whereas in rats subjected to water deprivation, more than 60% of the binding was observed in peak I. The general regulatory activity of the cytosol was unchanged in the experimental animals in comparison with intact animals. It is suggested that during dehydration there is an induction of the synthesis of the regulatory subunit of type I cAMP-dependent protein kinase in the renal papilla

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

Science.gov (United States)

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

The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

Science.gov (United States)

Jette, Nicholas; Lees-Miller, Susan P.

2015-01-01

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

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.

Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

KAUST Repository

Kwezi, Lusisizwe

2018-01-22

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

KAUST Repository

Kwezi, Lusisizwe; Wheeler, Janet I; Marondedze, Claudius; Gehring, Christoph A; Irving, Helen R

2018-01-01

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

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

Directory of Open Access Journals (Sweden)

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.

A role for barley calcium-dependent protein kinase CPK2a in the response to drought

Directory of Open Access Journals (Sweden)

Agata Cieśla

2016-10-01

Full Text Available Increasing the drought tolerance of crops is one of the most challenging goals in plant breeding. To improve crop productivity during periods of water deficit, it is essential to understand the complex regulatory pathways that adapt plant metabolism to environmental conditions. Among various plant hormones and second messengers, calcium ions are known to be involved in drought stress perception and signaling. Plants have developed specific calcium-dependent protein kinases that convert calcium signals into phosphorylation events. In this study we attempted to elucidate the role of a calcium-dependent protein kinase in the drought stress response of barley (Hordeum vulgare L., one of the most economically important crops worldwide. The ongoing barley genome project has provided useful information about genes potentially involved in the drought stress response, but information on the role of calcium-dependent kinases is still limited. We found that the gene encoding the calcium-dependent protein kinase HvCPK2a was significantly upregulated in response to drought. To better understand the role of HvCPK2a in drought stress signaling, we generated transgenic Arabidopsis plants that overexpressed the corresponding coding sequence. Overexpressing lines displayed drought sensitivity, reduced nitrogen balance index, an increase in total chlorophyll content and decreased relative water content. In addition, in vitro kinase assay experiments combined with mass spectrometry allowed HvCPK2a autophosphorylation sites to be identified. Our results suggest that HvCPK2a is a dual-specificity calcium-dependent protein kinase that functions as a negative regulator of the drought stress response in barley.

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

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...

Partial reconstitution of photoreceptor cGMP phosphodiesterase characteristics in cGMP phosphodiesterase-5.

Science.gov (United States)

Granovsky, A E; Artemyev, N O

2001-06-15

Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory gamma-subunits (Pgamma). The Pgamma block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pgamma and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pgamma and has a more than 100-fold lower k(cat) for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6alpha' enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6alpha') with Pgamma is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met(758), Phe(777), and Phe(781). The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6alpha'-specific glycine residues for the corresponding PDE5 alanine residues, Ala(608) and Ala(612). The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis.

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

Science.gov (United States)

Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

2014-06-25

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

Neuronal phosphorylated RNA-dependent protein kinase in Creutzfeldt-Jakob disease.

LENUS (Irish Health Repository)

Paquet, Claire

2009-02-01

The mechanisms of neuronal apoptosis in Creutzfeldt-Jakob disease (CJD) and their relationship to accumulated prion protein (PrP) are unclear. A recent cell culture study showed that intracytoplasmic PrP may induce phosphorylated RNA-dependent protein kinase (PKR(p))-mediated cell stress. The double-stranded RNA protein kinase PKR is a proapoptotic and stress kinase that accumulates in degenerating neurons in Alzheimer disease. To determine whether neuronal apoptosis in human CJD is associated with activation of the PKR(p) signaling pathway, we assessed in situ end labeling and immunocytochemistry for PrP, glial fibrillary acidic protein, CD68, activated caspase 3, and phosphorylated PKR (Thr451) in samples of frontal, occipital, and temporal cortex, striatum, and cerebellum from 6 patients with sporadic CJD and 5 controls. Neuronal immunostaining for activated PKR was found in all CJD cases. The most staining was in nuclei and, in contrast to findings in Alzheimer disease, cytoplasmic labeling was not detected. Both the number and distribution of PKR(p)-positive neurons correlated closely with the extent of neuronal apoptosis, spongiosis, astrocytosis, and microglial activation and with the phenotype and disease severity. There was no correlation with the type, topography, or amount of extracellular PrP deposits. These findings suggest that neuronal apoptosis in human CJD may result from PKR(p)-mediated cell stress and are consistent with recent studies supporting a pathogenic role for intracellular or transmembrane PrP.

The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

KAUST Repository

Meier, Stuart; Ruzvidzo, Oziniel; Morse, Monique; Donaldson, Lara; Kwezi, Lusisizwe; Gehring, Christoph A

2010-01-01

Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

KAUST Repository

Meier, Stuart

2010-01-26

Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

Roles of calcium/calmodulin-dependent kinase II in long-term memory formation in crickets.

Directory of Open Access Journals (Sweden)

Makoto Mizunami

Full Text Available Ca(2+/calmodulin (CaM-dependent protein kinase II (CaMKII is a key molecule in many systems of learning and memory in vertebrates, but roles of CaMKII in invertebrates have not been characterized in detail. We have suggested that serial activation of NO/cGMP signaling, cyclic nucleotide-gated channel, Ca(2+/CaM and cAMP signaling participates in long-term memory (LTM formation in olfactory conditioning in crickets, and here we show participation of CaMKII in LTM formation and propose its site of action in the biochemical cascades. Crickets subjected to 3-trial conditioning to associate an odor with reward exhibited memory that lasts for a few days, which is characterized as protein synthesis-dependent LTM. In contrast, animals subjected to 1-trial conditioning exhibited memory that lasts for only several hours (mid-term memory, MTM. Injection of a CaMKII inhibitor prior to 3-trial conditioning impaired 1-day memory retention but not 1-hour memory retention, suggesting that CaMKII participates in LTM formation but not in MTM formation. Animals injected with a cGMP analogue, calcium ionophore or cAMP analogue prior to 1-trial conditioning exhibited 1-day retention, and co-injection of a CaMKII inhibitor impaired induction of LTM by the cGMP analogue or that by the calcium ionophore but not that by the cAMP analogue, suggesting that CaMKII is downstream of cGMP production and Ca(2+ influx and upstream of cAMP production in biochemical cascades for LTM formation. Animals injected with an adenylyl cyclase (AC activator prior to 1-trial conditioning exhibited 1-day retention. Interestingly, a CaMKII inhibitor impaired LTM induction by the AC activator, although AC is expected to be a downstream target of CaMKII. The results suggest that CaMKII interacts with AC to facilitate cAMP production for LTM formation. We propose that CaMKII serves as a key molecule for interplay between Ca(2+ signaling and cAMP signaling for LTM formation, a new role of Ca

The calcium-dependent protein kinase CPK7 acts on root hydraulic conductivity.

Science.gov (United States)

Li, Guowei; Boudsocq, Marie; Hem, Sonia; Vialaret, Jérôme; Rossignol, Michel; Maurel, Christophe; Santoni, Véronique

2015-07-01

The hydraulic conductivity of plant roots (Lp(r)) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post-translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lp(r) of knockout Arabidopsis plants for four Ca(2+)-dependent protein kinases. cpk7 plants showed a 30% increase in Lp(r) because of a higher aquaporin activity. A quantitative proteomic analysis of wild-type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lp(r) of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7-dependent stability of specific membrane proteins. © 2014 John Wiley & Sons Ltd.

Nitric oxide signaling depends on biotin in Jurkat human lymphoma cells.

Science.gov (United States)

Rodriguez-Melendez, Rocio; Zempleni, Janos

2009-03-01

Biotin affects gene expression through a diverse array of cell signaling pathways. Previous studies provided evidence that cGMP-dependent signaling also depends on biotin, but the mechanistic sequence of cGMP regulation by biotin is unknown. Here we tested the hypothesis that the effects of biotin in cGMP-dependent cell signaling are mediated by nitric oxide (NO). Human lymphoid (Jurkat) cells were cultured in media containing deficient (0.025 nmol/L), physiological (0.25 nmol/L), and pharmacological (10 nmol/L) concentrations of biotin for 5 wk. Both levels of intracellular biotin and NO exhibited a dose-dependent relationship in regard to biotin concentrations in culture media. Effects of biotin on NO levels were disrupted by the NO synthase (NOS) inhibitor N-monomethyl-arginine. Biotin-dependent production of NO was linked with biotin-dependent expression of endothelial and neuronal NOS, but not inducible NOS. Previous studies revealed that NO is an activator of guanylate cyclase. Consistent with these previous observations, biotin-dependent generation of NO increased the abundance of cGMP in Jurkat cells. Finally, the biotin-dependent generation of cGMP increased protein kinase G activity. Collectively, the results of this study are consistent with the hypothesis that biotin-dependent cGMP signaling in human lymphoid cells is mediated by NO.

Evolutionary Paths of the cAMP-Dependent Protein Kinase (PKA) Catalytic Subunits

Science.gov (United States)

Søberg, Kristoffer; Jahnsen, Tore; Rognes, Torbjørn; Skålhegg, Bjørn S.; Laerdahl, Jon K.

2013-01-01

3′,5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods. PMID:23593352

Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

DEFF Research Database (Denmark)

Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller

2008-01-01

AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho......-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of c......AMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response....

Evolution of the cAMP-dependent protein kinase (PKA catalytic subunit isoforms.

Directory of Open Access Journals (Sweden)

Kristoffer Søberg

Full Text Available The 3',5'-cyclic adenosine monophosphate (cAMP-dependent protein kinase, or protein kinase A (PKA, pathway is one of the most versatile and best studied signaling pathways in eukaryotic cells. The two paralogous PKA catalytic subunits Cα and Cβ, encoded by the genes PRKACA and PRKACB, respectively, are among the best understood model kinases in signal transduction research. In this work, we explore and elucidate the evolution of the alternative 5' exons and the splicing pattern giving rise to the numerous PKA catalytic subunit isoforms. In addition to the universally conserved Cα1/Cβ1 isoforms, we find kinase variants with short N-termini in all main vertebrate classes, including the sperm-specific Cα2 isoform found to be conserved in all mammals. We also describe, for the first time, a PKA Cα isoform with a long N-terminus, paralogous to the PKA Cβ2 N-terminus. An analysis of isoform-specific variation highlights residues and motifs that are likely to be of functional importance.

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.

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

DEFF Research Database (Denmark)

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...

Functions of Calcium-Dependent Protein Kinases in Plant Innate Immunity

Directory of Open Access Journals (Sweden)

Xiquan Gao

2014-03-01

Full Text Available An increase of cytosolic Ca2+ is generated by diverse physiological stimuli and stresses, including pathogen attack. Plants have evolved two branches of the immune system to defend against pathogen infections. The primary innate immune response is triggered by the detection of evolutionarily conserved pathogen-associated molecular pattern (PAMP, which is called PAMP-triggered immunity (PTI. The second branch of plant innate immunity is triggered by the recognition of specific pathogen effector proteins and known as effector-triggered immunity (ETI. Calcium (Ca2+ signaling is essential in both plant PTI and ETI responses. Calcium-dependent protein kinases (CDPKs have emerged as important Ca2+ sensor proteins in transducing differential Ca2+ signatures, triggered by PAMPs or effectors and activating complex downstream responses. CDPKs directly transmit calcium signals by calcium binding to the elongation factor (EF-hand domain at the C-terminus and substrate phosphorylation by the catalytic kinase domain at the N-terminus. Emerging evidence suggests that specific and overlapping CDPKs phosphorylate distinct substrates in PTI and ETI to regulate diverse plant immune responses, including production of reactive oxygen species, transcriptional reprogramming of immune genes, and the hypersensitive response.

Functions of Calcium-Dependent Protein Kinases in Plant Innate Immunity

Science.gov (United States)

Gao, Xiquan; Cox, Kevin L.; He, Ping

2014-01-01

An increase of cytosolic Ca2+ is generated by diverse physiological stimuli and stresses, including pathogen attack. Plants have evolved two branches of the immune system to defend against pathogen infections. The primary innate immune response is triggered by the detection of evolutionarily conserved pathogen-associated molecular pattern (PAMP), which is called PAMP-triggered immunity (PTI). The second branch of plant innate immunity is triggered by the recognition of specific pathogen effector proteins and known as effector-triggered immunity (ETI). Calcium (Ca2+) signaling is essential in both plant PTI and ETI responses. Calcium-dependent protein kinases (CDPKs) have emerged as important Ca2+ sensor proteins in transducing differential Ca2+ signatures, triggered by PAMPs or effectors and activating complex downstream responses. CDPKs directly transmit calcium signals by calcium binding to the elongation factor (EF)-hand domain at the C-terminus and substrate phosphorylation by the catalytic kinase domain at the N-terminus. Emerging evidence suggests that specific and overlapping CDPKs phosphorylate distinct substrates in PTI and ETI to regulate diverse plant immune responses, including production of reactive oxygen species, transcriptional reprogramming of immune genes, and the hypersensitive response. PMID:27135498

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.

Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

Science.gov (United States)

Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

2015-09-08

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

2,5-hexanedione (HD) treatment alters calmodulin, Ca2+/calmodulin-dependent protein kinase II, and protein kinase C in rats' nerve tissues

International Nuclear Information System (INIS)

Wang Qingshan; Hou Liyan; Zhang Cuili; Zhao Xiulan; Yu Sufang; Xie, Ke-Qin

2008-01-01

Calcium-dependent mechanisms, particularly those mediated by Ca 2+ /calmodulin (CaM)-dependent protein kinase II (CaMKII), have been implicated in neurotoxicant-induced neuropathy. However, it is unknown whether similar mechanisms exist in 2,5-hexanedione (HD)-induced neuropathy. For that, we investigated the changes of CaM, CaMKII, protein kinase C (PKC) and polymerization ratios (PRs) of NF-L, NF-M and NF-H in cerebral cortex (CC, including total cortex and some gray), spinal cord (SC) and sciatic nerve (SN) of rats treated with HD at a dosage of 1.75 or 3.50 mmol/kg for 8 weeks (five times per week). The results showed that CaM contents in CC, SC and SN were significantly increased, which indicated elevation of Ca 2+ concentrations in nerve tissues. CaMKII contents and activities were also increased in CC and were positively correlated with gait abnormality, but it could not be found in SC and SN. The increases of PKC contents and activities were also observed in SN and were positively correlated with gait abnormality. Except for that of NF-M in CC, the PRs of NF-L, NF-M and NF-H were also elevated in nerve tissues, which was consistent with the activation of protein kinases. The results suggested that CaMKII might be partly (in CC but not in SC and SN) involved in HD-induced neuropathy. CaMKII and PKC might mediate the HD neurotoxicity by altering the NF phosphorylation status and PRs

Conformational dependence of a protein kinase phosphate transfer reaction

Science.gov (United States)

Labute, Montiago; Henkelman, Graeme; Tung, Chang-Shung; Fenimore, Paul; McMahon, Ben

2007-03-01

Atomic motions and energetics for a phosphate transfer reaction catalyzed by the cAMP-dependent protein kinase have been calculated using plane-wave density functional theory, starting from structures of proteins crystallized in both the reactant conformation (RC) and the transition-state conformation (TC). In TC, we calculate that the reactants and products are nearly isoenergetic with a 20-kJ/mol barrier, whereas phosphate transfer is unfavorable by 120 kJ/mol in the RC, with an even higher barrier. Our results demonstrate that the phosphate transfer reaction occurs rapidly and reversibly in a particular conformation of the protein, and that the reaction can be gated by changes of a few tenths of an angstrom in the catalytic site [1]. [1] G.H. Henkelman, M.X. LaBute, C.-S. Tung, P.W. Fenimore, B.H. McMahon, Proc. Natl. Acad. Sci. USA vol. 102, no. 43:15347-15351 (2005).

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

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.

The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence.

Science.gov (United States)

Alves de Castro, Patrícia; Dos Reis, Thaila Fernanda; Dolan, Stephen K; Oliveira Manfiolli, Adriana; Brown, Neil Andrew; Jones, Gary W; Doyle, Sean; Riaño-Pachón, Diego M; Squina, Fábio Márcio; Caldana, Camila; Singh, Ashutosh; Del Poeta, Maurizio; Hagiwara, Daisuke; Silva-Rocha, Rafael; Goldman, Gustavo H

2016-11-01

The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways. © 2016 John Wiley & Sons Ltd.

cGMP-Dependent Protein Kinase Inhibitors in Health and Disease

Directory of Open Access Journals (Sweden)

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.

TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism

DEFF Research Database (Denmark)

Bonefeld, Charlotte Menné; Rasmussen, B. A.; Lauritsen, J P

2003-01-01

of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based...

cGMP-dependent protein kinase type II knockout mice exhibit working memory impairments, decreased repetitive behavior, and increased anxiety-like traits.

