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Query: UNIPROT:P06889 (Mol)
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We have studied the effects of vasopressin and tetradecanoyl phorbol acetate (TPA) on cytosolic free Ca2+ ([Ca2+]i) and insulin release in HIT-T15 beta-cells. Saturable binding of [3H] [Arg8]-vasopressin to HIT cell microsomes indicated a single class of receptors with a dissociation constant (Kd) of 2.5 nM and a total number of binding sites (Bmax) equal to 120 fmol/mg protein. [Arg8]-vasopressin (0.1-100 nM) elicited dose-dependent insulin release from HIT cells by up to 25-fold. This increase was dependent on the presence of extracellular glucose and was blocked by omission of extracellular Ca2+ or addition of verapamil. The stimulation was biphasic; a rapid but short-lived large increase in release was followed by a smaller sustained rise. Vasopressin also evoked a marked, concentration-dependent increase in [Ca2+]i which was also biphasic; an initial spike was followed by a sustained elevation. This increase also required glucose and was blocked by the absence of extracellular Ca2+ or the addition of verapamil. Pretreatment of the cells with TPA overnight to deplete protein kinase C activity did not affect the [Ca2+]i or insulin responses to vasopressin. However, short-term exposure to TPA markedly reduced glucose-induced steady-state [Ca2+]i, despite potentiating glucose-stimulated insulin release sevenfold, and blocked the [Ca2+]i increase induced by vasopressin. These inhibitory effects of TPA were absent in protein kinase C-depleted cells and were prevented by staurosporine. TPA had no significant effect on vasopressin-induced insulin release. Vasopressin did not modify the activity of ATP-sensitive K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Endocrinol 1992 Apr
PMID:Stimulation of insulin release by vasopressin in the clonal beta-cell line, HIT-T15: the role of protein kinase C. 151 19

There is growing evidence that arachidonic acid (AA) and/or its metabolites may be involved in the control of insulin secretion. We have now investigated the effect of AA on insulin secretion from rat islets, and the possible involvement of protein kinase C (PKC) in this process. Exogenous AA stimulated insulin secretion from intact islets at a substimulatory concentration of glucose (2 mM), but did not further enhance glucose-induced (20mM) insulin secretion. AA-induced insulin secretion was temperature dependent. The secretory responses seen at 37 degrees C were totally abolished by reducing the incubation temperature to less than or equal to 34 degrees C. AA-induced insulin secretion was not dependent upon extracellular Ca2+ and was potentiated by omission of Ca2+ or bovine serum albumin from the media. PKC in rat islets can thus be stimulated by AA, but the stimulation of PKC is not required for AA-induced insulin secretion.
J Mol Endocrinol 1992 Apr
PMID:Arachidonic acid-induced insulin secretion from rat islets of Langerhans. 151 22

The compound 12-O-tetradecanoylphorbol-13-acetate (TPA) is extremely toxic to the P13 subclone of the Jurkat human T-cell leukemia line. By selecting for growth in the presence of TPA, we have isolated two TPA-resistant variants of these cells, P13-50 and P13-5/A8. Studies of protein kinase C (PKC) enzyme activity, immunoblot analyses, and assays for PKC mRNAs indicate that both of these variants express lower levels of PKC than do the parental P13 cells. We suggest that this protects them from the toxic effects of TPA. The P13-5/A8 cells are of particular interest because not only are they resistant to TPA toxicity but they actually require TPA for optimal growth. These cells have a more profound decrease in PKC expression that do P13-50 cells. In addition, P13-5/A8 cells display very little, if any, surface expression of CD45, a receptor-linked tyrosine protein phosphatase, and lck, a lymphocyte-specific tyrosine kinase. On the other hand, they express a very high level of interleukin-2 receptor. A model is proposed that suggests that these cells are dependent on TPA because they have defects in both the PKC and tyrosine kinase signal transduction pathways, and that TPA compensates for these defects by providing a strong stimulus to the residual level of PKC. This variant may be useful for studying the interactions between tyrosine kinase and PKC pathways in controlling the various functions of T lymphocytes.
Mol Cell Biol 1992 Jan
PMID:Altered expression of protein kinase C, lck, and CD45 in a 12-O-tetradecanoylphorbol-13-acetate-dependent leukemic T-cell variant that expresses a high level of interleukin-2 receptor. 153 Aug 79

