Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S1P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly(82) to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-alpha (TNFalpha), interleukin-1beta, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSK(G82D)). Basal SK activity was unaffected by expression of hSK(G82D). Expression of hSK(G82D) had no effect on TNFalpha-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSK(G82D) acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNFalpha activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation.
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PMID:Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation. A dominant-negative sphingosine kinase. 1094 34

AKT was originally identified as a proto-oncogene with a pleckstrin homology and Ser/Thr protein kinase domains. Recent studies revealed that AKT regulates a variety of cellular functions including cell survival, cell growth, cell differentiation, cell cycle progression, transcription, translation, and cellular metabolism. To clarify the substrate specificity of AKT, we have used an oriented peptide library approach to determine optimal amino acids at positions N-terminal and C-terminal to the site of phosphorylation. The predicted optimal peptide substrate (Arg-Lys-Arg-Xaa-Arg-Thr-Tyr-Ser*-Phe-Gly where Ser* is the phosphorylation site) has similarities to but is distinct from optimal substrates that we previously defined for related basophilic protein kinases such as protein kinase A, Ser/Arg-rich kinases, and protein kinase C family members. The positions most important for high V(max)/K(m) ratio were Arg-3>Arg-5>Arg-7. The substrate specificity of AKT was further investigated by screening a lambdaGEX phage HeLa cell cDNA expression library. All of the substrates identified by this procedure contained Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) motifs and were in close agreement with the motif identified by peptide library screening. The results of this study should help in prediction of likely AKT substrates from primary sequences.
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PMID:Peptide and protein library screening defines optimal substrate motifs for AKT/PKB. 1094 90

Phosphorylation of protein kinase C (PKC) provides an amplitude control that operates in conjunction with allosteric effectors. Under many conditions, PKC isotypes appear to be highly phosphorylated; however, the cellular inputs that maintain these phosphorylations are not characterized. In the present work, it is shown that there is a differential phosphorylation of PKCdelta in adherent versus suspension cultures of transfected HEK-293 cells. It is established that integrin activation is sufficient to trigger PKCdelta phosphorylation and that this signals through phosphoinositide 3-kinase (PI3-kinase) to stimulate the phosphorylation of two sites, T505 and S662. The loss of signal input to PKCdelta in suspension culture is dependent on the tumour suppressor gene PTEN, which encodes a bi-functional phosphotyrosine/phosphoinositide 3-phosphate phosphatase. In the PTEN(-/-) UM-UC-3 bladder carcinoma cell line grown in suspension, transfected PKCdelta no longer accumulates in a dephospho-form on serum removal. By contrast, in a UM-UC-3-derivative cell line stably expressing PTEN, PKCdelta does become dephosphorylated under these conditions. Employing the PTEN Gly(129)-->Glu mutant, which is selectively defective in lipid phosphatase activity, it was established that it is the lipid phosphatase activity that controls PKCdelta phosphorylation. The evidence indicates that PKCdelta phosphorylation and its latent activity are maintained in serum-deprived adherent cultures through integrin-matrix interactions. This control acts through a pathway involving a lipid product of PI3-kinase in a manner that can be suppressed by PTEN.
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PMID:Beta1-integrin and PTEN control the phosphorylation of protein kinase C. 1108 36

The regulatory pathways involved in the rapid response of the AP-1 transcription factor, c-fos, to mechanical load in human primary osteoblast-like (HOB) cells and the human MG-63 bone cell line were investigated using a four-point bending model. HOB and MG-63 cells showed upregulation of c-fos expression on fibronectin and collagen type I substrates; however, MG-63 cells did not respond on laminin YIGSR substrates. Addition of cytochalasin D and Arg-Gly-Asp peptides during loading did not inhibit the response, whereas addition of beta(1)-integrin antibodies inhibited the load response. The role of Ca(2+) signaling has been demonstrated by blocking upregulation with addition of 2 mM EGTA, which chelates extracellular Ca(2+), and gadolinium (10 microM), which inhibits stretch-activated channels. Addition of the Ca(2+) ionophore A-23187 induced upregulation without loading; however, addition of nifedipine (10 microM), the L-type channel blocker, failed to prevent the load response. Inhibitors of downstream pathways indicated the involvement of protein kinase C. Our results demonstrate a key involvement of Ca(2+) signaling pathways and integrin binding in the c-fos response to mechanical strain.
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PMID:Selected contribution: regulatory pathways involved in mechanical induction of c-fos gene expression in bone cells. 1109 Jun 8

