Effects of inhibitors and activators of protein kinase C on late erythroid progenitor (CFU-e) colony formation in vitro

Protein kinase C (PKC) plays a central role in external signal transduction for many cell types. To examine the involvement of PKC in the control of erythropoiesis, we tested the effects of PKC inhibitors on in vitro colony formation by late erythroid progenitors (CFU-e) from normal and Friend virus-infected mice. Inhibitors of PKC and other kinases (H-7 and H-8) inhibited CFU-e at concentrations which inhibit PKC. HA1004, an inhibitor of the cyclic nucleotide-dependent kinases and a weak inhibitor of PKC, had little effect on CFU-e. In the absence of erythropoietin, a combination of phorbol ester and Ca++ ionophore significantly increased normal CFU-e. These results suggest PKC plays a role in the transduction of regulatory signals for the growth of CFU-e.     

Phosphorylation of a 15- to 17-kDa protein correlated with lytic function in cytotoxic T lymphocytes

CTL are activated to lyse their targets through the interaction of the CTL-R and the appropriate Ag on the surface of the target cell. Experiments with tumor-promoting phorbol esters have suggested that the activation and translocation of protein kinase C (PKC) to the CTL membrane may be important in the activation process. We have studied the functional role of PKC in lytic signal transduction by correlating the phosphorylation of a set of CTL membrane proteins bound by the lectin Con A with lytic function in CTL clones. The data obtained indicate that the phosphorylation of a 15- to 17-kDa polypeptide in this subset is associated with the translocation of PKC to the membrane and the stimulation of lytic function. This suggests that the 15- to 17-kDa protein may be a physiologically relevant substrate for PKC translocated to the membrane as a result of Ag-specific perturbation of the CTL-R.     

Constitutive phosphorylation by protein kinase C regulates D1 dopamine receptor signaling

The D(1) dopamine receptor (D(1) DAR) is robustly phosphorylated by multiple protein kinases, yet the phosphorylation sites and functional consequences of these modifications are not fully understood. Here, we report that the D(1) DAR is phosphorylated by protein kinase C (PKC) in the absence of agonist stimulation. Phosphorylation of the D(1) DAR by PKC is constitutive in nature, can be induced by phorbol ester treatment or through activation of Gq-mediated signal transduction pathways, and is abolished by PKC inhibitors. We demonstrate that most, but not all, isoforms of PKC are capable of phosphorylating the receptor. To directly assess the functional role of PKC phosphorylation of the D(1) DAR, a site-directed mutagenesis approach was used to identify the PKC sites within the receptor. Five serine residues were found to mediate the PKC phosphorylation. Replacement of these residues had no effect on D(1) DAR expression or agonist-induced desensitization; however, G protein coupling and cAMP accumulation were significantly enhanced in PKC-null D(1) DAR. Thus, constitutive or heterologous PKC phosphorylation of the D(1) DAR dampens dopamine activation of the receptor, most likely occurring in a context-specific manner, mediated by the repertoire of PKC isozymes within the cell.     

Role of protein kinase C in induction of gene expression and inhibition of cell proliferation by interferon alpha.

Recent studies have suggested that protein kinase C (PKC) may be involved in the mechanism of signal transduction by which members of the interferon (IFN) family regulate gene expression and cell phenotype. We have investigated the role of PKC in the control of cell growth and gene expression by IFN alpha in Daudi cells. Treatment of these cells with two analogues of staurosporine, which are potent inhibitors of PKC, completely blocked the induction by IFN alpha of the mRNA for 2',5'-oligoadenylate synthetase and the 6-16 gene. These compounds also inhibited cell proliferation and thymidine incorporation in this system. In contrast, the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) did not significantly inhibit the induction of these genes by IFN alpha and had no effect on Daudi cell growth or thymidine incorporation in the presence or absence of IFN alpha. No effect of IFN alpha on total PKC activity could be observed, and there were no significant changes in the overall levels of individual PKC isoforms or their mRNA following IFN alpha treatment. In contrast, treatment of Daudi cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, which also inhibits cell proliferation, strongly down-regulated PKC. These data suggest that the activity of a PKC species, or a closely related enzyme, may be required both for continued cell proliferation and the response to IFN alpha in Daudi cells, but that IFN-induced growth inhibition does not involve overall down-regulation or change in activity of PKC.     

