zeta PKC induces phosphorylation and inactivation of I kappa B-alpha in vitro.

The zeta isotype of protein kinase C (zeta PKC), a distinct PKC unable to bind phorbol esters, is required during NF-kappa B activation as well as in mitogenic signalling in Xenopus oocytes and mammalian cells. To investigate the mechanism(s) for control of cellular functions by zeta PKC, this enzyme was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP), to allow immobilization on amylose beads to study signalling proteins in cell extracts that might form complex(es) with zeta PKC. The following evidence for interaction with the NF-kappa B/I kappa B pathway was obtained. MBP-zeta PKC, but not MBP, bound and activated a potentially novel I kappa B kinase of approximately 50 kDa molecular weight able to regulate I kappa B-alpha function. Activation of the I kappa B kinase was dependent on zeta PKC enzymatic activity and ATP, suggesting that zeta PKC controls, directly or indirectly, the activity of a functionally significant I kappa B kinase. Importantly, zeta PKC immunoprecipitates from TNF-alpha-stimulated NIH-3T3 fibroblasts displayed a higher I kappa B phosphorylating activity than untreated controls, indicating the in vivo relevance of these findings. We also show here that zeta PKC associates with and activates MKK-MAPK in vitro, suggesting that one of the mechanisms whereby overexpression of zeta PKC leads to deregulation of cell growth may be accounted for at least in part by activation of the MKK-MAPK complex. However, neither MKK nor MAPK is responsible for the putative I kappa B phosphorylating activity. These data provide a decisive step towards understanding the functions of zeta PKC.     

Biphasic effect of protein kinase C activators on spontaneous and glucocorticoid-induced apoptosis in primary mouse thymocytes

Spontaneous and glucocorticoid (fluocinolone acetonide, FA)-induced apoptosis of primary mouse thymocytes was inhibited by protein kinase C (PKC) activators such as bryostatin-1 and phorbol ester 12-O-tetradecanoyl-phorbol-13 acetate (TPA) within the first 2-4 h of incubation but was enhanced upon prolonged treatment. Only the anti-apoptotic but not the pro-apoptotic effect of TPA was completely suppressed by the PKC inhibitor Goe 6983 and moderately inhibited by Goe 6976. Immunoblot analysis revealed distinct PKC alpha, beta, delta, eta, theta, mu and zeta signals, a very faint PKCepsilon and no PKCgamma signal. Upon prolonged TPA treatment all PKC isoenzymes became downregulated, albeit at different rates (PKCdelta>alpha>mu>beta,theta>eta,zeta). No significant generation of caspase-derived catalytic PKC fragments, as found to be produced upon induction of apoptosis and to be pro-apoptotic in other systems, was observed in FA- or TPA-treated thymocytes. It is concluded that the early anti-apoptotic effect of TPA depends on the activation of n-type PKC isoenzymes, whereas stimulation of spontaneous and FA-induced apoptosis by TPA ensues, at least partially, from a downregulation (or inactivation) of anti-apoptotic PKC species, i.e. in primary thymocytes PKC activation is primarily involved in a negative regulation of apoptosis.     

Differential action of nerve growth factor and phorbol ester TPA on rat synaptosomal PKC isoenzymes.

The subcellular redistribution of protein kinase C family members (alpha, beta, gamma, delta, epsilon and zeta isoforms) was examined in response to treatment with 12-O-tetradecanoyl-phorbol-13 acetate (TPA) or nerve growth factor (NGF) in a synaptosomal-enriched P2 fraction from rat brain. Treatment with TPA affected members of the classical-PKC family (alpha, beta and gamma), resulting in a final loss of total protein of each isoenzyme. The kinetics of changes of members of the novel-PKC family are different, the delta isoform being translocated, but not down-regulated, while the epsilon isoform showing only a slight diminishing of immunoreactivity in the soluble and particulate fractions. The atypical-PKC zeta isoform was not translocated in response to TPA. Incubation with NGF induced a loss of immunoreactivity of the cytosolic alpha, beta and epsilon isoforms, but the membrane fractions of these isoforms were not appreciably affected. In contrast, a marked translocation from cytosol to membrane was observed in the case of the gamma and delta isoforms. The zeta isoform presented a slight translocation from the particulate fraction to the soluble fraction. Thus, the results show that the effects of TPA and NGF on PKC isoforms are not coincident in synaptosomes, the 6 isoform being activated and not down-regulated by both treatments, whereas the gamma isoform is only down-regulated in the case of TPA, but presents sustained translocation with NGF, indicating that PKC isoform-specific degradation pathways exist in synaptic terminals. The effects of NGF on PKC isoforms coexist with an increase in NGF-induced polyphosphoinositide hydrolysis, suggesting the participation of phospholipases.     