Science.gov (United States)

Wincott, Charlotte M; Abera, Sinedu; Vunck, Sarah A; Tirko, Natasha; Choi, Yoon; Titcombe, Roseann F; Antoine, Shannon O; Tukey, David S; DeVito, Loren M; Hofmann, Franz; Hoeffer, Charles A; Ziff, Edward B

2014-10-01

Neuronal activity regulates AMPA receptor trafficking, a process that mediates changes in synaptic strength, a key component of learning and memory. This form of plasticity may be induced by stimulation of the NMDA receptor which, among its activities, increases cyclic guanosine monophosphate (cGMP) through the nitric oxide synthase pathway. cGMP-dependent protein kinase type II (cGKII) is ultimately activated via this mechanism and AMPA receptor subunit GluA1 is phosphorylated at serine 845. This phosphorylation contributes to the delivery of GluA1 to the synapse, a step that increases synaptic strength. Previous studies have shown that cGKII-deficient mice display striking spatial learning deficits in the Morris Water Maze compared to wild-type littermates as well as lowered GluA1 phosphorylation in the postsynaptic density of the prefrontal cortex (Serulle et al., 2007; Wincott et al., 2013). In the current study, we show that cGKII knockout mice exhibit impaired working memory as determined using the prefrontal cortex-dependent Radial Arm Maze (RAM). Additionally, we report reduced repetitive behavior in the Marble Burying task (MB), and heightened anxiety-like traits in the Novelty Suppressed Feeding Test (NSFT). These data suggest that cGKII may play a role in the integration of information that conveys both anxiety-provoking stimuli as well as the spatial and environmental cues that facilitate functional memory processes and appropriate behavioral response. Published by Elsevier Inc.

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

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

Science.gov (United States)

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

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...

Quinolinone and pyridopyrimidinone inhibitors of DNA-dependent protein kinase.

Science.gov (United States)

Barbeau, Olivier R; Cano-Soumillac, Celine; Griffin, Roger J; Hardcastle, Ian R; Smith, Graeme C M; Richardson, Caroline; Clegg, William; Harrington, Ross W; Golding, Bernard T

2007-08-21

8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones with selected aryl and heteroaryl groups as the substituent have been synthesised as potential inhibitors of DNA-dependent protein kinase. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was utilised in the preparation of 8-substituted 2-morpholin-4-yl-quinolin-4-ones. For this purpose 8-bromo-2-morpholin-4-yl-quinolin-4-one was required as an intermediate. This compound was obtained by adapting a literature route in which thermal cyclocondensation of (2-bromoanilino)-morpholin-4-yl-5-methylene-2,2-dimethyl[1,3]dioxane-4,6-dione afforded 8-bromo-2-morpholin-4-yl-quinolin-4-one. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was also utilised to prepare 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones using 9-hydroxy-2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-one O-trifluoromethanesulfonate as an intermediate. 8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones were both inhibitors of DNA-dependent protein kinase. When the substituent was dibenzothiophen-4-yl, dibenzofuran-4-yl or biphen-3-yl, IC50 values in the low nanomolar range were observed. Interestingly, the pyridopyrimidinones and quinolinones were essentially equipotent with the corresponding 8-substituted 2-morpholin-4-yl-chromen-4-ones previously reported (I. R. Hardcastle, X. Cockcroft, N. J. Curtin, M. Desage El-Murr, J. J. J. Leahy, M. Stockley, B. T. Golding, L. Rigoreau, C. Richardson, G. C. M. Smith and R. J. Griffin, J. Med. Chem., 2005, 48, 7829-7846).

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

Science.gov (United States)

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.

Probes of the Mitochondrial cAMP-dependent Protein Kinase

Science.gov (United States)

Shell, Jennifer R.; Lawrence, David S.

2013-01-01

The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca2+, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R2C2) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). PMID:23410952

Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates

Directory of Open Access Journals (Sweden)

Amy eCurran

2011-08-01

Full Text Available The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs. While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16 and 34. Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ~70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites. Of these, 74 (27% were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

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

Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1

Science.gov (United States)

Child, Matthew A.; Garland, Megan; Foe, Ian; Madzelan, Peter; Treeck, Moritz; van der Linden, Wouter A.; Oresic Bender, Kristina; Weerapana, Eranthie; Wilson, Mark A.; Boothroyd, John C.; Reese, Michael L.

2017-01-01

ABSTRACT Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii. PMID:28246362

Characterization of CoPK02, a Ca2+/calmodulin-dependent protein kinase in mushroom Coprinopsis cinerea.

Science.gov (United States)

Yamashita, Masashi; Sueyoshi, Noriyuki; Yamada, Hiroki; Katayama, Syouichi; Senga, Yukako; Takenaka, Yasuhiro; Ishida, Atsuhiko; Kameshita, Isamu; Shigeri, Yasushi

2018-04-20

We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca 2+ /CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca 2+ /CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca 2+ -signaling in C. cinerea.

The role of DNA dependent protein kinase in synapsis of DNA ends.

Science.gov (United States)

Weterings, Eric; Verkaik, Nicole S; Brüggenwirth, Hennie T; Hoeijmakers, Jan H J; van Gent, Dik C

2003-12-15

DNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks the action of exonucleases and ligases. The DNA termini become accessible after autophosphorylation of DNA-PK(CS), which we demonstrate to require synapsis of DNA ends. Interestingly, the presence of DNA-PK prevents ligation of the two synapsed termini, but allows ligation to another DNA molecule. This alteration of the ligation route is independent of the type of ligase that we used, indicating that the intrinsic architecture of the DNA-PK complex itself is not able to support ligation of the synapsed DNA termini. We present a working model in which DNA-PK creates a stable molecular bridge between two DNA ends that is remodeled after DNA-PK autophosphorylation in such a way that the extreme termini become accessible without disrupting synapsis. We infer that joining of synapsed DNA termini would require an additional protein factor.

Protein and signaling networks in vertebrate photoreceptor cells

Directory of Open Access Journals (Sweden)

Karl-Wilhelm eKoch

2015-11-01

Full Text Available Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination. The photoexcitation and adaptation machinery in photoreceptor cells consists of protein complexes that can form highly ordered supramolecular structures and control the homeostasis and mutual dependence of the secondary messengers cGMP and Ca2+. The visual pigment in rod photoreceptors, the G protein-coupled receptor rhodopsin is organized in tracks of dimers thereby providing a signaling platform for the dynamic scaffolding of the G protein transducin. Illuminated rhodopsin is turned off by phosphorylation catalyzed by rhodopsin kinase GRK1 under control of Ca2+-recoverin. The GRK1 protein complex partly assembles in lipid raft structures, where shutting off rhodopsin seems to be more effective. Re-synthesis of cGMP is another crucial step in the recovery of the photoresponse after illumination. It is catalyzed by membrane bound sensory guanylate cyclases and is regulated by specific neuronal Ca2+-sensor proteins called GCAPs. At least one guanylate cyclase (ROS-GC1 was shown to be part of a multiprotein complex having strong interactions with the cytoskeleton and being controlled in a multimodal Ca2+-dependent fashion. The final target of the cGMP signaling cascade is a cyclic nucleotide-gated channel that is a hetero-oligomeric protein located in the plasma membrane and interacting with accessory proteins in highly organized microdomains. We summarize results and interpretations of findings related to the inhomogeneous organization of signaling units in photoreceptor outer segments.

The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants

KAUST Repository

Kwezi, Lusisizwe; Ruzvidzo, Oziniel; Wheeler, Janet I.; Govender, Kershini; Iacuone, Sylvana; Thompson, Philip E.; Gehring, Christoph A; Irving, Helen R.

2011-01-01

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5 μM and Vmax of 1800 nmol min-1 mg-1 of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg2+ or Mn2+. Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants

KAUST Repository

Kwezi, Lusisizwe

2011-04-19

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5 μM and Vmax of 1800 nmol min-1 mg-1 of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg2+ or Mn2+. Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein kinase C interaction with calcium: a phospholipid-dependent process.

LENUS (Irish Health Repository)

Bazzi, M D

1990-08-21

The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol\\/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.

Ca2+-calmodulin-dependent protein kinase expression and signalling in skeletal muscle during exercise

DEFF Research Database (Denmark)

Rose, Adam John; Kiens, Bente; Richter, Erik

2006-01-01

Ca2+ signalling is proposed to play an important role in skeletal muscle function during exercise. Here, we examined the expression of multifunctional Ca2+-calmodulin-dependent protein kinases (CaMK) in human skeletal muscle and show that CaMKII and CaMKK, but not CaMKI or CaMKIV, are expressed...

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

DNA-dependent protein kinase in nonhomologous end joining: a lock with multiple keys?

Science.gov (United States)

Weterings, Eric; Chen, David J

2007-10-22

The DNA-dependent protein kinase (DNA-PK) is one of the central enzymes involved in DNA double-strand break (DSB) repair. It facilitates proper alignment of the two ends of the broken DNA molecule and coordinates access of other factors to the repair complex. We discuss the latest findings on DNA-PK phosphorylation and offer a working model for the regulation of DNA-PK during DSB repair.

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

2009-03-01

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.

LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization1

Science.gov (United States)

Rutschmann, Frank; Stalder, Urs; Piotrowski, Markus; Oecking, Claudia; Schaller, Andreas

2002-01-01

The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. PMID:12011347

Protein Kinase C alpha (PKCα) dependent signaling mediates endometrial cancer cell growth and tumorigenesis

Science.gov (United States)

Haughian, James M.; Reno, Elaine M.; Thorne, Alicia M.; Bradford, Andrew P.

2009-01-01

Endometrial cancer is the most common invasive gynecologic malignancy, yet molecular mechanisms and signaling pathways underlying its etiology and pathophysiology remain poorly characterized. We sought to define a functional role for the protein kinase C (PKC) isoform, PKCα, in an established cell model of endometrial adenocarcinoma. Ishikawa cells depleted of PKCα protein grew slower, formed fewer colonies in anchorage-independent growth assays and exhibited impaired xenograft tumor formation in nude mice. Consistent with impaired growth, PKCα knockdown increased levels of the cyclin dependent kinase (CDK) inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Despite the absence of functional phosphatase and tensin homologue (PTEN) protein in Ishikawa cells, PKCα knockdown reduced Akt phosphorylation at serine 473 and concomitantly inhibited phosphorylation of the Akt target, glycogen synthase kinase-3β (GSK-3β). PKCα knockdown also resulted in decreased basal ERK phosphorylation and attenuated ERK activation following EGF stimulation. p21 and p27 expression was not increased by treatment of Ishikawa cells with ERK and Akt inhibitors, suggesting PKCα regulates CDK expression independently of Akt and ERK. Immunohistochemical analysis of grade 1 endometrioid adenocarcinoma revealed aberrant PKCα expression, with foci of elevated PKCα staining, not observed in normal endometrium. These studies demonstrate a critical role for PKCα signaling in endometrial tumorigenesis by regulating expression of CDK inhibitors p21 and p27 and activation of Akt and ERK dependent proliferative pathways. Thus, targeting PKCα may provide novel therapeutic options in endometrial tumors. PMID:19672862

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...

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.

Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

KAUST Repository

Kim, Jeong Joo

2016-04-09

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Kim et al. obtain the first crystal structure of the PKG I R domain bound with cGMP representing its activated state. It reveals a symmetric R dimer where cGMP molecules provide dimeric contacts. This R-R interaction prevents the high-affinity inhibitory interaction between R-C domain and sustains activation. © 2016 Elsevier Ltd.

Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

KAUST Repository

Kim, Jeong  Joo; Lorenz, Robin; Arold, Stefan T.; Reger, Albert  S.; Sankaran, Banumathi; Casteel, Darren  E.; Herberg, Friedrich  W.; Kim, Choel

2016-01-01

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Kim et al. obtain the first crystal structure of the PKG I R domain bound with cGMP representing its activated state. It reveals a symmetric R dimer where cGMP molecules provide dimeric contacts. This R-R interaction prevents the high-affinity inhibitory interaction between R-C domain and sustains activation. © 2016 Elsevier Ltd.

A cyclic GMP signalling module that regulates gliding motility in a malaria parasite.

Directory of Open Access Journals (Sweden)

Robert W Moon

2009-09-01

Full Text Available The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3', 5'-cyclic monophosphate (cGMP as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase delta (PDEdelta, unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase beta (GCbeta, showing that in ookinetes GCbeta is an important source for cGMP, and that PDEdelta is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito.

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.

The role of cAMP-dependent protein kinase A in bile canalicular plasma membrane biogenesis in hepatocytes

NARCIS (Netherlands)

Wojtal, Kacper Andrze

2007-01-01

cAMP-dependent protein kinase A is one of the most important enzymes in the eukaryotic cell. The function of this protein is strictly in a close relation to the signaling pathways, which trigger the production of intracellular secondary messenger –cAMP. As a consequence of PKA activation numerous

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

DEFF Research Database (Denmark)

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....

Genomic analysis of murine DNA-dependent protein kinase

International Nuclear Information System (INIS)

Fujimori, A.; Abe, M.

2003-01-01

Full text: The gene of catalytic subunit of DNA dependent protein kinase is responsible gene for SCID mice. The molecules play a critical role in non-homologous end joining including the V(D)J recombination. Contribution of the molecules to the difference of radiosensitivity and the susceptibility to cancer has been suggested. Here we show the entire nucleotide sequence of approximately 193 kbp and 84 kbp genomic regions encoding the entire DNA-PKcs gene in the mouse and chicken respectively. Retroposon was found in the intron 51 of mouse genomic DNA-PKcs gene but in human and chicken. Comparative analysis of these two species strongly suggested that only two genes, DNA-PKcs and MCM4, exist in the region of both species. Several conserved sequences and cis elements, however, were predicted. Recently, the orthologous region for the human DNA-PKcs locus was completed. The results of further comparative study will be discussed

Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

DEFF Research Database (Denmark)

Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan

2013-01-01

V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) ar......V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA......-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA...... when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA...

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.

Keratin 8 phosphorylation in vitro by cAMP-dependent protein kinase occurs within the amino- and carboxyl-terminal end domains.

Science.gov (United States)

Ando, S; Tokui, T; Yano, T; Inagaki, M

1996-04-05

We reported earlier that phosphorylation in vitro of keratin filaments reconstituted from rat type I keratin 18 and type II keratin 8 by cAPM-dependent protein kinase induces disassembly of the keratin filament structure. Keratin 8 rather than keratin 18 was the major target of the kinase. We have now identified the sites on rat keratin 8 for cAMP-dependent protein kinase. Sequential analysis of the purified phosphoropeptides, together with the known primary sequence, revealed that four major sites, Ser-12, Ser-23, Ser-36, and Ser-50, and three minor sites, Ser-8, Ser-33, Ser-42, are located in the amino-terminal head domain, while three minor sites, Ser-416, Ser-423 and Ser-425 locate in the carboxyl-terminal tail domain.

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

NARCIS (Netherlands)

Baker, D.A.; Stewart, L.B.; Large, J.M.; Bowyer, P.W.; Ansell, K.H.; Jimenez-Diaz, M.B.; Bakkouri, M. El; Birchall, K.; Dechering, K.J.; Bouloc, N.S.; Coombs, P.J.; Whalley, D.; Harding, D.J.; Smiljanic-Hurley, E.; Wheldon, M.C.; Walker, E.M.; Dessens, J.T.; Lafuente, M.J.; Sanz, L.M.; Gamo, F.J.; Ferrer, S.B.; Hui, R.; Bousema, T.; Angulo-Barturen, I.; Merritt, A.T.; Croft, S.L.; Gutteridge, W.E.; Kettleborough, C.A.; Osborne, S.A.

2017-01-01

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

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.

Cyclin-dependent kinase 5, a node protein in diminished tauopathy: a systems biology approach

Directory of Open Access Journals (Sweden)

John Fredy Castro-Alvarez

2014-09-01

Full Text Available Alzheimer's disease (AD is the most common cause of dementia worldwide. One of the main pathological changes that occurs in AD is the intracellular accumulation of hyperphosphorylated Tau protein in neurons. Cyclin-dependent kinase 5 (CDK5 is one of the major kinases involved in Tau phosphorylation, directly phosphorylating various residues and simultaneously regulating various substrates such as kinases and phosphatases that influence Tau phosphorylation in a synergistic and antagonistic way. It remains unknown how the interaction between CDK5 and its substrates promotes Tau phosphorylation, and systemic approaches are needed that allow an analysis of all the proteins involved. In this review, the role of the CDK5 signaling pathway in Tau hyperphosphorylation is described, an in silico model of the CDK5 signaling pathway is presented. The relationship among these theoretical and computational models shows that the regulation of Tau phosphorylation by PP2A and GSK3β is essential under basal conditions and also describes the leading role of CDK5 under excitotoxic conditions, where silencing of CDK5 can generate changes in these enzymes to reverse a pathological condition that simulates AD.

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

Sildenafil improves blood perfusion in steroid-induced avascular necrosis of femoral head in rabbits via a protein kinase G-dependent mechanism.