Oncogenic and activated wild-type ras-p21 proteins induce different isoforms of protein kinase C in mitogenic signal transduction

We have previously found that the protein kinase C (PKC) inhibitor, CGP 41 251, blocks oncogenic ras-p21 protein- and beta-PKC-induced oocyte maturation, but only weakly inhibits insulin-induced oocyte maturation (which requires activation of wild-type endogenous ras-p21). Because the dose-response curves for inhibition of oncogenic p21- and beta-PKC-induced oocyte maturation by CGP 41 251 superimpose and because the ras-p21-inactivating antibody, Y13-259, does not inhibit beta-PKC-induced oocyte maturation, we concluded that the oncogenic, but not wild-type, protein requires beta-PKC as a downstream target. Because multiple isoforms of PKC exist and several of these, such as epsilon-PKC, have been found to be important on ras signal transduction pathways, we have investigated which PKC isoforms are critical to each ras protein. For this purpose, we used PKC-isoform-specific inhibitors, which have been shown to inhibit selectively the function and translocation of PKC isoforms in vitro and in vivo. Specifically, the peptides KLFIMN, QEVIRN, and EAVSLKPT each inhibit beta-1, beta-2, and epsilon-PKC, respectively, but do not cross-inhibit other PKC isoforms. We find that the epsilon-PKC inhibitory peptide strongly blocks insulin- but not oncogenic ras-p21-induced oocyte maturation whereas the beta-2 inhibitory peptide more strongly inhibits oncogenic ras-p21-induced oocyte maturation, corroborating our previous studies. The beta-1 inhibitory peptide has little effect on either protein. We conclude that selective inhibition of individual PKC isoforms permits the distinction between signal transduction initiated by oncogenic and activated wild-type p21 proteins and implicate different specific PKC isoforms in mitogenic signal transduction by each of these proteins. The ability to dissect the role of individual PKC isozymes in this regulation is of therapeutic significance.     

CD3 receptor modulation in Jurkat leukemic cell line

CD3 antigen is a crucial molecule in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3(low) (217.6), CD3+(217.9) or CD3(low) (217.7). The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor--chelerythrine or calcineurin blocker--cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones.     

胰岛素影响自发性高血压大鼠血管平滑肌细胞增殖及肌动蛋白分布的丝裂原激酶的机制探讨

血管平滑肌细胞增殖的同时伴有细胞内肌动蛋白的改变,这种改变受PKC-MAPK信号转导途径调控,但目前机制尚不清楚。为探讨胰岛素对PKC-MAPK信号转导途径参与调控血管平滑肌细胞增殖及细胞内肌动蛋白分布的影响,本研究用PKC抑制剂预处理SHR在鼠体外培养的血管平滑肌细胞,观察预处理的血管平滑肌细胞经胰岛素刺激后细胞内DNA的合成、MAPK的活性、表达及细胞内肌动蛋白的分布。发现,胰岛素刺激后可使血管平滑肌细胞增殖,同时伴有[^3H]TdR掺入增加、MAPK活性及表达与对照组比较明显升高。这些作用可被PKC抑制剂阻断。胰岛素在刺激血管平滑肌细胞增殖的同时也使细胞内肌动蛋白重新分布,这一效应也可被PKC抑制剂阻断。 上述结果提示,胰岛素使血管平滑肌细胞增殖的效应可能与MAPK信号转导途径有关。     