Differential effects of free and liposome-associated 1-O-octadecyl-2-O-methylglycerophosphocholine on protein kinase C.

Incorporation of ET-18-OCH3 into well-characterized liposomes known as ELL-12 has eliminated its gastrointestinal and hemolytic toxicity without loss of growth inhibiting activity. ET-18-OCH3, but not ELL-12, blunted the increase in membrane protein kinase C (PKC) activity induced by 12-O-tetradecanoylphorbol 13-myristate (TPA) and markedly reduced levels of PKC alpha in NIH 3T3 fibroblasts. Furthermore, prolonged treatment with ELL-12 neither inhibited TPA-induced translocations of PKC alpha and PKC delta to the particulate fraction nor caused down-regulation, and did not affect the cellular distribution of TPA-insensitive PKC zeta. In Jurkat T cells, where ELL-12 markedly induced apoptosis that was blocked by an inhibitor of caspase-3-like activities, it had no effect on PKC activity or translocation induced by TPA. Thus, it seems unlikely that PKC is involved in the therapeutic effects of ELL-12.     

Purification and characterization of the zeta isoform of protein kinase C from bovine kidney.

The zeta isoform of protein kinase C (PKC zeta) was purified to near homogeneity from the cytosolic fraction of bovine kidney by successive chromatography on DEAE-Sephacel, heparin-Sepharose, phenyl-5PW, hydroxyapatite, and Mono Q. The purified enzyme had a molecular mass of 78 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was recognized by an antibody raised against a synthetic oligopeptide corresponding to the deduced amino acid sequence of rat PKC zeta. The enzymatic properties of PKC zeta were examined and compared with conventional protein kinase C purified from rat brain. The activity of PKC zeta was stimulated by phospholipid but was unaffected by phorbol ester, diacylglycerol, or Ca2+. PKC zeta did not bind phorbol ester, and autophosphorylation was not affected by phorbol ester. Unsaturated fatty acid activated PKC zeta, but this activation was neither additive nor synergistic with phospholipid. These results indicate that regulation of PKC zeta is distinct from that of other isoforms and suggest that hormone-stimulated increases in diacylglycerol and Ca2+ do not activate this isoform in cells. It is possible that PKC zeta belongs to another enzyme family, in which regulation is by a different mechanism from that for other isoforms of protein kinase C.     

Tumor necrosis factor induction of endothelial cell surface antigens is independent of protein kinase C activation or inactivation. Studies with phorbol myristate acetate and staurosporine.

We have investigated whether TNF-induced changes in human endothelial cell (EC) surface Ag expression are mediated by protein kinase C (PKC). This suggestion arose from the observations that PMA, a potent PKC activator, can mimic TNF by inducing expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1 (ICAM-1), and class I MHC molecules on human EC. However, in contrast to the actions of PMA, TNF neither causes membrane translocation of PKC nor induces the phosphorylation of the myristoylated alanine-rich C kinase substrate, two measures of PKC activation. Moreover, the PKC inhibitor staurosporine can block PMA-induced endothelial leukocyte adhesion molecule 1 expression at 4 h, but does not inhibit the actions of TNF. At 24 h, staurosporine itself induces intercellular adhesion molecule 1 and class I MHC, and acts additively with TNF. Twenty four hour treatment with PMA causes loss of PKC. We propose that at 24 h, staurosporine and PMA share a mechanism of action, namely diminution of PKC activity. However, 24 h treatment with TNF does not reduce the amount of PKC nor does it prevent activation of PKC by PMA. We conclude that TNF effects in EC are not mediated by PKC activation or inactivation.     

The transient increase of tight junction permeability induced by bryostatin 1 correlates with rapid downregulation of protein kinase C-alpha