The E1A gene of adenovirus type 5 (Ad5) induces morphological transformation and anchorage-independent growth in cloned rat embryo fibroblast (CREF) cells. In contrast, CREF cells transfected with a beta 1 protein kinase C (PKC) gene and expressing low-levels of beta 1 PKC display a CREF-like morphology and do not form colonies when grown in agar. The combination of Ad5 E1A and low-level beta 1 PKC expression in the same CREF cell line results in an enhanced ability to grow when suspended in agar. In Ad5 E1A and Ad5 E1A + low-level beta 1 PKC expressing CREF clones, the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further enhances anchorage-independence. In contrast, TPA does not induce CREF cells or transfected CREF cells expressing low-levels of beta 1 PKC to grow in agar. Low-level beta 1 PKC expression in transfected CREF cells is associated with a modest 1.2 to 1.6-fold increase in binding of [3H]-phorbol-12,13-dibutyrate (PDBu) and only a 2.3-fold increase in PKC enzymatic activity. In contrast, specific beta 1 PKC-retroviral vector transformed CREF clones (CREF-RV-PKC) display higher levels of PKC mRNA, PDBu binding and PKC enzymatic activity. A majority of CREF-RV-PKC clones exhibit a transformed morphology and grow more rapidly in monolayer culture, form macroscopic colonies in agar in the absence of TPA and in many independent clones TPA further enhances anchorage-independent growth. This effect is not directly related to the level of enhanced [3H]-PDBu binding. The present study indicates that the effect of beta 1 PKC on cellular phenotype in immortal rat embryo cells is complex and is affected by its mode of insertion into CREF cells, i.e. transfection versus retroviral insertion. In addition, the combination of a transfected Ad5 E1A and a beta 1 PKC gene in the same CREF clone results in an enhanced expression of the transformed phenotype in both the absence and presence of TPA.
Cell Mol Biol 1992 Feb
PMID:Alterations in cellular phenotype induced by the type 5 adenovirus E1A and the beta 1 protein kinase C genes in cloned rat embryo fibroblast cells. 153 67

Rat 6 fibroblasts that overproduce protein kinase C beta 1 (R6-PKC3 cells) are hypersensitive to complete transformation by the T24 H-ras oncogene; yet T24 H-ras-transformed R6-PKC3 cells are killed when exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) (W.-L. W. Hsiao, G. M. Housey, M. D. Johnson, and I. B. Weinstein, Mol. Cell. Biol. 9:2641-2647, 1989). Treatment of an R6-PKC3 subclone that harbors a T24 H-ras gene under the control of an inducible mouse metallothionein I promoter with ZnSO4 and TPA is extremely cytocidal. This procedure was used to isolate rare revertants that are resistant to this toxicity. Two revertant lines, R-1a and ER-1-2, continue to express very high levels of protein kinase C enzyme activity but, unlike the parental cells, do not grow in soft agar. Furthermore, these revertants are resistant to the induction of anchorage-independent growth by the v-src, v-H-ras, v-raf, and, in the case of the R-1a line, v-fos oncogenes. Both revertant lines, however, retain the ability to undergo morphological alterations when either treated with TPA or infected with a v-H-ras virus, thus dissociating anchorage independence from morphological transformation. The revertant phenotype of both R-1a and ER-1-2 cells is dominant over the transformed phenotype in somatic cell hybridizations. Interestingly, the revertant lines no longer induce the metallothionein I-T24 H-ras construct or the endogenous metallothionein I and II genes in response to three distinct agents: ZnSO4, TPA, and dexamethasone. The reduction in activity of metallothionein promoters seen in these revertants may reflect defects in signal transduction pathways that control the expression of genes mediating specific effects of protein kinase C and certain oncogenes in cell transformation.
Mol Cell Biol 1992 Jul
PMID:Novel revertants of H-ras oncogene-transformed R6-PKC3 cells. 153 85

Staurosporine is an antibiotic that specifically inhibits protein kinase C. Fourteen staurosporine- and temperature-sensitive (stt) mutants of Saccharomyces cerevisiae were isolated and characterized. These mutants were divided into ten complementation groups, and characterized for their cross-sensitivity to K-252a, neomycin, or CaCl2. The STT1 gene was cloned and sequenced. The nucleotide sequence of the STT1 gene revealed that STT1 is the same gene as PKC1. The STT1 gene conferred resistance to staurosporine on wild-type cells, when present on a high copy number plasmid. STT1/stt1::HIS3 diploid cells were more sensitive to staurosporine than STT1/STT1 diploid cells. Analysis of temperature-sensitive stt1 mutants showed that the STT1 gene product functioned in S or G2/M phase. These results suggest that a protein kinase (the STT1 gene product) is one of the essential targets of staurosporine in yeast cells.
Mol Gen Genet 1992 Feb
PMID:Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1. 153 90

The action of many agonists results in rapid production of sn-1,2-diacylglycerol (DAG). Using platelets as a model system, we previously identified a delayed phase of DAG accumulation that is temporally associated with secondary aggregation and secretion. In the present study, we examined the quantitative relationship between this delayed DAG accumulation and platelet aggregation and secretion. To quantitate the low levels of DAG in platelets, we used the sensitive DAG kinase assay and simultaneously compared DAG levels with aggregation and ATP secretion. In platelets stimulated by gamma-thrombin or collagen, there was a dose response between concentration of agonist and DAG accumulation. Significantly, a dose response was observed between DAG accumulation and extent of aggregation and secretion in platelets stimulated by either agonist. A concentration of either gamma-thrombin or collagen that caused secondary aggregation and secretion was associated with DAG accumulation above 0.2 pmol of DAG/nmol of phospholipid. Subthreshold concentrations of gamma-thrombin or collagen resulted in DAG levels less than 0.2 pmol/nmol of phospholipid. Thus, these data suggest that a to occur. Moreover, secretion was blocked when DAG production was blocked with aspirin or when protein kinase C was inhibited with sphingosine. We conclude that endogenously formed DAG plays a critical role in regulating secondary aggregation and secretion and, therefore, represents an important target for future antiplatelet agents.
Mol Pharmacol 1992 Feb
PMID:Quantitative analysis of diacylglycerol second messengers in human platelets: correlation with aggregation and secretion. 153 14