Genome-Wide Screening of Aluminum Tolerance in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genome-wide screening has identified 37 Al-tolerance genes in Saccharomyces cerevisiae. These genes can be roughly categorised into three groups on the basis of function, i.e., genes related to vesicle transport processes, signal transduction pathways, and protein mannosylation. The largest group is composed of genes related to vesicle transport processes; severe Al sensitivity was found in yeast strains lacking these genes. The retrograde transport of endosome-derived vesicles back to the Golgi apparatus is an important factor in determining the Al tolerance of the vesicle transport system. The PKC1-MAPK cascade signalling pathway is important in the Al tolerance of signal transduction. The lack of the gene implicated in this process leads to weakened cell wall architecture, rendering the yeast Al-sensitive. Alternatively, Al might attack the cell wall and/or plasma membrane, and, as signalling is prevented in cells devoid of the genes related to signalling processes, the cells may be unable to alleviate the damage. The genes for protein mannosylation are also associated with Al tolerance, demonstrating the importance of cell wall architecture. These genes are involved in cell integrity processes. An erratum to this article is available at .     

MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.     

The regulation of protein kinase C activity in normal, immortalized and transformed rat fibroblasts]

In view of recent studies showing that cell proliferation of E1Aad5+c-Ha-ras-transformed fibroblasts cannot be regulated by growth factors and phorbol eaters in contrast to normal and E1Aad5-immortalized cell lines, the present work was undertaken to examine the role of protein kinase C (PKC) in the mitogenic signal transduction machinery in rat embryonal fibroblasts. It is shown that PKC is activated by acidic growth factor and phorbol esters in all the three cell lines. These findings suggest the existence of an additional, not associated with PKC-, growth-signaling pathway in E1Aad5-Ha-ras-transformed rat embryonal fibroblasts.     

Activity of novel protein kinase C and distribution of protein kinase C theta in subcellular fractions of normal and Duchenne muscular dystrophic muscle

Phospholipid-dependent, Ca(2+)-independent isoenzymes termed novel protein kinase C or nPKC, include PKC delta, epsilon, eta, theta and mu. Status and role of nPKC and PKC theta in Duchenne muscular dystrophic (DMD) condition is unknown. In the present study, we have shown that most of the nPKC isoforms are translocated to the membrane fraction of DMD tissue specimen. It is well established that translocation plays a key role in signal transduction by individual PKC isoforms. In our experiment, the increased association of nPKC isoform PKC theta to membrane was further confirmed by Western blot. Increased expression of PKC theta mRNA was identified by dot blot analysis. The above results suggest that, the alterations in nPKC location and increased expression of PKC theta observed is a result of modification of PKC-mediated signal transduction and cell function.     

Okadaic acid,sphingosine, and phorbol ester reversibly modulate heat induction on protein kinase Fa/GSK-3α in A431 cells

Exposure of A431 cells to a rapid and sudden increase from 37°C to 46°C for 30 min could induce an increase in protein level and cellular activity of protein (kinase Fa /GSK-3α) up to ∼200% of control level. However, when cells were first treated with 500 nM tumor promoter phorbol ester TPA at 37°C for 30 min to activate cellular protein kinase C (PKC) or with 400 nM okadaic acid at 37°C for 30 min to inhibit cellular protein phosphatases followed by heat shock at 46°C for another 30 min, the heat induction on kinase Fa /GSK-3α was found to be completed blocked. In sharp contrast, when cells were first treated with 1 μM TPA at 37°C for 24 h or with 5 μM sphingosine at 37°C for 30 min to down-regulate cellular PKC, the heat induction on kinase Fa /GSK-3α was found to be reversely promoted up to ∼ 250% of control level, demonstrating that kinase Fa /GSK-3α may not represent a constitutively active/mitogen-inactivated protein kinase as previously conceived. Taken together, the results provide initial evidence that TPA/sphingosine and okadaic acid could reversibly modulate the heat induction on kinase Fa /GSK-3α in A431 cells, suggesting that phosphorylation/dephosphorylation mechanisms are involved in the regulation of the heat-shock induction of kinase Fa /GSK-3α, representing a new mode of signal transduction for the regulation of this multisubstrate protein kinase and a new mode of signaling pathway modulating the heat-induction process. © 1996 Wiley-Liss, Inc.     