Science.gov (United States)

Song, Qichun; Ni, Jianlong; Jiang, Hongyuan; Shi, Zhibin

2017-10-01

The aim of the study were to evaluate the effect of sildenafil against avascular necrosis of femoral head (ANFH) in a rabbit model, and to study the role of protein kinase G (PKG) pathway and vascular endothelial growth factor (VEGF) in ANFH. Three weeks after inducing ANFH with methylprednisolone injection, 45 female adult New Zealand white rabbits were divided into three groups and treated as follows: group SI received daily intraperitoneal sildenafil with a dose of 10 mg/kg per day; group SD received daily sildenafil identically to group SI plus auricular vein injection DT3 (a specific PKG inhibitor); group NS received only normal saline. The blood perfusion function in the femoral head was measured by perfusion MRI and ink artery infusion. Bilateral femora heads were examined histopathologically for the presence of osteonecrosis; VEGF of tissue was examined by Western blot analysis; cGMP level and PKG activity were also measured. The incidence of ANFH in SI group was significantly lower than that observed in NS and SD groups (p < 0.05). VEGF in SI group was increased compared to NS group. cGMP level and PKG activity were also significantly different between NS and SI group (p < 0.05). However, these effects of sildenafil in SD group were all markedly inhibited by the administration of DT3 compared to SI group. Sildenafil appear to increase the perfusion of femoral head by up-regulating VEGF through PKG pathway. The increased perfusion of femoral head could prevent ANFH. Copyright © 2017 Turkish Association of Orthopaedics and Traumatology. Production and hosting by Elsevier B.V. All rights reserved.

Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development.

Science.gov (United States)

Pagnussat, Gabriela Carolina; Lanteri, María Luciana; Lombardo, María Cristina; Lamattina, Lorenzo

2004-05-01

Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002, 2003). However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)- and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA- and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogen-activated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA + cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process

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...

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....

Non-degradative Ubiquitination of Protein Kinases.

Directory of Open Access Journals (Sweden)

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.

Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

KAUST Repository

Qia, Zhi; Verma, Rajeev K.; Gehring, Christoph A; Yamaguchi, Yube; Zhao, Yichen; Ryan, Clarence A.; Berkowitz, Gerald A.

2010-01-01

receptor- like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2- dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33

DNA-dependent protein kinase (DAN-PK), a key enzyme in the re-ligation of DNA double-strand breaks

International Nuclear Information System (INIS)

Hennequin, C.; Averbeck, D.

1999-01-01

Repair pathways of DNA are now defined and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEH) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEj implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridization in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catalytic subunit (DNA-PK cs ) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immuno-deficient (scid) mice are deficient in DNA-PK cs : this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylate Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival. (authors)

Molecular modelling of calcium dependent protein kinase 4 (CDPK4) from Plasmodium falciparum

CSIR Research Space (South Africa)

Tsekoa, Tsepo L

2009-10-01

Full Text Available eukaryotic protein kinases (ePKs) as defined in model organisms. A novel family of phylogenetically distinct ePK-related genes in P. falciparum has been identified. These kinases (up to 20 in number [2], designated the FIKK family due to a conserved amino...]. The protein kinase complement of Plasmodium falciparum, the main infectious agent of lethal malaria in humans, has been analysed in detail [2, 3]. These analyses revealed that the P. falciparum kinome comprises as many as 65 sequences related to typical...

Role of Nitric Oxide, Nitric Oxide Synthase, Soluble Guanylyl Cyclase, and cGMP-Dependent Protein Kinase I in Mouse Stem Cell Cardiac Development

Directory of Open Access Journals (Sweden)

Valentina Spinelli

2016-01-01

Full Text Available Introduction and Aim. Nitric oxide (NO can trigger cardiac differentiation of embryonic stem cells (ESCs, indicating a cardiogenic function of the NO synthetizing enzyme(s (NOS. However, the involvement of the NO/NOS downstream effectors soluble guanylyl cyclase (sGC and cGMP activated protein kinase I (PKG-I is less defined. Therefore, we assess the involvement of the entire NO/NOS/sGC/PKG-I pathway during cardiac differentiation process. Methods. Mouse ESCs were differentiated toward cardiac lineages by hanging drop methodology for 21 days. NOS/sGC/PKG-I pathway was studied quantifying genes, proteins, enzymatic activities, and effects of inhibition during differentiation. Percentages of beating embryoid bodies (mEBs were evaluated as an index of cardiogenesis. Results and Discussion. Genes and protein expression of enzymes were increased during differentiation with distinctive kinetics and proteins possessed their enzymatic functions. Exogenous administered NO accelerated whereas the blockade of PKG-I strongly slowed cardiogenesis. sGC inhibition was effective only at early stages and NOS blockade ineffective. Of NOS/sGC/PKG-I pathway, PKG-I seems to play the prominent role in cardiac maturation. Conclusion. We concluded that exogenous administered NO and other pharmacological strategies able to increase the activity of PKG-I provide new tools to investigate and promote differentiation of cardiogenic precursors.

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

Science.gov (United States)

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.

Correlative intravital imaging of cGMP signals and vasodilation in mice

Directory of Open Access Journals (Sweden)

Martin eThunemann

2014-10-01

Full Text Available Cyclic guanosine monophosphate (cGMP is an important signaling molecule and drug target in the cardiovascular system. It is well known that stimulation of the vascular nitric oxide (NO-cGMP pathway results in vasodilation. However, the spatiotemporal dynamics of cGMP signals themselves and the cGMP concentrations within specific cardiovascular cell types in health, disease, and during pharmacotherapy with cGMP-elevating drugs are largely unknown. To facilitate the analysis of cGMP signaling in vivo, we have generated transgenic mice that express fluorescence resonance energy transfer (FRET-based cGMP sensor proteins. Here, we describe two models of intravital FRET/cGMP imaging in the vasculature of cGMP sensor mice: (1 epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2 ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber. Both methods allow simultaneous monitoring of NO-induced cGMP transients and vasodilation in living mice. Detailed protocols of all steps necessary to perform and evaluate intravital imaging experiments of the vasculature of anesthetized mice including surgery, imaging, and data evaluation are provided. An image segmentation approach is described to estimate FRET/cGMP changes within moving structures such as the vessel wall during vasodilation. The methods presented herein should be useful to visualize cGMP or other biochemical signals that are detectable with FRET-based biosensors, such as cyclic adenosine monophosphate or Ca2+, and to correlate them with respective vascular responses. With further refinement and combination of transgenic mouse models and intravital imaging technologies, we envision an exciting future, in which we are able to ‘watch’ biochemistry, (patho physiology, and pharmacotherapy in the context of a living mammalian organism.

Contraction-associated translocation of protein kinase C in rat skeletal muscle

DEFF Research Database (Denmark)

Richter, Erik; Cleland, P J; Rattigan, S

1987-01-01

Electrical stimulation of the sciatic nerve of the anaesthetized rat in vivo led to a time-dependent translocation of protein kinase C from the muscle cytosol to the particulate fraction. Maximum activity of protein kinase C in the particulate fraction occurred after 2 min of intermittent short...... tetanic contractions of the gastrocnemius-plantaris-soleus muscle group and coincided with the loss of activity from the cytosol. Translocation of protein kinase C may imply a role for this kinase in contraction-initiated changes in muscle metabolism....

Protein kinase substrate identification on functional protein arrays

Directory of Open Access Journals (Sweden)

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

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

Directory of Open Access Journals (Sweden)

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.

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

Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase.

Science.gov (United States)

Polya, G M; Chandra, S; Condron, R

1993-02-01

A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cation-exchange column. This RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by denaturation in the presence of guanidinium HCl and mercaptoethanol and subsequent reverse-phase HPLC on a C8 column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Ca(2+)-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA preparation, RCA1, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 microM, respectively). N-terminal sequencing by sequential Edman degradation of RCA1, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3' (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent to the sites of proteolytic precursor cleavage.

Phagocytosis by macrophages mediated by receptors for denatured proteins - dependence on tyrosine protein kinases

Directory of Open Access Journals (Sweden)

M.R. Hespanhol

2002-03-01

Full Text Available Previous studies have demonstrated that some components of the leukocyte cell membrane, CR3 (Mac-1, CD11b/CD18 and p150/95, are able to bind to denatured proteins. Thus, it is of interest to know which effector functions of these cells can be triggered by these receptors when they interact with particles or surfaces covered with denatured proteins. In the present study we analyzed their possible role as mediators of phagocytosis of red cells covered with denatured bovine serum albumin (BSA by mouse peritoneal macrophages. We observed that a macrophages are able to recognize (bind to these red cells, b this interaction can be inhibited by denatured BSA in the fluid phase, c there is no phagocytosis of these particles by normal macrophages, d phagocytosis mediated by denatured BSA can be, however, effectively triggered in inflammatory macrophages induced by glycogen or in macrophages activated in vivo with LPS, and e this phagocytic capacity is strongly dependent on the activity of tyrosine protein kinases in its signal transduction pathway, as demonstrated by using three kinds of enzyme inhibitors (genistein, quercetin and herbimycin A.

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

Energy Technology Data Exchange (ETDEWEB)

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

Calcium-Dependent Protein Kinases in Phytohormone Signaling Pathways

Directory of Open Access Journals (Sweden)

Wuwu Xu

2017-11-01

Full Text Available Calcium-dependent protein kinases (CPKs/CDPKs are Ca2+-sensors that decode Ca2+ signals into specific physiological responses. Research has reported that CDPKs constitute a large multigene family in various plant species, and play diverse roles in plant growth, development, and stress responses. Although numerous CDPKs have been exhaustively studied, and many of them have been found to be involved in plant hormone biosynthesis and response mechanisms, a comprehensive overview of the manner in which CDPKs participate in phytohormone signaling pathways, regulating nearly all aspects of plant growth, has not yet been undertaken. In this article, we reviewed the structure of CDPKs and the mechanism of their subcellular localization. Some CDPKs were elucidated to influence the intracellular localization of their substrates. Since little work has been done on the interaction between CDPKs and cytokinin signaling pathways, or on newly defined phytohormones such as brassinosteroids, strigolactones and salicylic acid, this paper mainly focused on discussing the integral associations between CDPKs and five plant hormones: auxins, gibberellins, ethylene, jasmonates, and abscisic acid. A perspective on future work is provided at the end.

Differential Contribution of the Guanylyl Cyclase-Cyclic GMP-Protein Kinase G Pathway to the Proliferation of Neural Stem Cells Stimulated by Nitric Oxide

Directory of Open Access Journals (Sweden)

Bruno P. Carreira

2012-02-01

Full Text Available Nitric oxide (NO is an important inflammatory mediator involved in the initial boost in the proliferation of neural stem cells following brain injury. However, the mechanisms underlying the proliferative effect of NO are still unclear. The aim of this work was to investigate whether cyclic GMP (cGMP and the cGMP-dependent kinase (PKG are involved in the proliferative effect triggered by NO in neural stem cells. For this purpose, cultures of neural stem cells isolated from the mouse subventricular zone (SVZ were used. We observed that long-term exposure to the NO donor (24 h, NOC-18, increased the proliferation of SVZ cells in a cGMP-dependent manner, since the guanylate cyclase inhibitor, ODQ, prevented cell proliferation. Similarly to NOC-18, the cGMP analogue, 8-Br-cGMP, also increased cell proliferation. Interestingly, shorter exposures to NO (6 h increased cell proliferation in a cGMP-independent manner via the ERK/MAP kinase pathway. The selective inhibitor of PKG, KT5823, prevented the proliferative effect induced by NO at 24 h but not at 6 h. In conclusion, the proliferative effect of NO is initially mediated by the ERK/MAPK pathway, and at later stages by the GC/cGMP/PKG pathway. Thus, our work shows that NO induces neural stem cell proliferation by targeting these two pathways in a biphasic manner.

Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

Science.gov (United States)

Amador, Erick; López-Pacheco, Karla; Morales, Nataly; Coria, Roberto; López-Villaseñor, Imelda

2017-04-01

Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

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

Directory of Open Access Journals (Sweden)

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.

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

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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 Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels

NARCIS (Netherlands)

van Tiel, Claudia M.; Westerman, Jan; Paasman, Marten A.; Hoebens, Martha M.; Wirtz, Karel W. A.; Snoek, Gerry T.

2002-01-01

Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165)

Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

Science.gov (United States)

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

Protein kinase C-dependent regulation of connexin43 gap junctions and hemichannels

DEFF Research Database (Denmark)

Alstrøm, Jette Skov; Stroemlund, Line Waring; Nielsen, Morten Schak

2015-01-01

Connexin43 (Cx43) generates intercellular gap junction channels involved in, among others, cardiac and brain function. Gap junctions are formed by the docking of two hemichannels from neighbouring cells. Undocked Cx43 hemichannels can upon different stimuli open towards the extracellular matrix...... and allow transport of molecules such as fluorescent dyes and ATP. A range of phosphorylated amino acids have been detected in the C-terminus of Cx43 and their physiological role has been intensively studied both in the gap junctional form of Cx43 and in its hemichannel configuration. We present the current...... knowledge of protein kinase C (PKC)-dependent regulation of Cx43 and discuss the divergent results....

How protein kinases co-ordinate mitosis in animal cells.

Science.gov (United States)

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.

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases.

Science.gov (United States)

Melchior, Aurélie; Denys, Agnès; Deligny, Audrey; Mazurier, Joël; Allain, Fabrice

2008-02-01

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.

Activation of protein kinase C inhibits synthesis and release of decidual prolactin

International Nuclear Information System (INIS)

Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

1986-01-01

Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

International Nuclear Information System (INIS)

Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

2010-01-01

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca 2+ -dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

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.

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

Science.gov (United States)

Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

2002-11-01

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

The role of cGMP as a mediator of lipolysis in bovine oocytes and its effects on embryo development and cryopreservation.

Directory of Open Access Journals (Sweden)

Kátia R L Schwarz

Full Text Available This study aimed to determine the influence of cyclic guanosine 3'5'-monophosphate (cGMP and cGMP-dependent kinase (PKG during in vitro maturation (IVM on lipolysis-related parameters in bovine cumulus-oocyte complexes (COCs, and on embryo development and cryosurvival. COCs were matured with cGMP/PKG modulators and assessed for metaphase II rates (MII, cGMP levels, lipid content in oocytes (OO, transcript abundance for genes involved in lipolysis (ATGL and lipid droplets (PLIN2 in cumulus cells (CC and OO, and presence of phosphorylated (active hormone sensitive lipase (HSLser563 in OO. Embryo development, lipid contents and survival to vitrification were also assessed. Phosphodiesterase 5 inhibition (PDE5; cGMP-hydrolyzing enzyme with 10-5M sildenafil (SDF during 24 h IVM increased cGMP in COCs (56.9 vs 9.5 fMol/COC in untreated controls, p<0.05 and did not affect on maturation rate (84.3±6.4% MII. Fetal calf serum (FCS in IVM medium decreased cGMP in COCs compared to bovine serum albumin (BSA + SDF (19.6 vs 66.5 fMol/COC, respectively, p<0.05. FCS increased lipid content in OO (40.1 FI, p<0.05 compared to BSA (34.6 FI, while SDF decreased (29.8 and 29.6 FI, with BSA or FCS, respectively p<0.05. PKG inhibitor (KT5823 reversed this effect (38.9 FI, p<0.05. ATGL and PLIN2 transcripts were detected in CC and OO, but were affected by cGMP and PKG only in CC. HSLser563 was detected in OO matured with or without modulators. Reduced lipid content in embryos were observed only when SDF was added during IVM and IVC (27.6 FI compared to its use in either or none of the culture periods (34.2 FI, p<0.05. Survival to vitrification was unaffected by SDF. In conclusion, cGMP and PKG are involved in lipolysis in OO and possibly in CC and embryos; serum negatively affects this pathway, contributing to lipid accumulation, and cGMP modulation may reduce lipid contents in oocytes and embryos, but without improving embryo cryotolerance.

Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response

KAUST Repository

Almadanim, M. Cecí lia; Alexandre, Bruno M.; Rosa, Margarida T.G.; Sapeta, Helena; Leitã o, Antó nio E.; Ramalho, José C.; Lam, TuKiet T.; Negrã o, Só nia; Abreu, Isabel A.; Oliveira, M. Margarida

2017-01-01

Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here

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

Competitive protein binding assay

International Nuclear Information System (INIS)

Kaneko, Toshio; Oka, Hiroshi

1975-01-01

The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

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.