The present study specifically addresses the role of protein kinase C (PKC) activation in human endothelial cell Ca2+ mobilization, a response that is functionally coupled to the production of the potent arachidonate (AA) metabolite, prostacyclin (PGI2). Phorbol 12-myristate 13-acetate (PMA), alpha-thrombin, and sodium fluoride (NaF), a direct G-protein activator, produced a rapid and time-dependent translocation of PKC from the cytosol to the membrane. Activation of PKC by brief pretreatment of human umbilical vein endothelial cell (HUVEC) monolayers with PMA resulted in the inhibition of NaF-induced inositol phosphate increases and attenuation of both alpha-thrombin- and NaF-activated increases in intracellular Ca2+ (Ca2+i). Ca2+ mobilization induced by ionophore A23187 was not affected by PKC preactivation, suggesting PKC-dependent negative feedback inhibition of phosphatidylinositol (PI)-specific phospholipase C (PLC). Agonist-stimulated AA release and PGI2 synthesis in PMA-pretreated cultured human endothelial cells, however, was potentiated, and the enhanced PGI2 synthesis produced by A23187, NaF, and alpha-thrombin was dependent upon the dose of PMA. Treatment of HUVEC monolayers with an intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid-acetoxymethylester (BAPTA-AM), dramatically reduced alpha-thrombin-, NaF-, and A23187-induced PGI2 synthesis, demonstrating the importance of Ca2+i availability in PGI2 synthesis. BAPTA pretreatment did not inhibit PMA-induced PKC activation, and BAPTA-mediated inhibition of agonist-stimulated PGI2 synthesis was partially attenuated by prior PMA pretreatment. Staurosporine, a potent PKC inhibitor, at concentrations that inhibited PKC-induced phosphorylation of histone-1, augmented both alpha-thrombin- and NaF-induced production of inositol phosphates but markedly inhibited alpha-thrombin-, NaF-, and A23187-induced PGI2 synthesis. The downregulation of PKC activity by prolonged PMA treatment (18 h) produced similar inhibition of PGI2 synthesis by these agonists (approximately 50% inhibition). These studies indicate that the integrated phospholipase A2 and PLC activities are under complex regulation by factors that include both PKC activation and [Ca2+i]. PKC exerts dual effects on prostaglandin synthesis via negative regulation of Gp-coupled PI-specific PLC and positive feedback regulation of AA release and PGI2 synthesis. PKC is thus a critical determinant in the regulation of human endothelial cell prostaglandin synthesis by both receptor-mediated and G-protein-dependent cellular activation.
Am J Respir Cell Mol Biol 1992 Mar
PMID:Role of protein kinase C in the regulation of prostaglandin synthesis in human endothelium. 154 Mar 95

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Consistent with this, overexpression of eIF-4E in fibroblasts results in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylation on a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor (NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympathetic neurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment, resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermal growth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhance eIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarily a protein kinase C-independent response. The NGF-induced enhancement of eIF-4E phosphorylation is abrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating that eIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effects translation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation may play a role in switching the pattern of gene expression during the differentiation of PC12 cells.
Mol Cell Biol 1992 Mar
PMID:Phosphorylation of translation initiation factor eIF-4E is induced in a ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation. 154 5

The tumor promoters phorbol esters are thought to induce changes in cell growth and gene expression by direct activation of protein kinase C (PKC). However, the molecular mechanisms by which PKC molecules transduce signals into the cell nucleus are unknown. In this study, we provide evidence for a direct target for phorbol esters in the nucleus. We demonstrate that the new PKC-related family member, PKC-L, recently isolated by us, is expressed specifically in the cell nucleus. Localization of PKC-L in the cell nucleus is shown both by immunofluorescence staining and by subcellular fractionation experiments of several human cell lines, including the human epidermoid carcinoma line A431. Treatment of these cells by phorbol esters does not induce the down-regulation of PKC-L, in contrast to their effect on classical PKC family members. This is the only PKC isoenzyme described so far that resides permanently and specifically in the cell nucleus. PKC-L may function as an important link in tumor promoting, e.g., as a nuclear regulator of gene expression that changes the phosphorylation state of transcriptional components such as the AP-1 complex.
Mol Cell Biol 1992 Mar
PMID:The protein kinase C-related PKC-L(eta) gene product is localized in the cell nucleus. 154 11


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