Ca^2＋与细胞凋亡

Ｃａ＾２＋在某些因素诱导的细胞凋亡中起着重要信使作用。细胞内Ｃａ＾２＋浓度上升可来源于胞外Ｃａ＾２＋内汉、内库钙动员或者二者兼之。     

Evidence for a role of protein kinase C in FGF signal transduction in the developing chick limb bud

In developing limbs, numerous signaling molecules have been identified but less is known about the mechanisms by which such signals direct patterning. We have explored signal transduction pathways in the chicken limb bud. A cDNA encoding RACK1, a protein that binds and stabilizes activated protein kinase C (PKC), was isolated in a screen for genes induced by retinoic acid (RA) in the chick wing bud. Fibroblast growth factor (FGF) also induced RACK1 and such induction of RACK1 expression was accompanied by a significant augmentation in the number of active PKC molecules and an elevation of PKC enzymatic activity. This suggests that PKCs mediate signal transduction in the limb bud. Application of chelerythrine, a potent PKC inhibitor, to the presumptive wing region resulted in buds that did not express sonic hedgehog (Shh) and developed into wings that were severely truncated. This observation suggests that the expression of Shh depends on PKCs. Providing ectopic SHH protein, RA or ZPA grafts overcome the effects of blocking PKC with chelerythrine and resulted in a rescue of the wing morphology. Taken together, these findings suggest that the responsiveness of Shh to FGF is mediated, at least in part, by PKCs.     

Protein tyrosine phosphorylation and p56lck modification in IL-2 or phorbol ester-activated human natural killer cells.

Protein tyrosine kinases play fundamental roles in the transduction of signals that regulate cell growth, differentiation, and functional responses to a diversity of external stimuli. It is therefore likely that understanding protein tyrosine kinase activity in NK cells will be crucial in further defining the intracellular regulation of their unique and specialized functions. We investigated the role of protein tyrosine phosphorylation in receptor-mediated signal transduction using stimuli known to play major roles in regulating NK cell activation. Immunoblot analyses with antiphosphotyrosine antibodies demonstrated that IL-2, a potent stimulus for NK cell proliferation and an agent that enhances NK cytotoxic function, induced the tyrosine phosphorylation of at least eight proteins in clonal CD16+/CD3-human NK cells. In contrast, IL-4, which modulates NK cell function without inducing proliferation, had no apparent effect on protein tyrosine phosphorylation. Because protein kinase C (PKC) activation plays a prominent, yet distinct role in NK cell-mediated cytolytic reactions, we next investigated whether PKC activation affects NK cell protein tyrosine phosphorylation. Surprisingly, PKC-activating agents, including the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and 4 beta-phorbol 12, 13-didecanoate, as well as the synthetic diacylglycerol,1-oleoyl-2-acetylglycerol, also induced the tyrosine phosphorylation of a distinct set of proteins. The 4 beta-phorbol 12,13-didecanoate homolog, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, also failed to induce protein tyrosine phosphorylation. Further, the PKC inhibitor, 1-O-hexadecyl-2-O-methylglycerol blocked tyrosine phosphorylation induced by 1-oleoyl-2-acetylglycerol. In subsequent studies, both CD8+ and CD8- NK clones were found to express the src-family tyrosine kinase, p56lck, which was detected by immunoblot analysis with anti-p56lck antiserum. In both types of clonal NK cell lines, IL-2 and 12-O-tetradecanoyl-phorbol appeared to stimulate the differential phosphorylation of p56lck as evidenced by the appearance of higher molecular mass isoforms on SDS-polyacrylamide gels. Thus, our results identify and characterize a potential role for tyrosine phosphorylation and for the lymphocyte-specific tyrosine kinase p56lck in the signaling events that regulate NK cell activation.