Genome-wide Identification and Expression Analysis of Calcium-dependent Protein Kinase and Its Closely Related Kinase Genes in Capsicum annuum

Directory of Open Access Journals (Sweden)

hanyang ecai

2015-09-01

Full Text Available As Ca2+ sensors and effectors, calcium-dependent protein kinases (CDPKs play important roles in regulating the downstream components of calcium signaling, which are ubiquitously involved in plant growth, development, and response to environmental cues. However, no CDPKs have been characterized in Capsicum annuum thus far. Herein, a comprehensive analysis of genes encoding pepper CDPKs and CDPK-related protein kinases (CRKs was performed, and 31 CDPK genes and five closely related kinase genes were identified, which were phylogenetically divided into four distinct subfamilies and unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures were found to be shared by pepper CDPKs within the same subfamily, and the expansion of the CaCPK family in pepper was found to be due to segmental duplication events. Five CDPKs in the Capsicum annuum variety CM334 were found to be mutated in the Chiltepin variety, and one CDPK present in CM334 was lost in Chiltepin. The majority of CDPK and CRK genes were expressed in different pepper tissues and developmental stages, and 10, 12, and eight CDPK genes were transcriptionally modified by salt, heat, and Ralstonia solanacearum stresses, respectively. Furthermore, these genes were found to respond specifically to one stress as well as respond synergistically to two stresses or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum. Our results lay the foundation for future functional characterization of pepper CDPK and its closely related gene families.

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

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

Directory of Open Access Journals (Sweden)

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.

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....

Cocoa Procyanidins Suppress Transformation by Inhibiting Mitogen-activated Protein Kinase Kinase*S⃞

Science.gov (United States)

Kang, Nam Joo; Lee, Ki Won; Lee, Dong Eun; Rogozin, Evgeny A.; Bode, Ann M.; Lee, Hyong Joo; Dong, Zigang

2008-01-01

Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation. PMID:18519570

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...

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.

Adenosine monophosphate-activated protein kinase from the mud ...

Indian Academy of Sciences (India)

2016-12-01

Dec 1, 2016 ... to the understanding of the molecular mechanism of acclimation to cold hardiness in S. ... have shown that the stress associated with cold temperature ..... vation by cyclic-AMP-dependent protein kinase, studied using.

Propofol reduced myocardial contraction of vertebrates partly by mediating the cyclic AMP-dependent protein kinase phosphorylation pathway

International Nuclear Information System (INIS)

Sun, Xiaotong; Zhang, Xinyu; Bo, Qiyu; Meng, Tao; Lei, Zhen; Li, Jingxin; Hou, Yonghao; Yu, Xiaoqian; Yu, Jingui

2016-01-01

Propofol inhibits myocardial contraction in a dose dependent manner. The present study is designed to examine the effect of propofol on PKA mediated myocardial contraction in the absence of adrenoreceptor agonist. The contraction of isolated rat heart was measured in the presence or absence of PKA inhibitor H89 or propofol, using a pressure transducer. The levels of cAMP and PKA kinase activity were detected by ELISA. The mRNA and total protein or phosphorylation level of PKA and downstream proteins were tested in the presence or absence of PKA inhibitor H89 or propofol, using RT-PCR, QPCR and western blotting. The phosphorylation level of PKA was examined thoroughly using immunofluorescence and PKA activity non-radioactive detection kit. Propofol induced a dose-dependent negative contractile response on the rat heart. The inhibitory effect of high concentration propofol (50 μM) with 45% decease of control could be partly reversed by the PKA inhibitor H89 (10 μM) and the depressant effect of propofol decreased from 45% to 10%. PKA kinase activity was inhibited by propofol in a dose-dependent manner. Propofol also induced a decrease in phosphorylation of PKA, which was also inhibited by H89, but did not alter the production of cAMP and the mRNA levels of PKA. The downstream proteins of PKA, PLN and RyR2 were phosphorylated to a lesser extent with propofol or H89 than control. These results demonstrated that propofol induced a negative myocardial contractile response partly by mediating the PKA phosphorylation pathway.

The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling

CSIR Research Space (South Africa)

Wheeler, J

2017-06-01

Full Text Available ) with the ll PickUp Injection mode using the loading pump at 15 ll min�1 flow rate for 3 min. Samples were then loaded on a RSLC, 75 lm 9 500 mm, nanoVi- per, C18, 2 lm, 100 �A column (Acclaim, PepMap) retrofitted to an EASY-spray source with a flow rate of 300... receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling Janet I. Wheeler1,2,†, Aloysius Wong3,4, Claudius Marondedze3,5, Arnoud J. Groen5, Lusisizwe Kwezi1,6, Lubna Freihat1, Jignesh Vyas1, Misjudeen A. Raji7, Helen R. Irving1...

An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

International Nuclear Information System (INIS)

Meier, K.; Klein, C.

1988-01-01

The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

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.

Structural Bioinformatics and Protein Docking Analysis of the Molecular Chaperone-Kinase Interactions: Towards Allosteric Inhibition of Protein Kinases by Targeting the Hsp90-Cdc37 Chaperone Machinery

Directory of Open Access Journals (Sweden)

Gennady Verkhivker

2013-11-01

Full Text Available A fundamental role of the Hsp90-Cdc37 chaperone system in mediating maturation of protein kinase clients and supporting kinase functional activity is essential for the integrity and viability of signaling pathways involved in cell cycle control and organism development. Despite significant advances in understanding structure and function of molecular chaperones, the molecular mechanisms and guiding principles of kinase recruitment to the chaperone system are lacking quantitative characterization. Structural and thermodynamic characterization of Hsp90-Cdc37 binding with protein kinase clients by modern experimental techniques is highly challenging, owing to a transient nature of chaperone-mediated interactions. In this work, we used experimentally-guided protein docking to probe the allosteric nature of the Hsp90-Cdc37 binding with the cyclin-dependent kinase 4 (Cdk4 kinase clients. The results of docking simulations suggest that the kinase recognition and recruitment to the chaperone system may be primarily determined by Cdc37 targeting of the N-terminal kinase lobe. The interactions of Hsp90 with the C-terminal kinase lobe may provide additional “molecular brakes” that can lock (or unlock kinase from the system during client loading (release stages. The results of this study support a central role of the Cdc37 chaperone in recognition and recruitment of the kinase clients. Structural analysis may have useful implications in developing strategies for allosteric inhibition of protein kinases by targeting the Hsp90-Cdc37 chaperone machinery.

Xanthene derivatives increase glucose utilization through activation of LKB1-dependent AMP-activated protein kinase.

Directory of Open Access Journals (Sweden)

Yonghoon Kwon

Full Text Available 5' AMP-activated protein kinase (AMPK is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl-thioureido]-ethyl}-amide (Xn and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl-thioureido]-ethyl}-amide (Xc elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4. Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.

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

Directory of Open Access Journals (Sweden)

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.

In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

KAUST Repository

Raji, Misjudeen; Gehring, Christoph A

2017-01-01

Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

KAUST Repository

Raji, Misjudeen

2017-05-31

Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

Detection of protein kinase activity by renaturation in sodium dodecyl sulfate-polyacrylamide gels

International Nuclear Information System (INIS)

Anostario, M. Jr.; Harrison, M.L.; Geahlen, R.L.

1986-01-01

The authors have developed a procedure for identifying protein kinase activity in protein samples following electrophoresis on SDS-polyacrylamide gels. Proteins are allowed to renature directly in the gel by removal of detergent. The gel is then incubated with [γ- 32 P]ATP to allow renatured protein kinases to autophosphorylate or to phosphorylate various substrates which can be incorporated into the gel. The positions of the radiolabeled proteins can then be detected by autoradiography. With this technique, using purified catalytic subunit of cAMP-dependent protein kinase, enzyme concentrations as low as 0.01 μg can be detected on gels containing 1.0 mg/ml casein. The procedure is also applicable for the determination of active subunits of multisubunit protein kinases. For example, when the two subunits of casein kinase II are separated by SDS-polyacrylamide gel electrophoresis and allowed to renature, only the larger α subunit shows activity. This procedure can also be used to detect and distinguish kinases present in heterogeneous mixtures. Starting with a particulate fraction from LSTRA, a murine T cell lymphoma, several distinct enzymes were detected, including a 30,000 Dalton protein with protein-tyrosine kinase activity. This same enzyme has also been detected in T lymphocytes and other T lymphoid cell lines

DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

Science.gov (United States)

Dolman, M Emmy M; van der Ploeg, Ida; Koster, Jan; Bate-Eya, Laurel Tabe; Versteeg, Rogier; Caron, Huib N; Molenaar, Jan J

2015-01-01

Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

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

Science.gov (United States)

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.

Cyclic Nucleotide Signalling in Kidney Fibrosis

Directory of Open Access Journals (Sweden)

Elisabeth Schinner

2015-01-01

Full Text Available Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP and cyclic guanosine monophosphate (cGMP were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure.

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.

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

-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. 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. © FASEB.

Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

International Nuclear Information System (INIS)

Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

1986-01-01

Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca 2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32 P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32 P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

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

Directory of Open Access Journals (Sweden)

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.

Receptor-interacting protein (RIP) kinase family

Science.gov (United States)

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

The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

Science.gov (United States)

Schleede, Justin; Blair, Seth S

2015-10-01

The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

A framework for classification of prokaryotic protein kinases.

Directory of Open Access Journals (Sweden)

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

Identification of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel target of bisphenol A.

Science.gov (United States)

Ito, Yuki; Ito, Takumi; Karasawa, Satoki; Enomoto, Teruya; Nashimoto, Akihiro; Hase, Yasuyoshi; Sakamoto, Satoshi; Mimori, Tsuneyo; Matsumoto, Yoshihisa; Yamaguchi, Yuki; Handa, Hiroshi

2012-01-01

Bisphenol A (BPA) forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER) and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80), is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM), high doses of BPA were required before cellular effects were observed (100-300 μM). The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR)-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient). Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.

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

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

Directory of Open Access Journals (Sweden)

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.

Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

Science.gov (United States)

Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

1997-01-01

A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

Directory of Open Access Journals (Sweden)

M Emmy M Dolman

Full Text Available Tumor cells might resist therapy with ionizing radiation (IR by non-homologous end-joining (NHEJ of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK. The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

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

OpenAIRE

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...

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

Directory of Open Access Journals (Sweden)

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

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

Science.gov (United States)

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

Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases

NARCIS (Netherlands)

Wit, René J.W. de; Hekstra, Doeke; Jastorff, Bernd; Stec, Wojciech J.; Baraniak, Janina; Driel, Roel van; Haastert, Peter J.M. van

1984-01-01

A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP-binding sites in mammalian protein kinase type I. In addition, the activation of the kinase by these analogs was monitored. The binding

A Calcium-Dependent Protein Kinase Is Systemically Induced upon Wounding in Tomato Plants1

Science.gov (United States)

Chico, José Manuel; Raíces, Marcela; Téllez-Iñón, María Teresa; Ulloa, Rita María

2002-01-01

A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks. PMID:11788771

Cyclic guanosine monophosphate in the regulation of the cell function

Directory of Open Access Journals (Sweden)

Małgorzata Zbrojkiewicz

2016-12-01

Full Text Available Intracellular concentration of cGMP depends on the activity of guanylate cyclase, responsible for its synthesis, on the activity of cyclic nucleotide degrading enzymes - phosphodiesterases (PDEs. There are two forms of guanylate cyclase: the membrane-bound cyclase and the soluble form. The physiological activators of the membrane guanylate cyclase are natriuretic peptides (NPs, and of the cytosolic guanylate cyclase - nitric oxide (NO and carbon monoxide (CO. Intracellular cGMP signaling pathways arise from its direct effect on the activity of G protein kinases, phosphodiesterases and cyclic nucleotide dependent cation channels. It has been shown in recent years that cGMP can also affect other signal pathways in cell signaling activity involving Wnt proteins and sex hormones. The increased interest in the research on the role of cGMP, resulted also in the discovery of its role in the regulation of phototransduction in the eye, neurotransmission, calcium homeostasis, platelet aggregation, heartbeat, bone remodeling, lipid metabolism and the activity of the cation channels. Better understanding of the mechanisms of action of cGMP in the regulation of cell function can create new opportunities for the cGMP affecting drugs use in the pharmacotherapy.

Differential expression of mRNAs for protein kinase inhibitor isoforms in mouse brain.

OpenAIRE

Seasholtz, A F; Gamm, D M; Ballestero, R P; Scarpetta, M A; Uhler, M D

1995-01-01

Many neurotransmitters are known to regulate neuronal cell function by means of activation of cAMP-dependent protein kinase (PKA) and phosphorylation of neuronal substrate proteins, including transcription factors and ion channels. Here, we have characterized the gene expression of two isoforms of a protein kinase inhibitor (PKI) specific for PKA in mouse brain by RNase protection and in situ hybridization histochemistry. The studies demonstrate that the PKI alpha isoform is abundant in many ...

Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth.

Directory of Open Access Journals (Sweden)

Hitomi Kurokawa

Full Text Available cAMP-dependent protein kinase (PKA has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R-overexpressing tachyzoites showed a significant growth defect.Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

Protein kinase C activation induces conductance changes in Hermissenda photoreceptors like those seen in associative learning.

Science.gov (United States)

Farley, J; Auerbach, S

Phosphorylation of ion channels has been suggested as one molecular mechanism responsible for learning-produced long-term changes in neuronal excitability. Persistent training-produced changes in two distinct K+ currents (IA (ref. 2), IK-Ca (refs 3,4)) and a voltage-dependent calcium current (ICa; refs 3,4) have previously been shown to occur in type B photoreceptors of Hermissenda, as a result of associative learning. But the identity of the phosphorylation pathway(s) responsible for these changes has not as yet been determined. Injections of cyclic AMP-dependent protein kinase reduce a K+ current (IK) in B cells which is different from those changed by training, but fails to reduce IA and IK-Ca. Phosphorylase b kinase (an exogenous calcium/calmodulin-dependent kinase) reduces IA, but whether IK-Ca and ICa are changed in the manner of associative training is not yet known. Another protein kinase present in high concentrations in both mammalian brain and molluscan nervous systems is protein kinase C, which is both calcium- and phospholipid-sensitive. We now present evidence that activation of protein kinase C by the tumour promoter phorbol ester (PDB) and intracellular injection of the enzyme induce conductance changes similar to those caused by associative training in Hermissenda B cells (that is a reduction of IA and IK-Ca, and enhancement of ICa). These results represent the first direct demonstration that protein kinase C affects membrane K+ ion conductance mechanisms.

Inhibition of protein kinase C induces differentiation in Neuro-2a cells

International Nuclear Information System (INIS)

Minana, M.D.; Felipo, V.; Grisolia, S.

1990-01-01

1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 μM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 μM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased ∼7-fold after 48 hr with 500 μM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

Science.gov (United States)

Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

2015-01-30

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

Directory of Open Access Journals (Sweden)

Yasuko Kitagishi

2015-01-01

Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

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

Science.gov (United States)

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.

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

Science.gov (United States)

Roskoski, Robert

2015-10-01

Protein kinases play a predominant regulatory role in nearly every aspect of cell biology and they can modify the function of a protein in almost every conceivable way. Protein phosphorylation can increase or decrease enzyme activity and it can alter other biological activities such as transcription and translation. Moreover, some phosphorylation sites on a given protein are stimulatory while others are inhibitory. The human protein kinase gene family consists of 518 members along with 106 pseudogenes. Furthermore, about 50 of the 518 gene products lack important catalytic residues and are called protein pseudokinases. The non-catalytic allosteric interaction of protein kinases and pseudokinases with other proteins has added an important regulatory feature to the biochemistry and cell biology of the protein kinase superfamily. With rare exceptions, a divalent cation such as Mg2+ is required for the reaction. All protein kinases exist in a basal state and are activated only as necessary by divergent regulatory stimuli. The mechanisms for switching between dormant and active protein kinases can be intricate. Phosphorylase kinase was the first protein kinase to be characterized biochemically and the mechanism of its regulation led to the discovery of cAMP-dependent protein kinase (protein kinase A, or PKA), which catalyzes the phosphorylation and activation of phosphorylase kinase. This was the first protein kinase cascade or signaling module to be elucidated. The epidermal growth factor receptor-Ras-Raf-MEK-ERK signaling module contains protein-tyrosine, protein-serine/threonine, and dual specificity protein kinases. PKA has served as a prototype of this enzyme family and more is known about this enzyme than any other protein kinase. The inactive PKA holoenzyme consists of two regulatory and two catalytic subunits. After binding four molecules of cAMP, the holoenzyme dissociates into a regulatory subunit dimer (each monomer binds two cAMP) and two free and active

Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

KAUST Repository

Qia, Zhi

2010-11-18

A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogendefense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca 2+ permeable channels in mesophyll cells, resulting in cytosolic Ca 2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor- like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2- dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca 2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca 2+ conductance and resulting cytosolic Ca 2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen- defense genes in a Ca 2+-dependent manner.

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

Science.gov (United States)

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.

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum.

Science.gov (United States)

Abdi, Abdirahman; Eschenlauer, Sylvain; Reininger, Luc; Doerig, Christian

2010-10-01

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal "sterile alpha-motif" domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

Science.gov (United States)

Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

2014-01-01

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

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...

Nicotine shifts the temporal activation of hippocampal protein kinase A and extracellular signal-regulated kinase 1/2 to enhance long-term, but not short-term, hippocampus-dependent memory.

Science.gov (United States)

Gould, Thomas J; Wilkinson, Derek S; Yildirim, Emre; Poole, Rachel L F; Leach, Prescott T; Simmons, Steven J

2014-03-01

Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 h but not 2 h post-training, delineating time points for STM (2 h) and LTM (4 h and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 h but not 2 h post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning, and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories. Copyright © 2014 Elsevier Inc. All rights reserved.