Gonadotropin-releasing hormone (GnRH), acting via the GnRH receptor, elicited rapid extracellular acidification responses in mouse gonadotrope-derived alphaT3-1 cells as measured by the Cytosensor microphysiometer, which indirectly monitors cellular metabolic rates. GnRH increased the extracellular acidification rate of the cells in a dose-dependent manner (EC(50) = 1.81 +/- 0.24 nM). The GnRH-stimulated acidification rate could be attenuated by protein kinase C (PKC) down-regulation, extracellular Ca2+ depletion, and the voltage-sensitive Ca2+ channel (VSCC) blocker nifedipine, indicating that the acidification response is activated by both Ca2+ and PKC-mediated pathways. Upon continuous exposure to 100 nM GnRH or periodic stimulation by 10 nM GnRH at 40 min intervals, homologous desensitization was more pronounced in the absence of extracellular Ca2+, suggesting that desensitization of GnRH activity may be mediated via depletion of intracellular Ca2+ stores. We have also compared the potency of eight GnRH analogs on alphaT3-1 cells. No acidification response was detected for GnRH free acid, consistent with the idea that the C-terminal amide is a critical structural determinant for GnRH activity. Replacement of Gly-NH(2) at the C-terminus by N-ethylamide dramatically reduced the EC(50) value, suggesting that substitution of the Gly-NH(2) moiety by N-ethylamide increases the potency of GnRH analogs. Substitution of Gly at position 6 by D-Trp significantly reduced the EC(50) value, whereas D-Lys at the same position slightly increased the EC(50) value, implying that either an aromatic amino acid or a non-basic amino acid at position 6 may be essential for potent GnRH agonists. In summary, our results demonstrate that the Cytosensor microphysiometer can be used to evaluate the actions of GnRH and GnRH analogs in alphaT3-1 cells in a real-time and noninvasive manner. This silicon-based microphysiometric system should provide new information on the structure-function studies of GnRH and is an invaluable tool for the screening of new GnRH agonists and antagonists in the future.
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PMID:Real-time analysis of the activities of GnRH and GnRH analogs in alphaT3-1 cells by the Cytosensor microphysiometer. 1113 59

Integrins mediate cell-extracellular matrix connections and are particularly important during neuronal development. We here investigated the regulatory role of extracellular matrix (ECM) proteins on the synaptic transmission at developing motoneurons. Synaptic currents were recorded from innervated myocytes of 1-day-old Xenopus cultures by whole-cell recordings. Soluble fibronectin and laminin had no significant effect on the frequency of spontaneous synaptic currents (SSCs) by themselves and markedly increased SSC frequency in the presence of low concentration of protein kinase C (PKC) activators. Pretreatment with Gly-Arg-Gly-Asp-Ser peptide inhibited the SSC increasing action of 12-o-tetradecanoyl-phorbol-13-acetate (TPA, 0.5 microM) plus fibronectin, but not that of TPA plus laminin. Genistein but not cytochalasin D inhibited the SSC increasing action of TPA plus fibronectin or laminin. High concentration of TPA (5 microM) markedly increased the SSC frequency by itself and occluded the SSC increasing action of fibronectin. Very low concentration of TPA (0.05 microM) markedly enhanced the SSC frequency when the cells were plated onto fibronectin- or laminin-coated substratum for 1 day. The SSC frequency increased markedly right after a train stimulation, which was defined as post-train potentiation (PTrP), when the cultures were plated onto fibronectin substratum and chronically treated with brain-derived neurotrophic factor (BDNF). The PTrP phenomenon is not observed upon chronic treatment with neurotrophin-3, glial cell line-derived neurotrophic factor, or ciliary neurotrophic factor. Our results suggest that the activation of PKC and tyrosine kinase but not actin reorganization plays a role in the SSC potentiating action of fibronectin. BDNF exerts synergistic effects in increasing synaptic transmission in neurons grown on fibronectin substratum. ECMs in concert with neurotrophic factor may play a role in regulating synaptic function at developing motoneurons.
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PMID:Regulation of acetylcholine release by extracellular matrix proteins at developing motoneurons in Xenopus cell cultures. 1117 Jan 82

The protein kinase C (PKC)gamma isoform is a major pool of the PKC family in the mammalian spinal cord. PKCgamma is distributed strategically in the superficial layers of the dorsal horn and, thus, may serve as an important biochemical substrate in sensory signal processing including pain. Here we report that mu-opioid receptor-mediated analgesia/antinociception and activation of G-proteins in the spinal cord are enhanced in PKCgamma knockout mice. In contrast, delta- and kappa-opioidergic and ORL-1 receptor-mediated activation of G-proteins in PKCgamma knockout mice was not altered significantly relative to the wild-type mice. Deletion of PKCgamma had no significant effect on the mRNA product of spinal mu-opioid receptors but caused an increase of maximal binding of the mu-opioid receptor agonist [3H][d-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin in spinal cord membranes obtained from PKCgamma knockout mice. These findings suggest that deletion of PKCgamma genes protects the functional mu-opioid receptors from degradation by phosphorylation. More importantly the present data provide direct evidence that PKCgamma constitutes an essential pathway through which phosphorylation of mu-opioid receptors occurs.
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PMID:Enhanced mu-opioid responses in the spinal cord of mice lacking protein kinase Cgamma isoform. 1127 52