Participation of intracellular signal transduction in the radio-adaptive response induced by low-dose X-irradiation in human embryonic cells

International Nuclear Information System (INIS)

Ishii, Keiichiro; Hoshi, Yuko; Iwasaki, Toshiyasu; Watanabe, Masami.

1996-01-01

To elucidate the induction mechanism of radio-adaptive response in normal cells, we searched the literatures of the intracellular signal transduction. Furthermore, we examined the induction of radio-adaptive response with or without inhibitors of several kinds of protein kinase. The major results obtained were as follows; (1) According to the literature survey it is revealed that there are 4 intracellular signal transduction pathways which are possibly involved in the induction of radio-adaptive response: pathways depending on cAMP, calcium, cGMP, or protein-tyrosine kinase. (2) Addition of either inhibitor of protein-tyrosine kinase or protein kinase C to the cell culture medium during the low-dose X-irradiation inhibited the induction of radio-adaptive response. However, the addition of inhibitor of cAMP-dependent protein kinase, cGMP-dependent protein kinase, or Ca 2+ -calmodulin kinase II failed to inhibit the induction of radio-adaptive response. (3) These results suggest that the signal induced in cells by low-dose X-irradiation was transduced from protein-tyrosine kinase to protein kinase C via either pathway of phosphatidylinositol 3-kinase or splitting of profilin binding phosphatidylinositol 4,5-bisphosphate. (author)

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.

IL-4 induces cAMP and cGMP in human monocytic cells

Directory of Open Access Journals (Sweden)

B. Dugas

1995-01-01

Full Text Available Human monocytes, preincubated with IFN-γ respond to IL-4 by a cGMP increase through activation of an inducible NO synthase. Here, IL-4 was found to induce an accumulation of cGMP (1 – 3 min and cAMP (20 – 25 min in unstimulated monocytes. This was impaired with NOS inhibitors, but also with EGTA and calcium/calmodulin inhibitors. These results suggest that: (1 IL-4 may stimulate different NOS isoforms in resting and IFN-γ activated monocytes, and (2 cAMP accumulation may be partially dependent on the NO pathway. By RT-PCR, a type III constitutive NOS mRNA was detected in U937 monocytic cells. IL-4 also increased the [Ca2+]i in these cells. Different NOS may thus be expressed in monocytic cells depending on their differentiation and the signals they receive.

The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

Directory of Open Access Journals (Sweden)

Justin Schleede

2015-10-01

Full Text Available The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog. However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

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

Bradykinin and vasopressin activate phospholipase D in rat Leydig cells by a protein kinase C-dependent mechanism

DEFF Research Database (Denmark)

Vinggaard, Anne Marie; Hansen, Harald S.

1993-01-01

of PMA and vasopressin (AVP), PMA and bradykinin, or AVP and bradykinin produced no additive phosphatidylethanol or choline response, suggesting that AVP, bradykinin and PMA stimulated phospholipase D catalysed phosphatidylcholine hydrolysis by a similar protein kinase C-dependent mechanism. Furthermore......, LH (10 ng/ml), insulin (500 nmol/l), GH (100 ng/ml), interleukin-1ß (5 U/ml) and platelet-activating factor (200 nmol/l) were found not to activate phospholipase D, whereas the Ca ionophore A23187 (10 µmol/l) stimulated phosphatidylethanol formation, suggesting that Ca might be a regulator...

Effect of microinjections of subunits of cAMP-dependent protein kinase on development, proliferation, and RNA synthesis in early embryos of the loach Misgurnus fossilis L

International Nuclear Information System (INIS)

Glukhov, A.I.; Benyumov, A.O.; Nesterova, M.V.; Severin, E.S.; Gazaryan, K.G.

1986-01-01

The effect of the catalytic and regulatory subunits of cAMP-dependent protein kinase type II on development, proliferation, and RNA synthesis was studied in loach embryos. It was found that injection of the catalytic subunit in a physiological concentration leads to a disturbance in the course of development and inhibits proliferation and RNA synthesis in the embryos. An increase in the concentration of this protein above the physiological level leads to death of the embryos in the first hours of development. Injection of the regulatory subunit stimulated the incorporation of labeled uridine into the acid-insoluble fraction of the embryos, beginning with the gastrula stage. The cell nuclei of loach embryos injected with subunits of protein kinase type II were transplanted into activated loach egg cells: subunits of protein kinase type I had no effect on the ability of nuclei of undetermined loach embryo cells to provide de novo development and their effect was reversible

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.

Calcium-dependent protein kinase 21 phosphorylates 14-3-3 proteins in response to ABA signaling and salt stress in rice.

Science.gov (United States)

Chen, Yixing; Zhou, Xiaojin; Chang, Shu; Chu, Zhilin; Wang, Hanmeng; Han, Shengcheng; Wang, Yingdian

2017-12-02

The calcium-dependent protein kinases (CDPKs) are a class of plant-specific kinase that directly bind Ca 2+ and mediate the calcium-signaling pathways to play important physiological roles in growth and development. The rice genome contains 31 CDPK genes, one of which, OsCPK21, is known to modulate the abscisic acid (ABA) and salt stress responses in this crop; however, the molecular mechanisms underlying this regulation are largely unknown. In the present study, we performed yeast two-hybrid screening, glutathione S-transferase pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation assays to confirm the interaction between OsCPK21 and one of its putative targets, Os14-3-3 (OsGF14e). We used an in vitro kinase assay and site-directed mutagenesis to verify that OsCPK21 phosphorylates OsGF14e at Tyr-138. We used real-time PCR to reveal that several ABA and salt inducible genes were more highly expressed in the OsCPK21-OE and OsGF14e WT-OE plants than in the mutant OsGF14e Y138A-OE and wild-type plants. These results suggest that OsCPK21 phosphorylates OsGF14e to facilitate the response to ABA and salt stress. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4 includes the regulatory PSTAIRE helix

Directory of Open Access Journals (Sweden)

Grassmann Ralph

2005-09-01

Full Text Available Abstract Background The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1 is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4. Results To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity. Conclusion Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.

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...

Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

International Nuclear Information System (INIS)

Yang, Weng-Lang; Ravatn, Roald; Kudoh, Kazuya; Alabanza, Leah; Chin, Khew-Voon

2010-01-01

The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R 2 C 2 . The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RIα, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RIα, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RIα subunit of PKA may have functions independent of the kinase. We show here that the RIα subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RIα results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RIα and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RIα modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RIα with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

Deficient plasticity in the primary visual cortex of alpha-calcium/calmodulin-dependent protein kinase II mutant mice.

Science.gov (United States)

Gordon, J A; Cioffi, D; Silva, A J; Stryker, M P

1996-09-01

The recent characterization of plasticity in the mouse visual cortex permits the use of mutant mice to investigate the cellular mechanisms underlying activity-dependent development. As calcium-dependent signaling pathways have been implicated in neuronal plasticity, we examined visual cortical plasticity in mice lacking the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha CaMKII). In wild-type mice, brief occlusion of vision in one eye during a critical period reduces responses in the visual cortex. In half of the alpha CaMKII-deficient mice, visual cortical responses developed normally, but visual cortical plasticity was greatly diminished. After intensive training, spatial learning in the Morris water maze was severely impaired in a similar fraction of mutant animals. These data indicate that loss of alpha CaMKII results in a severe but variable defect in neuronal plasticity.

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.

Nitric Oxide Binds to and Modulates the Activity of a Pollen Specific Arabidopsis Diacylglycerol Kinase

KAUST Repository

Wong, Aloysius Tze

2014-06-01

Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5\\'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered

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 ...

Lipid composition of cAMP-dependent protein kinase mutants of Aspergillus niger.

Science.gov (United States)

Jernejc, Katarina; Bencina, Mojca

2003-08-29

Lipid composition of cAMP-dependent protein kinase (PKA) Aspergillus niger mutants with overexpressed or deleted genes for either regulatory and/or the catalytic subunit of PKA was analyzed. Disruption of the gene encoding the PKA regulatory subunit resulted in 20% less total lipids, 30% less neutral lipids, four times more glycolipids and two-fold higher triacylglycerol lipase activity compared to the control strain. Concomitantly a five-fold decrease in phosphatidylcholine, accompanied with 1.5-, 1.8- and 2.8-fold increases in phosphatidylethanolamine, lysophosphatidylethanolamine and phosphatidylinositol, was determined, respectively. The lack of PKA activity, due to the disruption of a gene encoding the PKA catalytic subunit, resulted in a 1.6-times increase in total lipids with two times more neutral lipids associated with lower triacylglycerol lipase activity and a decrease in phospholipids. The mutants with unrestricted PKA activity synthesized twice as much citric acid as the control strain and three times more than strains lacking PKA activity. The results indicate the involvement of cAMP-mediated PKA activity in regulation of lipid biosynthesis as well as citric acid synthesis.

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

Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

Science.gov (United States)

Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

2014-11-01

Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

Science.gov (United States)

Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

2018-05-03

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

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.

Heterologous desensitization of adenylate cyclase from pigeon erythrocytes under the action of the catalytic subunit of cAMP-dependent protein kinase

International Nuclear Information System (INIS)

Popov, K.M.; Bulargina, T.V.; Severin, E.S.

1985-01-01

Preincubation of the plasma membranes from pigeon erythrocytes with the catalytic subunit of cAMP-dependent protein kinase leads to desensitization of adenylate cyclase of the erythrocytes. The adenylate cyclase activity, measured in the presence of 10 μM isoproterenol and 50 μM GTP-γ-S, is decreased by 40% in 10 min of incubation, while the activity in the presence of 50 μM GTP-γ-S is decreased by 35% in 20 min. The decrease in the adenylate cyclase activity is due to an increase in the lag phase of activation of the enzyme in the presence of a GTP analog stable to hydrolysis and a decrease in the activity in the steady-state phase of activation. Heterologous desensitization of adenylate cyclase under the action of cAMP-dependent protein kinase is coupled with a decrease in the number of β-adrenoreceptors capable of passing into a state of high affinity for antagonists in the absence of guanylic nucleotides. The influence of the catalytic subunit on adenylate cyclase entirely models the process of desensitization of the enzyme absorbed in the influence of isoproterenol or cAMP on erythrocytes

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)

Nicotine reward and affective nicotine withdrawal signs are attenuated in calcium/calmodulin-dependent protein kinase IV knockout mice.

Directory of Open Access Journals (Sweden)

Kia J Jackson

Full Text Available The influx of Ca(2+ through calcium-permeable nicotinic acetylcholine receptors (nAChRs leads to activation of various downstream processes that may be relevant to nicotine-mediated behaviors. The calcium activated protein, calcium/calmodulin-dependent protein kinase IV (CaMKIV phosphorylates the downstream transcription factor cyclic AMP response element binding protein (CREB, which mediates nicotine responses; however the role of CaMKIV in nicotine dependence is unknown. Given the proposed role of CaMKIV in CREB activation, we hypothesized that CaMKIV might be a crucial molecular component in the development of nicotine dependence. Using male CaMKIV genetically modified mice, we found that nicotine reward is attenuated in CaMKIV knockout (-/- mice, but cocaine reward is enhanced in these mice. CaMKIV protein levels were also increased in the nucleus accumbens of C57Bl/6 mice after nicotine reward. In a nicotine withdrawal assessment, anxiety-related behavior, but not somatic signs or the hyperalgesia response are attenuated in CaMKIV -/- mice. To complement our animal studies, we also conducted a human genetic association analysis and found that variants in the CaMKIV gene are associated with a protective effect against nicotine dependence. Taken together, our results support an important role for CaMKIV in nicotine reward, and suggest that CaMKIV has opposing roles in nicotine and cocaine reward. Further, CaMKIV mediates affective, but not physical nicotine withdrawal signs, and has a protective effect against nicotine dependence in human genetic association studies. These findings further indicate the importance of calcium-dependent mechanisms in mediating behaviors associated with drugs of abuse.

Roles of Apicomplexan protein kinases at each life cycle stage.

Science.gov (United States)

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.

Structural basis of divergent cyclin-dependent kinase activation by Spy1/RINGO proteins

Energy Technology Data Exchange (ETDEWEB)

McGrath, Denise A.; Fifield, Bre-Anne; Marceau, Aimee H.; Tripathi, Sarvind; Porter, Lisa A.; Rubin, Seth M. (UCSC); (Windsor)

2017-06-30

Cyclin-dependent kinases (Cdks) are principal drivers of cell division and are an important therapeutic target to inhibit aberrant proliferation. Cdk enzymatic activity is tightly controlled through cyclin interactions, posttranslational modifications, and binding of inhibitors such as the p27 tumor suppressor protein. Spy1/RINGO (Spy1) proteins bind and activate Cdk but are resistant to canonical regulatory mechanisms that establish cell-cycle checkpoints. Cancer cells exploit Spy1 to stimulate proliferation through inappropriate activation of Cdks, yet the mechanism is unknown. We have determined crystal structures of the Cdk2-Spy1 and p27-Cdk2-Spy1 complexes that reveal how Spy1 activates Cdk. We find that Spy1 confers structural changes to Cdk2 that obviate the requirement of Cdk activation loop phosphorylation. Spy1 lacks the cyclin-binding site that mediates p27 and substrate affinity, explaining why Cdk-Spy1 is poorly inhibited by p27 and lacks specificity for substrates with cyclin-docking sites. We identify mutations in Spy1 that ablate its ability to activate Cdk2 and to proliferate cells. Our structural description of Spy1 provides important mechanistic insights that may be utilized for targeting upregulated Spy1 in cancer.

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

Interaction of the regulatory subunit of the cAMP-dependent protein kinase with PATZ1 (ZNF278)

Energy Technology Data Exchange (ETDEWEB)

Yang, Weng-Lang [Long Island Jewish Medical Center, North Shore University Hospital, Manhasset, NY 11030 (United States); Ravatn, Roald [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Kudoh, Kazuya [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Department of Obstetrics and Gynecology, National Defense Medical College, Tokorozawa, Saitama (Japan); Alabanza, Leah [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States); Chin, Khew-Voon, E-mail: khew-voon.chin@utoledo.edu [Department of Medicine, University of Toledo, College of Medicine, Toledo, OH 43614 (United States)

2010-01-15

The effects of cAMP in cell are predominantly mediated by the cAMP-dependent protein kinase (PKA), which is composed of two genetically distinct subunits, catalytic (C) and regulatory (R), forming a tetrameric holoenzyme R{sub 2}C{sub 2}. The only known function for the R subunit is that of inhibiting the activity of the C subunit kinase. It has been shown that overexpression of RI{alpha}, but not the C subunit kinase, is associated with neoplastic transformation. In addition, it has also been demonstrated that mutation in the RI{alpha}, but not the C subunit is associated with increased resistance to the DNA-damaging anticancer drug cisplatin, thus suggesting that the RI{alpha} subunit of PKA may have functions independent of the kinase. We show here that the RI{alpha} subunit interacts with a BTB/POZ domain zinc-finger transcription factor, PATZ1 (ZNF278), and co-expression with RI{alpha} results in its sequestration in the cytoplasm. The cytoplasmic/nuclear translocation is inducible by cAMP. C-terminus deletion abolishes PATZ1 interaction with RI{alpha} and results in its localization in the nucleus. PATZ1 transactivates the cMyc promoter and the presence of cAMP and co-expression with RI{alpha} modulates its transactivation. Moreover, PATZ1 is aberrantly expressed in cancer. Taken together, our results showed a potentially novel mechanism of cAMP signaling mediated through the interaction of RI{alpha} with PATZ1 that is independent of the kinase activity of PKA, and the aberrant expression of PATZ1 in cancer point to its role in cell growth regulation.

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...

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

Calcium ion binding properties of Medicago truncatula calcium/calmodulin-dependent protein kinase.

Science.gov (United States)

Swainsbury, David J K; Zhou, Liang; Oldroyd, Giles E D; Bornemann, Stephen

2012-09-04

A calcium/calmodulin-dependent protein kinase (CCaMK) is essential in the interpretation of calcium oscillations in plant root cells for the establishment of symbiotic relationships with rhizobia and mycorrhizal fungi. Some of its properties have been studied in detail, but its calcium ion binding properties and subsequent conformational change have not. A biophysical approach was taken with constructs comprising either the visinin-like domain of Medicago truncatula CCaMK, which contains EF-hand motifs, or this domain together with the autoinhibitory domain. The visinin-like domain binds three calcium ions, leading to a conformational change involving the exposure of hydrophobic surfaces and a change in tertiary but not net secondary or quaternary structure. The affinity for calcium ions of visinin-like domain EF-hands 1 and 2 (K(d) = 200 ± 50 nM) was appropriate for the interpretation of calcium oscillations (~125-850 nM), while that of EF-hand 3 (K(d) ≤ 20 nM) implied occupancy at basal calcium ion levels. Calcium dissociation rate constants were determined for the visinin-like domain of CCaMK, M. truncatula calmodulin 1, and the complex between these two proteins (the slowest of which was 0.123 ± 0.002 s(-1)), suggesting the corresponding calcium association rate constants were at or near the diffusion-limited rate. In addition, the dissociation of calmodulin from the protein complex was shown to be on the same time scale as the dissociation of calcium ions. These observations suggest that the formation and dissociation of the complex between calmodulin and CCaMK would substantially mirror calcium oscillations, which typically have a 90 s periodicity.

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...

Photoaffinity labeling of cAMP-dependent protein kinase by 4-azido-2-nitrophenyladenylyl pyrophosphate

International Nuclear Information System (INIS)

Johnson, D.R.; Ho, H.T.; Wong, S.S.

1986-01-01

A photoaffinity analogue of ATP, 4-azido-2-nitrophenyl-adenylyl pyrophosphate (ANAP) has been synthesized to investigate the topographical interaction between the catalytic and the regulatory subunits of the bovine heart type II cAMP-dependent protein kinase. The synthesis involves coupling of 4-azido-2-nitrophenyl phosphate with adenosine 5'-monophosphomorpholidate. ANAP has an absorption maximum at 260 nm (molar absorptivity = 35.4 x 10 3 M -1 cm -1 ) and a shoulder at 320 nm. Kinetically, ANAP inhibits the enzyme competitively against ATP with a Ki of 0.37 mM. The catalytic subunit is inactivated by ANAP upon photolysis in the presence of magnesium ion. ATP protects the enzyme from photoinactivation but the regulatory subunit does not. Gel electrophoretic analysis of the enzyme labeled by [ 14 C]ANAP shows that the photoincorporated ANAP is associated mainly with the catalytic subunit, even when the regulator dimer is in twelve fold excess. Little or no ANAP is found incorporated into the regulator subunit. The data suggest that the photoreactive portion of ANAP does not lie within reach of the regulatory protein when the analogue is bound to the catalytic subunit

The role of Ca2+/calmodulin-dependent protein kinase II and calcineurin in TNF-α-induced myocardial hypertrophy

International Nuclear Information System (INIS)

Wang, Gui-Jun; Wang, Hong-Xin; Yao, Yu-Sheng; Guo, Lian-Yi; Liu, Pei

2012-01-01

We investigated whether Ca 2+ /calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN) are involved in myocardial hypertrophy induced by tumor necrosis factor α (TNF-α). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-α (100 µg/L), and Ca 2+ signal transduction was blocked by several antagonists, including BAPTA (4 µM), KN-93 (0.2 µM) and cyclosporin A (CsA, 0.2 µM). Protein content, protein synthesis, cardiomyocyte volumes, [Ca 2+ ] i transients, CaMKIIδ B and CaN were evaluated by the Lowry method, [ 3 H]-leucine incorporation, a computerized image analysis system, a Till imaging system, and Western blot analysis, respectively. TNF-α induced a significant increase in protein content in a dose-dependent manner from 10 µg/L (53.56 µg protein/well) to 100 µg/L (72.18 µg protein/well), and in a time-dependent manner from 12 h (37.42 µg protein/well) to 72 h (42.81 µg protein/well). TNF-α (100 µg/L) significantly increased the amplitude of spontaneous [Ca 2+ ] i transients, the total protein content, cell size, and [ 3 H]-leucine incorporation in cultured cardiomyocytes, which was abolished by 4 µM BAPTA, an intracellular Ca 2+ chelator. The increases in protein content, cell size and [ 3 H]-leucine incorporation were abolished by 0.2 µM KN-93 or 0.2 µM CsA. TNF-α increased the expression of CaMKIIδ B by 35.21% and that of CaN by 22.22% compared to control. These effects were abolished by 4 µM BAPTA, which itself had no effect. These results suggest that TNF-α induces increases in [Ca 2+ ] i , CaMKIIδ B and CaN and promotes cardiac hypertrophy. Therefore, we hypothesize that the Ca 2+ /CaMKII- and CaN-dependent signaling pathways are involved in myocardial hypertrophy induced by TNF-α

Induction of rat hepatic zinc thionein by phorbol ester-mediated protein kinase C pathway

Energy Technology Data Exchange (ETDEWEB)

Garrett, S.H.; Funk, A.E.; Brady, F.O.

1986-05-01

Metallothionein (MT) exists in rat liver mainly as a zinc protein. The levels of this protein fluctuate in response to a variety of internal and external stimuli. Among these inducers of MT are metals, glucocorticoids, catecholamines, and polypeptide hormones. Metals and glucocorticoids are primary inducers of MT, while the others operate either via adenylate cyclase/cAMP/cAMP-dependent protein kinase, or via phospholipase C/inositol 1,4,5-triphosphate, diacylglycerol/Ca/sup 2 +/-dependent protein kinase, protein kinase C. The authors have examined the role of the protein kinase C pathway in the induction of MT by using a phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), to activate it. In vivo TPA is a good inducer of Zn/sub 7/-MT with an ED/sub 0.5/ of 26.5 nmoles/kg b.w. Maximal levels reached were about 7..mu..g Zn in MT/g liver, an induction increase of 8 to 10-fold. An inactive compound, 4..beta..-phorbol, and the vehicle (DMSO) did not stimulate the synthesis of Zn/sub 7/-MT. This induction by TPA requires de novo protein synthesis, as demonstrated by a cycloheximide/(/sup 35/S)-cysteine experiment. TPA stimulated Zn incorporation by 8.6-fold and (/sup 35/S)-cysteine incorporation by 4.8-fold during an 11h induction. These increases were blocked 100% by treatment with cycloheximide at -1 and +5h. These experiments have been repeated in cultured hepatocytes, using (/sup 35/S)-cysteine incorporation, slab SDS-PAGE, and autoradiography to quantitate MT levels.

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

Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase

International Nuclear Information System (INIS)

Buechler, J.A.; Taylor, S.S.

1988-01-01

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [ 14 C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified

Cardiac hyporesponsiveness in severe sepsis is associated with nitric oxide-dependent activation of G protein receptor kinase.

Science.gov (United States)

Dal-Secco, Daniela; DalBó, Silvia; Lautherbach, Natalia E S; Gava, Fábio N; Celes, Mara R N; Benedet, Patricia O; Souza, Adriana H; Akinaga, Juliana; Lima, Vanessa; Silva, Katiussia P; Kiguti, Luiz Ricardo A; Rossi, Marcos A; Kettelhut, Isis C; Pupo, André S; Cunha, Fernando Q; Assreuy, Jamil

2017-07-01

G protein-coupled receptor kinase isoform 2 (GRK2) has a critical role in physiological and pharmacological responses to endogenous and exogenous substances. Sepsis causes an important cardiovascular dysfunction in which nitric oxide (NO) has a relevant role. The present study aimed to assess the putative effect of inducible NO synthase (NOS2)-derived NO on the activity of GRK2 in the context of septic cardiac dysfunction. C57BL/6 mice were submitted to severe septic injury by cecal ligation and puncture (CLP). Heart function was assessed by isolated and perfused heart, echocardiography, and β-adrenergic receptor binding. GRK2 was determined by immunofluorescence and Western blot analysis in the heart and isolated cardiac myocytes. Sepsis increased NOS2 expression in the heart, increased plasma nitrite + nitrate levels, and reduced isoproterenol-induced isolated ventricle contraction, whole heart tension development, and β-adrenergic receptor density. Treatment with 1400W or with GRK2 inhibitor prevented CLP-induced cardiac hyporesponsiveness 12 and 24 h after CLP. Increased labeling of total and phosphorylated GRK2 was detected in hearts after CLP. With treatment of 1400W or in hearts taken from septic NOS2 knockout mice, the activation of GRK2 was reduced. 1400W or GRK2 inhibitor reduced mortality, improved echocardiographic cardiac parameters, and prevented organ damage. Therefore, during sepsis, NOS2-derived NO increases GRK2, which leads to a reduction in β-adrenergic receptor density, contributing to the heart dysfunction. Isolated cardiac myocyte data indicate that NO acts through the soluble guanylyl cyclase/cGMP/PKG pathway. GRK2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction. NEW & NOTEWORTHY The main novelty presented here is to show that septic shock induces cardiac hyporesponsiveness to isoproterenol by a mechanism dependent on nitric oxide and mediated by G protein-coupled receptor kinase isoform 2. Therefore

Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress.

Directory of Open Access Journals (Sweden)

Christine R Collins

2013-05-01

Full Text Available The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV. Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

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.

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

Science.gov (United States)

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.

A-kinase anchoring protein 150 in the mouse brain is concentrated in areas involved in learning and memory

NARCIS (Netherlands)

Ostroveanu, Anghelus; Van der Zee, Eddy A.; Dolga, Amalia M.; Luiten, Paul G. M.; Eisel, Ulrich L. M.; Nijholt, Ingrid M.

2007-01-01

A-kinase anchoring proteins (AKAPs) form large macromolecular signaling complexes that specifically target cAMP-dependent protein kinase (PKA) to unique subcellular compartments and thus, provide high specificity to PKA signaling. For example, the AKAP79/150 family tethers PKA, PKC and PP2B to

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...

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

OpenAIRE

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...

Vanadium induces dopaminergic neurotoxicity via protein kinase Cdelta dependent oxidative signaling mechanisms: Relevance to etiopathogenesis of Parkinson's disease

International Nuclear Information System (INIS)

Afeseh Ngwa, Hilary; Kanthasamy, Arthi; Anantharam, Vellareddy; Song, Chunjuan; Witte, Travis; Houk, Robert; Kanthasamy, Anumantha G.

2009-01-01

Environmental exposure to neurotoxic metals through various sources including exposure to welding fumes has been linked to an increased incidence of Parkinson's disease (PD). Welding fumes contain many different metals including vanadium typically present as particulates containing vanadium pentoxide (V 2 O 5 ). However, possible neurotoxic effects of this metal oxide on dopaminergic neuronal cells are not well studied. In the present study, we characterized vanadium-induced oxidative stress-dependent cellular events in cell culture models of PD. V 2 O 5 was neurotoxic to dopaminergic neuronal cells including primary nigral dopaminergic neurons and the EC 50 was determined to be 37 μM in N27 dopaminergic neuronal cell model. The neurotoxic effect was accompanied by a time-dependent uptake of vanadium and upregulation of metal transporter proteins Tf and DMT1 in N27 cells. Additionally, vanadium resulted in a threefold increase in reactive oxygen species generation, followed by release of mitochondrial cytochrome c into cytoplasm and subsequent activation of caspase-9 (> fourfold) and caspase-3 (> ninefold). Interestingly, vanadium exposure induced proteolytic cleavage of native protein kinase Cdelta (PKCδ, 72-74 kDa) to yield a 41 kDa catalytically active fragment resulting in a persistent increase in PKCδ kinase activity. Co-treatment with pan-caspase inhibitor Z-VAD-FMK significantly blocked vanadium-induced PKCδ proteolytic activation, indicating that caspases mediate PKCδ cleavage. Also, co-treatment with Z-VAD-FMK almost completely inhibited V 2 O 5 -induced DNA fragmentation. Furthermore, PKCδ knockdown using siRNA protected N27 cells from V 2 O 5 -induced apoptotic cell death. Collectively, these results demonstrate that vanadium can exert neurotoxic effects in dopaminergic neuronal cells via caspase-3-dependent PKCδ cleavage, suggesting that metal exposure may promote nigral dopaminergic degeneration.

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...

Radioimmunoassay of inactive creatine kinase B protein in human plasma

Energy Technology Data Exchange (ETDEWEB)

Burnam, M H; Shell, W E [California Univ., Los Angeles (USA). School of Medicine

1981-08-27

The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. /sup 125/I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 ..mu..g equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity.

Radioimmunoassay of inactive creatine kinase B protein in human plasma

International Nuclear Information System (INIS)

Burnam, M.H.; Shell, W.E.

1981-01-01

The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. 125 I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 μg equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity. (Auth.)

Phosphodiesterase 9A regulates central cGMP and modulates responses to cholinergic and monoaminergic perturbation in vivo.

Science.gov (United States)

Kleiman, Robin J; Chapin, Douglas S; Christoffersen, Curt; Freeman, Jody; Fonseca, Kari R; Geoghegan, Kieran F; Grimwood, Sarah; Guanowsky, Victor; Hajós, Mihály; Harms, John F; Helal, Christopher J; Hoffmann, William E; Kocan, Geralyn P; Majchrzak, Mark J; McGinnis, Dina; McLean, Stafford; Menniti, Frank S; Nelson, Fredrick; Roof, Robin; Schmidt, Anne W; Seymour, Patricia A; Stephenson, Diane T; Tingley, Francis David; Vanase-Frawley, Michelle; Verhoest, Patrick R; Schmidt, Christopher J

2012-05-01

Cyclic nucleotides are critical regulators of synaptic plasticity and participate in requisite signaling cascades implicated across multiple neurotransmitter systems. Phosphodiesterase 9A (PDE9A) is a high-affinity, cGMP-specific enzyme widely expressed in the rodent central nervous system. In the current study, we observed neuronal staining with antibodies raised against PDE9A protein in human cortex, cerebellum, and subiculum. We have also developed several potent, selective, and brain-penetrant PDE9A inhibitors and used them to probe the function of PDE9A in vivo. Administration of these compounds to animals led to dose-dependent accumulation of cGMP in brain tissue and cerebrospinal fluid, producing a range of biological effects that implied functional significance for PDE9A-regulated cGMP in dopaminergic, cholinergic, and serotonergic neurotransmission and were consistent with the widespread distribution of PDE9A. In vivo effects of PDE9A inhibition included reversal of the respective disruptions of working memory by ketamine, episodic and spatial memory by scopolamine, and auditory gating by amphetamine, as well as potentiation of risperidone-induced improvements in sensorimotor gating and reversal of the stereotypic scratching response to the hallucinogenic 5-hydroxytryptamine 2A agonist mescaline. The results suggested a role for PDE9A in the regulation of monoaminergic circuitry associated with sensory processing and memory. Thus, PDE9A activity regulates neuronal cGMP signaling downstream of multiple neurotransmitter systems, and inhibition of PDE9A may provide therapeutic benefits in psychiatric and neurodegenerative diseases promoted by the dysfunction of these diverse neurotransmitter systems.

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.

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

Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

Science.gov (United States)

Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

1997-01-01

A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes.

Science.gov (United States)

Li, Yuwei; Ahrens, Molly J; Wu, Amy; Liu, Jennifer; Dudley, Andrew T

2011-01-01

For tissues that develop throughout embryogenesis and into postnatal life, the generation of differentiated cells to promote tissue growth is at odds with the requirement to maintain the stem cell/progenitor cell population to preserve future growth potential. In the growth plate cartilage, this balance is achieved in part by establishing a proliferative phase that amplifies the number of progenitor cells prior to terminal differentiation into hypertrophic chondrocytes. Here, we show that endogenous calcium/calmodulin-dependent protein kinase II (CamkII, also known as Camk2) activity is upregulated prior to hypertrophy and that loss of CamkII function substantially blocks the transition from proliferation to hypertrophy. Wnt signaling and Pthrp-induced phosphatase activity negatively regulate CamkII activity. Release of this repression results in activation of multiple effector pathways, including Runx2- and β-catenin-dependent pathways. We present an integrated model for the regulation of proliferation potential by CamkII activity that has important implications for studies of growth control and adult progenitor/stem cell populations.

PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

Directory of Open Access Journals (Sweden)

Ines Teichert

2014-09-01

Full Text Available Mitogen-activated protein kinase (MAPK pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1. We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

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

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.

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

Science.gov (United States)

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.

EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma.

Science.gov (United States)

Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

2016-01-01

Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase-dependent

The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

Science.gov (United States)

Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

1996-02-01

We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes.

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.

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

Oral protein kinase c β inhibition using ruboxistaurin

DEFF Research Database (Denmark)

Aiello, Lloyd Paul; Vignati, Louis; Sheetz, Matthew J

2011-01-01

To evaluate efficacy, safety, and causes of vision loss among 813 patients (1,392 eyes) with moderately severe to very severe nonproliferative diabetic retinopathy from the Protein Kinase C β Inhibitor-Diabetic Retinopathy Study and Protein Kinase C β Inhibitor-Diabetic Retinopathy Study 2 ruboxi...