We investigated the role of protein kinase C (PKC) in cell mu-opioid receptor (MOR) internalization and MOR-mediated acute tolerance in vivo. When Chinese hamster ovary cells expressing MOR were exposed to [D-Ala(2),MePhe(4),Gly-ol(5)]-enkephalin (DAMGO), receptor internalization was observed at 30 min. Incubation with morphine failed to induce receptor internalization. When calphostin C, a PKC inhibitor, was added, receptor internalization was observed as early as 10 min after morphine stimulation. The MOR internalization induced by DAMGO or morphine in the presence of calphostin C was dynamin dependent, because it was abolished 2 d after pretreatment with recombinant adenovirus to express a dominant interfering dynamin mutant (K44A/dynamin adenovirus). On the other hand, in a peripheral nociception test in mice, the nociceptive flexor response after intraplantar injection (i.pl.) of bradykinin was markedly inhibited by DAMGO (i.pl.). DAMGO analgesia was not affected by 2 hr prior injection (i.pl.) of DAMGO. Marked acute tolerance was observed after pretreatment with dynamin antisense oligodeoxynucleotide or K44A/dynamin adenovirus. The DAMGO-induced acute tolerance under such pretreatments was inhibited by calphostin C. Together, these findings suggest that PKC desensitizes MOR or has a role in the development of acute tolerance through MOR by inhibiting internalization mechanisms as a resensitization process.
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PMID:Protein kinase C-mediated inhibition of mu-opioid receptor internalization and its involvement in the development of acute tolerance to peripheral mu-agonist analgesia. 1131 80

Glycine release was facilitated by the activation of presynaptic ATP receptors (P(2X)-type) in a preparation of dissociated trigeminal nucleus pars caudalis neurons in which the native synaptic boutons were preserved. The action of ATP was completely blocked by substance P (SP) without alteration of the miniature IPSC (mIPSC) amplitude distribution. SP itself had no effect on mIPSC frequency or amplitude. The inhibitory effect of SP on ATP action was blocked by CP99994, indicating that the SP receptors are of the neurokinin-1 type. The ATP-induced facilitation of the mIPSC frequency was unaffected by Cd(2+). Moreover, SP did not inhibit the increase in mIPSC frequency induced high K(+) application, suggesting that SP did not modulate voltage-dependent calcium channels or subsequent steps in the release process. KT5720 and phorbol 12-myristate 13-acetate did not block SP action, indicating that neither the cAMP-protein kinase A nor the protein kinase C pathway mediates the SP effects. However, in the presence of N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7), SP was no longer able to inhibit the ATP-induced stimulation of mIPSC frequency. 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine also suppressed the SP action, suggesting that SP modulates P(2X) receptors via a Ca(2+)/calmodulin-dependent protein kinase II-mediated pathway. In conventional whole-cell mode, the presence of W-7 in the patch pipette did not affect the SP inhibitory action. Thus, SP is not likely to be generating its modulation through the production of a retrograde signal (involving calmodulin) from the postsynaptic cell to the presynaptic boutons. These results are the first demonstration of the modulation of one presynaptic receptor by another. Because SP inhibits the ATP stimulation of glycine release, SP may play a significant role in hyperalgesia or chronic pain.
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PMID:Substance P abolishes the facilitatory effect of ATP on spontaneous glycine release in neurons of the trigeminal nucleus pars caudalis. 1131 82

The purpose of this work was to investigate the effect of Arg-Gly-Asp-Ser (RGDS) peptide on platelet aggregation, protein phosphorylation, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activity during platelet activation. Experiments were performed on ADP activated rat platelets in vivo. Results showed that 50 mumol/L ADP, in addition to inducing platelet aggregation, obviously enhanced not only PKC and MAPK activities but also 95 and 66 kD protein phosphorylation. When platelets and ADP were incubated together with 50, 100, 200 mumol/L RGDS peptide it was found that the latter markedly inhibited ADP activated platelet aggregation and activation of PKC and MAPK, both in a concentration-dependently manner. RGDS peptide also inhibited 95 and 66 kD protein phosphorylation concentration-dependently and went positively with its activation of PKC and MAPK. The above result suggested that the antithrombotic effect of RGDS peptide was probably mediated through its effect on intracellular signal transduction in the ADP activation of platelets.
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PMID:[Effect of Arg-Gly-Asp-Ser (RGDS) peptide on ADP-induced signal transduction of activated rat platelet]. 1132 34


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