Regulation of p73 by Hck through kinase-dependent and independent mechanisms

Directory of Open Access Journals (Sweden)

Radha Vegesna

2007-05-01

Full Text Available Abstract Background p73, a p53 family member is a transcription factor that plays a role in cell cycle, differentiation and apoptosis. p73 is regulated through post translational modifications and protein interactions. c-Abl is the only known tyrosine kinase that phosphorylates and activates p73. Here we have analyzed the role of Src family kinases, which are involved in diverse signaling pathways, in regulating p73. Results Exogenously expressed as well as cellular Hck and p73 interact in vivo. In vitro binding assays show that SH3 domain of Hck interacts with p73. Co-expression of p73 with Hck or c-Src in mammalian cells resulted in tyrosine phosphorylation of p73. Using site directed mutational analysis, we determined that Tyr-28 was the major site of phosphorylation by Hck and c-Src, unlike c-Abl which phosphorylates Tyr-99. In a kinase dependent manner, Hck co-expression resulted in stabilization of p73 protein in the cytoplasm. Activation of Hck in HL-60 cells resulted in tyrosine phosphorylation of endogenous p73. Both exogenous and endogenous Hck localize to the nuclear as well as cytoplasmic compartment, just as does p73. Ectopically expressed Hck repressed the transcriptional activity of p73 as determined by promoter assays and semi-quantitative RT-PCR analysis of the p73 target, Ipaf and MDM2. SH3 domain- dependent function of Hck was required for its effect on p73 activity, which was also reflected in its ability to inhibit p73-mediated apoptosis. We also show that Hck interacts with Yes associated protein (YAP, a transcriptional co-activator of p73, and shRNA mediated knockdown of YAP protein reduces p73 induced Ipaf promoter activation. Conclusion We have identified p73 as a novel substrate and interacting partner of Hck and show that it regulates p73 through mechanisms that are dependent on either catalytic activity or protein interaction domains. Hck-SH3 domain-mediated interactions play an important role in the inhibition of p73

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

Cyclic AMP-dependent protein kinase interferes with GTP γS stimulated IP3 formation in differentiated HL-60 cell membranes

International Nuclear Information System (INIS)

Misaki, Naoyuki; Imaizumi, Taro; Watanabe, Yashuiro

1989-01-01

The effects of addition of activated cyclic AMP-dependent protein kinase (PKA) on the function of islet-activating protein (IAP)-sensitive GTP-binding (G) protein were studied in the plasma membranes of 3 H-inositol-labeled differentiated human leukemic (HL-60) cells. Pretreatment of the membranes with activated PKA in the presence of MgATP for 15 min. at 37 degree C decreased GTP γS-stimulated inositol trisphosphate (IP 3 ) formation by about 30%, but had no influence on Ca 2+ -stimulated IP 3 formation. And autoradiography in the phosphorylation experiments of solubilized HL-60 cell membranes by PKA showed some 32 P incorporated bands, and among them one of the major bands showed the migration at 40 kDa supporting that the G protein coupling with PI response was phosphorylated by PKA. These results showed that pretreatment with activated PKA inhibited the mediating function of the G protein between the fMLP receptor and phospholipase C by its phosphorylation

Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

Science.gov (United States)

Yoshizaki, G.; Shusa, M.; Takeuchi, T.; Patino, R.

2002-01-01

Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage; whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH [17,20??-dihydroxy-4-pregnen-3-one or 17,20??,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.

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...

p56Lck and p59Fyn Regulate CD28 Binding to Phosphatidylinositol 3-Kinase, Growth Factor Receptor-Bound Protein GRB-2, and T Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation

Science.gov (United States)

Raab, Monika; Cai, Yun-Cai; Bunnell, Stephen C.; Heyeck, Stephanie D.; Berg, Leslie J.; Rudd, Christopher E.

1995-09-01

T-cell activation requires cooperative signals generated by the T-cell antigen receptor ξ-chain complex (TCRξ-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, ξ-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Characterization of pathogenic germline mutations in human Protein Kinases

Directory of Open Access Journals (Sweden)

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.

Science.gov (United States)

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

Calcium/Calmodulin-dependent Protein Kinase II is a Ubiquitous Molecule in Human Long-term Memory Synaptic Plasticity: A Systematic Review

Science.gov (United States)

Ataei, Negar; Sabzghabaee, Ali Mohammad; Movahedian, Ahmad

2015-01-01

Background: Long-term memory is based on synaptic plasticity, a series of biochemical mechanisms include changes in structure and proteins of brain's neurons. In this article, we systematically reviewed the studies that indicate calcium/calmodulin kinase II (CaMKII) is a ubiquitous molecule among different enzymes involved in human long-term memory and the main downstream signaling pathway of long-term memory. Methods: All of the observational, case–control and review studies were considered and evaluated by the search engines PubMed, Cochrane Central Register of Controlled Trials and ScienceDirect Scopus between 1990 and February 2015. We did not carry out meta-analysis. Results: At the first search, it was fined 1015 articles which included “synaptic plasticity” OR “neuronal plasticity” OR “synaptic density” AND memory AND “molecular mechanism” AND “calcium/calmodulin-dependent protein kinase II” OR CaMKII as the keywords. A total of 335 articles were duplicates in the databases and eliminated. A total of 680 title articles were evaluated. Finally, 40 articles were selected as reference. Conclusions: The studies have shown the most important intracellular signal of long-term memory is calcium-dependent signals. Calcium linked calmodulin can activate CaMKII. After receiving information for learning and memory, CaMKII is activated by Glutamate, the most important neurotransmitter for memory-related plasticity. Glutamate activates CaMKII and it plays some important roles in synaptic plasticity modification and long-term memory. PMID:26445635

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

Science.gov (United States)

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...

SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair

DEFF Research Database (Denmark)

McCord, Ronald A; Michishita, Eriko; Hong, Tao

2009-01-01

-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor......-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA......, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA...

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

Directory of Open Access Journals (Sweden)

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.

rad-Dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint

NARCIS (Netherlands)

Walworth, N.C.; Bernards, R.A.

1996-01-01

Exposure of eukaryotic cells to agents that generate DNA damage results in transient arrest of progression through the cell cycle. In fission yeast, the DNA damage checkpoint associated with cell cycle arrest before mitosis requires the protein kinase p56chk1. DNA damage induced by ultraviolet

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

Science.gov (United States)

Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

2011-11-25

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

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.

Science.gov (United States)

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.

Characterization of the cGMP-dependent protein kinase SmcGK1 of Schistosoma mansoni

Directory of Open Access Journals (Sweden)

Silke Leutner

2011-06-01

Full Text Available Schistosomes are trematode parasites and of worldwide medical importance for humans and animals. Growth and development of these parasites require a specific host environment, but also permanent communication processes between the two genders. Accumulating molecular evidence indicates that the responsible interactions are mediated by signal transduction processes. Conserved signaling molecules were identified, and first approaches made for their characterization. However, no representative of the conserved family of cGMP-dependent protein kinases (cGKs has been described in this parasite yet. Within the Schistosoma mansoni genome data-set we identified cGK homologs, of which one was investigated in more detail in this study. We present the cloning of SmcGK1, whose sequence shows homology to cGKs of higher eukaryotes. SmcGK1 was found to be gender-independently transcribed in adult schistosomes. The occurrence of SmcGK1 sense and antisense transcripts suggests that the expression of this gene is controlled at the post-transcriptional level. In situ hybridization experiments demonstrated a gonad-preferential expression profile in both genders indicating a role of SmcGK1, at least during sexual development of schistosomes. Using a cGK-specific inhibitor to treat adult schistosomes in vitro finally resulted in a multifaceted phenotype including slow motion, oocyte congestion, and reduced egg production.Esquistossomos são parasitas trematodos de importância médica em todo o mundo para o homem e os animais. O crescimento e o desenvolvimento destes parasitas requerem um ambiente específico do hospedeiro, mas também um processo de comunicação permanente entre parasitas dos dois sexos. Evidência molecular tem se acumulado e indica que as interações são mediadas por processos de transdução de sinal. Moléculas sinalizadoras conservadas foram identificadas, e as primeiras abordagens têm sido feitas para sua caracterização. Contudo, não foi

Protein kinase C signaling and cell cycle regulation

Directory of Open Access Journals (Sweden)

Adrian R Black

2013-01-01

Full Text Available A link between T cell proliferation and the protein kinase C (PKC family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks, cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1→S and/or G2→M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in

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

Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

Directory of Open Access Journals (Sweden)

Mathieu Brochet

2014-03-01

Full Text Available Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²⁺ from intracellular stores to activate stage-specific Ca²⁺-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²⁺ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²⁺ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca²⁺ effectors, PKG emerges as a unifying factor to control multiple cellular Ca²⁺ signals essential for malaria parasite development and transmission.

Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

Directory of Open Access Journals (Sweden)

Lee A Borthwick

Full Text Available Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-dependent protein kinase A (PKA and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2 forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A. Overlay (Far-Western and Surface Plasmon Resonance (SPR analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727. Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability.

Science.gov (United States)

Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D; Silkworth, Whitney; Ross, Nathan T; Scherer, Christina A; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A; Stern, Andrew M; Schreiber, Stuart L; Golub, Todd R

2012-02-21

Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.

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

Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

International Nuclear Information System (INIS)

Stein, J.C.

1985-01-01

Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several 37 P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the 32 P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex

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.

Tau-Induced Ca2+/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation.

Science.gov (United States)

Wei, Yu-Ping; Ye, Jin-Wang; Wang, Xiong; Zhu, Li-Ping; Hu, Qing-Hua; Wang, Qun; Ke, Dan; Tian, Qing; Wang, Jian-Zhi

2018-04-01

Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer's disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca 2+ concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca 2+ concentration with a simultaneous increase in the phosphorylation of Ca 2+ /calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca 2+ /CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca 2+ /calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca 2+ /CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca 2+ concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.

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...

Raf Kinase Inhibitory Protein protects cells against locostatin-mediated inhibition of migration.

Directory of Open Access Journals (Sweden)

Anne N Shemon

2009-06-01

Full Text Available Raf Kinase Inhibitory Protein (RKIP, also PEBP1, a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function.We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/- mouse embryonic fibroblasts (MEFs to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/- MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle.These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.

Raf Kinase Inhibitory Protein protects cells against locostatin-mediated inhibition of migration.

Science.gov (United States)

Shemon, Anne N; Eves, Eva M; Clark, Matthew C; Heil, Gary; Granovsky, Alexey; Zeng, Lingchun; Imamoto, Akira; Koide, Shohei; Rosner, Marsha Rich

2009-06-24

Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function. We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/-)) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/-) MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.

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.

Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

International Nuclear Information System (INIS)

Watkins, D.T.

1991-01-01

The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing [32P]ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules

Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response

KAUST Repository

Almadanim, M. Cecília

2017-01-19

Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analyzing OsCPK17 knockout, silencing, and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose phosphate synthase OsSPS4, and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

Protein kinase C involvement in focal adhesion formation

DEFF Research Database (Denmark)

Woods, A; Couchman, J R

1992-01-01

Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have...... still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form....... Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h...

Parathyroid Hormone Activates Phospholipase C (PLC)-Independent Protein Kinase C Signaling Pathway via Protein Kinase A (PKA)-Dependent Mechanism: A New Defined Signaling Route Would Induce Alternative Consideration to Previous Conceptions.

Science.gov (United States)

Tong, Guojun; Meng, Yue; Hao, Song; Hu, Shaoyu; He, Youhua; Yan, Wenjuan; Yang, Dehong

2017-04-20

BACKGROUND Parathyroid hormone (PTH) is an effective anti-osteoporosis agent, after binding to its receptor PTHR1, several signaling pathways, including cAMP/protein kinase A (PKA) and phospholipase C (PLC)/protein kinase C (PKC), are initiated through G proteins; with the cAMP/PKA pathway as the major pathway. Earlier studies have reported that PTHR1 might also activate PKC via a PLC-independent mechanism, but this pathway remains unclear. MATERIAL AND METHODS In HEK293 cells, cAMP accumulation was measured with ELISA and PKC was measured with fluorescence resonance energy transfer (FRET) analysis using CKAR plasmid. In MC3T3-E1 cells, real-time PCR was performed to examine gene expressions. Then assays for cell apoptosis, cell differentiation, alkaline phosphatase activity, and mineralization were performed. RESULTS The FRET analysis found that PTH(1-34), [G1,R19]PTH(1-34) (GR(1-34), and [G1,R19]PTH(1-28) (GR(1-28) were all activated by PKC. The PKC activation ability of GR(1-28) was blocked by cAMP inhibitor (Rp-cAMP) and rescued with the addition of active PKA-α and PKA-β. The PKC activation ability of GR(1-34) was partially inhibited by Rp-cAMP. In MC3T3-E1 cells, gene expressions of ALP, CITED1, NR4a2, and OSX that was regulated by GR(1-28) were significantly changed by the pan-PKC inhibitor Go6983. After pretreatment with Rp-cAMP, the gene expressions of ALP, CITED1, and OPG were differentially regulated by GR(1-28) or GR(1-34), and the difference was blunted by Go6983. PTH(1-34), GR(1-28), and GR(1-34) significantly decreased early apoptosis and augmented osteoblastic differentiation in accordance with the activities of PKA and PKC. CONCLUSIONS PLC-independent PKC activation induced by PTH could be divided into two potential mechanisms: one was PKA-dependent and associated with PTH(1-28); the other was PKA-independent and associated with PTH(29-34). We also found that PTH could activate PLC-independent PKC via PKA-dependent mechanisms.

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

OpenAIRE

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

2016-01-01

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 T308 before S473 autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present ...

The role of c-AMP-dependent protein kinase in spinal cord and post synaptic dorsal column neurons in a rat model of visceral pain

OpenAIRE

Wu, Jing; Su, Guangxiao; Ma, Long; Zhang, Xuan; Lei, Yongzhong; Lin, Qing; Nauta, Haring J.W.; Li, Junfa; Fang, Li

2007-01-01

Visceral noxious stimulation induces central neuronal plasticity changes and suggests that the c-AMP-dependent protein kinase (PKA) signal transduction cascade contributes to long-term changes in nociceptive processing at the spinal cord level. Our previous studies reported the clinical neurosurgical interruption of post synaptic dorsal column neuron (PSDC) pathway by performing midline myelotomy effectively alleviating the intractable visceral pain in patients with severe pain. However, the ...

HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

Science.gov (United States)

Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

2008-01-01

Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1).

Science.gov (United States)

Tabish, M; Clegg, R A; Rees, H H; Fisher, M J

1999-04-01

The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896-6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523-527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5'-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.

Myosin light chain kinase phosphorylation in tracheal smooth muscle

International Nuclear Information System (INIS)

Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E.

1990-01-01

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32 P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+

Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

Directory of Open Access Journals (Sweden)

Grant S. Nichols

2015-01-01

Full Text Available Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX, the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is “off” in adults.

Phosphorylation of protein kinase C sites Ser42/44 decreases Ca2+-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes

NARCIS (Netherlands)

Wijnker, P.J.M.; Sequeira Oliveira, V.; Witjas-Paalberends, E.R.; Foster, D.B.; dos Remedios, C.G.; Murphy, A.M.; Stienen, G.J.M.; van der Velden, J.

2014-01-01

Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal

Interacting factors and cellular localization of SR protein-specific kinase Dsk1

International Nuclear Information System (INIS)

Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

2012-01-01

Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A) + RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G 2 phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

Interacting factors and cellular localization of SR protein-specific kinase Dsk1

Energy Technology Data Exchange (ETDEWEB)

Tang, Zhaohua, E-mail: ztang@jsd.claremont.edu [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Lin, Ren-Jang [Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010 (United States); Murray, Johanne; Carr, Antony [Genome Damage and Stability Center, University of Sussex, Falmer, BN1 9RQ (United Kingdom)

2012-10-01

Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

MARK/Par1 Kinase Is Activated Downstream of NMDA Receptors through a PKA-Dependent Mechanism.

Directory of Open Access Journals (Sweden)

Laura P Bernard

Full Text Available The Par1 kinases, also known as microtubule affinity-regulating kinases (MARKs, are important for the establishment of cell polarity from worms to mammals. Dysregulation of these kinases has been implicated in autism, Alzheimer's disease and cancer. Despite their important function in health and disease, it has been unclear how the activity of MARK/Par1 is regulated by signals from cell surface receptors. Here we show that MARK/Par1 is activated downstream of NMDA receptors in primary hippocampal neurons. Further, we show that this activation is dependent on protein kinase A (PKA, through the phosphorylation of Ser431 of Par4/LKB1, the major upstream kinase of MARK/Par1. Together, our data reveal a novel mechanism by which MARK/Par1 is activated at the neuronal synapse.

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.

The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

International Nuclear Information System (INIS)

Cross, Janet V; Foss, Frank W; Rady, Joshua M; Macdonald, Timothy L; Templeton, Dennis J

2007-01-01

Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of 'Phase 2' enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. These results demonstrate that MEKK1 is directly modified and inhibited by

Isolation and characterization of cAMP-free and cAMP-bound forms of bovine heart type II cAMP-dependent protein kinase

International Nuclear Information System (INIS)

Cobb, C.E.

1986-01-01

Bovine heart type II cAMP-dependent protein kinase holoenzyme (cAMP-PK) was purified to homogeneity as determined by denaturing SDS-PAGE. An HPLC-DEAE purification step resolved two distinct peaks of cAMP-dependent kinase activity, which were designated Peak 1 and Peak 2 based on their order of elution. They had the same Stoke's radii and had very similar sedimentation coefficients. As determined by densitometric scanning of SDS-PAGE brands, by their mobility on denaturing PAGE, and by the ratios of equilibrium [ 3 H] cAMP binding to maximal kinase activity, the subunit stoichiometry of the two peaks was the same. In a cAMP assay it was found that Peak 1 holoenzyme was cAMP-free, but half of the Peak 2 holoenzyme cAMP binding sites contained cAMP. Dissociation assays indicated that the cAMP was equally distributed in binding Site 1 and Site 2 of Peak 2. Although SDS-PAGE analysis ruled out conversions by proteolysis or autophosphorylation-dephosphorylation, Peak 1 could be partially converted to Peak 2 by the addition of subsaturating amounts of cAMP, and Peak 2 could be partially converted to Peak 1 by aging. The interconvertibility of the two holoenzyme peaks strongly suggested that the difference between the two peaks was caused by the presence of cAMP in Peak 2

A kinase-dependent feedforward loop affects CREBB stability and long term memory formation.

Science.gov (United States)

Lee, Pei-Tseng; Lin, Guang; Lin, Wen-Wen; Diao, Fengqiu; White, Benjamin H; Bellen, Hugo J

2018-02-23

In Drosophila , long-term memory (LTM) requires the cAMP-dependent transcription factor CREBB, expressed in the mushroom bodies (MB) and phosphorylated by PKA. To identify other kinases required for memory formation, we integrated Trojan exons encoding T2A-GAL4 into genes encoding putative kinases and selected for genes expressed in MB. These lines were screened for learning/memory deficits using UAS-RNAi knockdown based on an olfactory aversive conditioning assay. We identified a novel, conserved kinase, Meng-Po ( MP , CG11221 , SBK1 in human), the loss of which severely affects 3 hr memory and 24 hr LTM, but not learning. Remarkably, memory is lost upon removal of the MP protein in adult MB but restored upon its reintroduction. Overexpression of MP in MB significantly increases LTM in wild-type flies showing that MP is a limiting factor for LTM. We show that PKA phosphorylates MP and that both proteins synergize in a feedforward loop to control CREBB levels and LTM. key words: Drosophila, Mushroom bodies, SBK1, deGradFP, T2A-GAL4, MiMIC.

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

Science.gov (United States)

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

Mitogen-activated protein kinases mediate Mycobacterium ...

Indian Academy of Sciences (India)

2012-01-19

Jan 19, 2012 ... CD44, an adhesion molecule, has been reported to be a binding site for ... receptors in mediating mitogen-activated protein kinase activation. ... surface expression and tumour necrosis factor-alpha levels, ... Abbreviations used: Abs, antibodies; ANOVA, analysis of variance; AP-1, activator protein -1; BCG, ...

Calcium binding properties of calcium dependent protein kinase 1 (CaCDPK1) from Cicer arietinum.

Science.gov (United States)

Dixit, Ajay Kumar; Jayabaskaran, Chelliah

2015-05-01

Calcium plays a crucial role as a secondary messenger in all aspects of plant growth, development and survival. Calcium dependent protein kinases (CDPKs) are the major calcium decoders, which couple the changes in calcium level to an appropriate physiological response. The mechanism by which calcium regulates CDPK protein is not well understood. In this study, we investigated the interactions of Ca(2+) ions with the CDPK1 isoform of Cicer arietinum (CaCDPK1) using a combination of biophysical tools. CaCDPK1 has four different EF hands as predicted by protein sequence analysis. The fluorescence emission spectrum of CaCDPK1 showed quenching with a 5 nm red shift upon addition of calcium, indicating conformational changes in the tertiary structure. The plot of changes in intensity against calcium concentrations showed a biphasic curve with binding constants of 1.29 μM and 120 μM indicating two kinds of binding sites. Isothermal calorimetric (ITC) titration with CaCl2 also showed a biphasic curve with two binding constants of 0.027 μM and 1.7 μM. Circular dichroism (CD) spectra showed two prominent peaks at 208 and 222 nm indicating that CaCDPK1 is a α-helical rich protein. Calcium binding further increased the α-helical content of CaCDPK1 from 75 to 81%. Addition of calcium to CaCDPK1 also increased fluorescence of 8-anilinonaphthalene-1-sulfonic acid (ANS) indicating exposure of hydrophobic surfaces. Thus, on the whole this study provides evidence for calcium induced conformational changes, exposure of hydrophobic surfaces and heterogeneity of EF hands in CaCDPK1. Copyright © 2015 Elsevier GmbH. All rights reserved.

Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

Science.gov (United States)

Deegan, Tom D; Yeeles, Joseph Tp; Diffley, John Fx

2016-05-02

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase.

Science.gov (United States)

Habjan, Matthias; Pichlmair, Andreas; Elliott, Richard M; Overby, Anna K; Glatter, Timo; Gstaiger, Matthias; Superti-Furga, Giulio; Unger, Hermann; Weber, Friedemann

2009-05-01

Rift Valley fever virus (RVFV) continues to cause large outbreaks of acute febrile and often fatal illness among humans and domesticated animals in Africa, Saudi Arabia, and Yemen. The high pathogenicity of this bunyavirus is mainly due to the viral protein NSs, which was shown to prevent transcriptional induction of the antivirally active type I interferons (alpha/beta interferon [IFN-alpha/beta]). Viruses lacking the NSs gene induce synthesis of IFNs and are therefore attenuated, whereas the noninducing wild-type RVFV strains can only be inhibited by pretreatment with IFN. We demonstrate here in vitro and in vivo that a substantial part of the antiviral activity of IFN against RVFV is due to a double-stranded RNA-dependent protein kinase (PKR). PKR-mediated virus inhibition, however, was much more pronounced for the strain Clone 13 with NSs deleted than for the NSs-expressing strain ZH548. In vivo, Clone 13 was nonpathogenic for wild-type (wt) mice but could regain pathogenicity if mice lacked the PKR gene. ZH548, in contrast, killed both wt and PKR knockout mice indiscriminately. ZH548 was largely resistant to the antiviral properties of PKR because RVFV NSs triggered the specific degradation of PKR via the proteasome. The NSs proteins of the related but less virulent sandfly fever Sicilian virus and La Crosse virus, in contrast, had no such anti-PKR activity despite being efficient suppressors of IFN induction. Our data suggest that RVFV NSs has gained an additional anti-IFN function that may explain the extraordinary pathogenicity of this virus.

Protein kinase C, focal adhesions and the regulation of cell migration

DEFF Research Database (Denmark)

Fogh, Betina S; Multhaupt, Hinke A B; Couchman, John Robert

2014-01-01

in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles...... and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components...

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

Science.gov (United States)

Lu, Y. T.; Hidaka, H.; Feldman, L. J.

1996-01-01

Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [35S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 microM KN-93, but binding is not affected by 5 microM KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 microM KN-93, but not by 5 microM KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.

Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons

Directory of Open Access Journals (Sweden)

Yuan Jian

2010-06-01

Full Text Available Abstract Background The downstream of tyrosine kinase/docking protein (Dok adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk receptor family, which has three members (TrkA, TrkB and TrkC, are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. Results In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST precipitation assays and coimmunoprecipitation (Co-IP experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3 stimulation. Conclusions We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.

Effects of Biotin Supplementation in the Diet on Adipose Tissue cGMP Concentrations, AMPK Activation, Lipolysis, and Serum-Free Fatty Acid Levels.

Science.gov (United States)

Boone-Villa, Daniel; Aguilera-Méndez, Asdrubal; Miranda-Cervantes, Adriana; Fernandez-Mejia, Cristina

2015-10-01

Several studies have shown that pharmacological concentrations of biotin decrease hyperlipidemia. The molecular mechanisms by which pharmacological concentrations of biotin modify lipid metabolism are largely unknown. Adipose tissue plays a central role in lipid homeostasis. In the present study, we analyzed the effects of biotin supplementation in adipose tissue on signaling pathways and critical proteins that regulate lipid metabolism, as well as on lipolysis. In addition, we assessed serum fatty acid concentrations. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (control: 1.76 mg biotin/kg; supplemented: 97.7 mg biotin/kg diet) over 8 weeks postweaning. Compared with the control group, biotin-supplemented mice showed an increase in the levels of adipose guanosine 3',5'-cyclic monophosphate (cGMP) (control: 30.3±3.27 pmol/g wet tissue; supplemented: 49.5±3.44 pmol/g wet tissue) and of phosphorylated forms of adenosine 5'-monophosphate-activated protein kinase (AMPK; 65.2%±1.06%), acetyl-coenzyme A (CoA), carboxylase-1 (196%±68%), and acetyl-CoA carboxylase-2 (78.1%±18%). Serum fatty acid concentrations were decreased (control: 1.12±0.04 mM; supplemented: 0.91±0.03 mM), and no change in lipolysis was found (control: 0.29±0.05 μmol/mL; supplemented: 0.33±0.08 μmol/mL). In conclusion, 8 weeks of dietary biotin supplementation increased adipose tissue cGMP content and protein expression of the active form of AMPK and of the inactive forms of acetyl-CoA carboxylase-1 and acetyl-CoA carboxylase-2. Serum fatty acid levels fell, and no change in lipolysis was observed. These findings provide insight into the effects of biotin supplementation on adipose tissue and support its use in the treatment of dyslipidemia.

Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

International Nuclear Information System (INIS)

Mukai, Atsushi; Hashimoto, Naohiro

2008-01-01

Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells

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.

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

Science.gov (United States)

Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

2005-01-01

CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

Synthetic peptides and ribosomal proteins as substrate for 60S ribosomal protein kinase from yeast cells

DEFF Research Database (Denmark)

Grankowski, N; Gasior, E; Issinger, O G

1993-01-01

Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential...... phosphorylation sites in natural substrates, i.e., acidic ribosomal proteins. The superiority of certain kinetic parameters for 60S kinase obtained with the native whole 80S ribosomes over those of the isolated fraction of acidic ribosomal proteins indicates that the affinity of 60S kinase to the specific protein...

Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

KAUST Repository

Kim, Dongjin; Ntui, Valentine Otang; Zhang, Nianshu; Xiong, Liming

2015-01-01

Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

KAUST Repository

Kim, Dongjin

2015-10-09

Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

Auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism

Science.gov (United States)

Cai, Weiming; Hu, Liwei; Hu, Xiangyang; Cui, Dayong; Cai, Weiming

Gravitropism is the asymmetric growth or curvature of plant organs in response to gravistimulation. There is a complex signal transduction cascade which involved in the differential growth of plants in response to changes in the gravity vector. The role of auxin in gravitropism has been demonstrated by many experiments, but little is known regarding the molecular details of such effects. In our studies before, mediation of the gravitropic bending of soybean roots and rice leaf sheath bases by nitric oxide, cGMP and gibberellins, are induced by auxin. The asymmetrical distribution of nitric oxide, cGMP and gibberellins resulted from the asymmetrical synthesis of them in bending sites. In soybean roots, inhibitions of NO and cGMP synthesis reduced differential NO and cGMP accumulation respectively, which both of these effects can lead to the reduction of gravitropic bending. Gibberellin-induced OsXET, OsEXPA4 and OsRWC3 were also found involved in the gravitropic bending. These data indicated that auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism. More experiments need to prove the more detailed mechanism of them.

[SP600125-induced polyploidization of megakaryocytic leukemia cell lines by ribosomal protein S6 kinase 1 depends on the degree of cell differentiation].

Science.gov (United States)

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

2016-10-01

Objective To investigate regulatory role of ribosomal protein S6 kinase 1 (S6K1) in the polyploidization of different megakaryocytic leukemia cell lines at the different differentiation stages. Methods Megakaryocytic leukemia cell lines (Dami, Meg-01 and HEL cells) were induced towards polyploidization by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. The SP600125-inducing process was blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor. The phenotype (CD41a, CD42a and CD42b) and DNA ploidy were detected by flow cytometry. The expression and phosphorylation of S6K1 and related proteins were detected by Western blotting. Results SP600125 induced polyploidization and increased the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in Dami, Meg-01 and HEL cells. However, the effect of SP600125 on polyploidization of the three cell lines was different, with the strongest effect on Dami cells and the weakest on Meg-01 cells. Moreover, SP600125 increased the phosphorylation of S6K1 Thr421/Ser424 and decreased the phosphorylation of Thr389 in Dami cells. However, it only increased the phosphorylation of Thr389 in HEL cells and had no effect on the phosphorylation of S6K1 in Meg-01 cells. Interestingly, H-89 only partially blocked the polyploidization of Dami cells, although it decreased the phosphorylation of 4E-BP1 in all SP600125-induced three cell lines. Noticeably, H-89 decreased the phosphorylation of S6K1 Thr421/Ser424 and increased the phosphorylation of Thr389 in Dami cells. However, H-89 had no effect on the phosphorylation of Thr421/Ser424, although it increased the phosphorylation of Thr389 in Meg-01 and HEL cells. Phenotypic analysis showed that the three cell lines were at different levels of differentiation in megakaryocytic lineage, with the highest differentiation in Dami and the lowest in Meg-01 cells. Conclusion SP600125-induced polyploidization of megakaryocytic leukemia cell lines is dependent on the effect

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

Science.gov (United States)

Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

2011-07-14

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Casein kinase 1 regulates sterol regulatory element-binding protein (SREBP) to control sterol homeostasis.

Science.gov (United States)

Brookheart, Rita T; Lee, Chih-Yung S; Espenshade, Peter J

2014-01-31

Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ε, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.

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

Directory of Open Access Journals (Sweden)

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.

Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

Science.gov (United States)

Bhattacharya, Sujoy; Ray, Ramesh M.; Johnson, Leonard R.

2014-01-01

Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF- /CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner. PMID:24242917

Expression of MMPs is dependent on the activity of mitogen-activated protein kinase in chondrosarcoma.

Science.gov (United States)

Yao, Min; Wang, Xiaomei; Zhao, Yufeng; Wang, Xiaomeng; Gao, Feng

2017-02-01

Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) serve an important role in chondrosarcoma. The present study investigated whether the expression of MMPs was dependent on the activity of mitogen-activated protein kinase (MAPK) in chondrosarcoma. Surgical pathological specimens were collected to detect MMP-1, MMP-13, TIMP-1, type II collagen and phosphorylated MAPK levels in normal cartilage, enchondroma and chondrosarcoma tissues. The expression of MMP‑1, MMP‑13, TIMP‑1 and type II collagen was investigated utilizing MAPK inhibitors in chondrosarcoma cells. It was noted that the expression levels of MMP‑1, MMP‑13 and TIMP‑1 were increased in chondrosarcoma with the activity of MAPK. After chondrosarcoma cells were pretreated with MAPK inhibitors, the levels of MMP‑1, MMP‑13 and TIMP‑1 were inhibited. Furthermore, MMP‑1 and MMP‑13 are essential in regulating the degradation of type II collagen and decomposing cartilage matrix major. The high expression levels of MMP‑1 and MMP‑13 in chondrosarcoma expedite the invasion by chondrosarcoma cells and their expression can be depressed by MAPK inhibitors.

Asymmetric expression of protein kinase CK2 subunits in human kidney tumors

DEFF Research Database (Denmark)

Stalter, G; Siemer, S; Becht, E

1994-01-01

of protein kinase CK2 alpha in tumors/normal tissue (T/N) was 1.58 and that of the protein kinase CK2 beta (T/N) was 2.65. The data suggest that the generally described increase in protein kinase CK2 activity in tumor cells may to some extent result from a deregulation in subunit biosynthesis or degradation...

Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

KAUST Repository

Dumbrack, Roland

2016-01-01

Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine

Phosphorylation of the Transient Receptor Potential Ankyrin 1 by Cyclin-dependent Kinase 5 affects Chemo-nociception

OpenAIRE

Hall, Bradford E.; Prochazkova, Michaela; Sapio, Matthew R.; Minetos, Paul; Kurochkina, Natalya; Binukumar, B. K.; Amin, Niranjana D.; Terse, Anita; Joseph, John; Raithel, Stephen J.; Mannes, Andrew J.; Pant, Harish C.; Chung, Man-Kyo; Iadarola, Michael J.; Kulkarni, Ashok B.

2018-01-01

Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and th...

Cellular reprogramming through mitogen-activated protein kinases

Directory of Open Access Journals (Sweden)

Justin eLee

2015-10-01

Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

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 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.

Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

Energy Technology Data Exchange (ETDEWEB)

Jackson, Nicole M.; Ceresa, Brian P., E-mail: brian.ceresa@louisville.edu

2016-08-15

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as a plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.

The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin

DEFF Research Database (Denmark)

Raaf, Jennifer; Klopffleisch, Karsten; Issinger, Olaf-Georg

2008-01-01

The Ser/Thr kinase CK2 (former name: casein kinase 2) is a heterotetrameric enzyme composed of two catalytic chains (CK2alpha) attached to a dimer of noncatalytic subunits. Together with the cyclin-dependent kinases and the mitogen-activated protein kinases, CK2alpha belongs to the CMGC family of...

The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

Directory of Open Access Journals (Sweden)

Macdonald Timothy L

2007-09-01

Full Text Available Abstract Background Dietary isothiocyanates (ITCs are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway. Methods The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases. Results ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected. Conclusion These results

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

Science.gov (United States)