The role of PKC-alpha in altered epithelial barrier permeability following the activation of PKC by TPA (12-O-tetradecanoyl phorbol 13-acetate) and bryostatin 1 in LLC-PK1 cells was investigated in this study. Like TPA, bryostatin 1 binds to and activates PKC but unlike TPA, it is not a tumor promoter. TPA at 10(-7) M induced a sustained 95% decrease in transepithelial electrical resistance (R(t)) across LLC-PK1 epithelial cell sheets, while 10(-7) M bryostatin 1 caused only a 30% decrease in R(t), which spontaneously reversed after 5 h. Simultaneous exposure of cell sheets to 10(-7) M TPA and 10(-7) M bryostatin 1 blunted the increase in epithelial permeability observed with 10(-7) M TPA alone. Co-incubation of cell sheets with bryostatin 1 and MG-132, a proteasomal inhibitor, caused a further decrease in R(t) at the 6-h time point and inhibited the recovery in R(t) seen with bryostatin 1 alone at this time point. TPA caused a rapid translocation of PKC-alpha from the cytosol to the membrane of the cell where it remained elevated. Bryostatin 1 treatment resulted in a slower translocation of PKC-alpha from the cytosol to the membrane and a much more rapid downregulation of PKC-alpha, with disappearance from this compartment after only 6 h. The classical PKC inhibitor Go6976 prevented the decrease in R(t) seen with TPA. Treatment of cells with TPA and bryostatin 1 resulted in a PKC-alpha translocation and downregulation profile which more closely resembled that seen with bryostatin 1 alone. Co-incubation of cells with MG-132 and bryostatin 1 caused a slower downregulation of PKC-alpha from the membrane fraction. Bryostatin 1 treatment of cells expressing a dominant/negative form of PKC-alpha resulted in a slower and less extensive decrease in R(t) compared to the corresponding control cells. For both TPA and bryostatin 1, the level of PKC-alpha in the membrane-associated fraction of the treated cells correlated closely with increased transepithelial permeability. Due to its transient effect on tight junction permeability, bryostatin 1 offers a novel pharmacological tool to investigate junctional physiology.     

Ceramide recruits and activates protein kinase C zeta (PKC zeta) within structured membrane microdomains

We have previously demonstrated that hexanoyl-D-erythro-sphingosine (C(6)-ceramide), an anti-mitogenic cell-permeable lipid metabolite, limited vascular smooth muscle growth by abrogating trauma-induced Akt activity in a stretch injury model of neointimal hyperplasia. Furthermore, ceramide selectively and directly activated protein kinase C zeta (PKC zeta) to suppress Akt-dependent mitogenesis. To further analyze the interaction between ceramide and PKC zeta, the ability of ceramide to localize within highly structured lipid microdomains (rafts) and activate PKC zeta was investigated. Using rat aorta vascular smooth muscle cells (A7r5), we now demonstrate that C(6)-ceramide treatment results in an increased localization and phosphorylation of PKC zeta within caveolin-enriched lipid microdomians to inactivate Akt. In addition, ceramide specifically reduced the association of PKC zeta with 14-3-3, a scaffold protein localized to less structured regions within membranes. Pharmacological disruption of highly structured lipid microdomains resulted in abrogation of ceramide-activated, PKC zeta-dependent Akt inactivation, whereas molecular strategies suggest that ceramide-dependent PKC zeta phosphorylation of Akt3 at Ser(34) was necessary for ceramide-induced vascular smooth muscle cell growth arrest. Taken together, these data demonstrate that structured membrane microdomains are necessary for ceramide-induced activation of PKC zeta and resultant diminished Akt activity, leading to vascular smooth muscle cell growth arrest.     

Biochemical characterization of the eta chain of the T-cell receptor. A unique subunit related to zeta

The T-cell antigen receptor is a multisubunit complex consisting of at least seven chains. Based upon structural and genetic considerations, we have divided these chains into three groups. The alpha and beta subunits (Ti) are the clonotypic chains responsible for antigen recognition. Three chains that are invariant among all T-cells define the CD3 complex. These include the CD3 gamma, delta, and epsilon chains. The zeta chain is a distinct component that, like the CD3 chains, is invariant among all T-cells. In the majority of receptors, zeta is found as a disulfide-linked homodimer. We have recently shown that approximately 10% of zeta is disulfide-linked to a chain which we have called eta. A preliminary model has been proposed, suggesting that there are two subclasses of receptors, depending upon the presence within the complex of either the zeta-zeta homodimer or the zeta-eta heterodimer. Evidence has been presented that these two subclasses may perform distinct signaling functions. In this paper the eta chain is characterized to determine whether it is structurally related to the zeta chain and, in particular, whether it might represent a post-translational modification of zeta. We can identify specific antigenic epitopes that are shared by both zeta and eta. However, not all antibodies raised against zeta can directly recognize eta. The apparent molecular mass of eta is 22 kDa, whereas zeta has a molecular mass of 16 kDa. We are unable to demonstrate any post-translational covalent modifications of eta to explain the difference in apparent molecular weight. These include phosphorylation, glycosylation, or sulfation. Amino acid incorporation studies demonstrate that the amino acid composition of eta is distinct from that of zeta. All of the eta in a T-cell is found in association with the rest of the components of the T-cell receptor. In addition, our anti-eta antibodies allow us to directly recognize human eta, which has an apparent molecular mass of approximately 23 kDa. Thus, eta and zeta appear to be related but distinct proteins, and we would propose that eta is the second member of the zeta group of components of the T-cell receptor.     

小鼠受精卵早期发育过程中PKC对cdc2和cdc25C活性的影响

为研究小鼠受精卵细胞早期发育过程中PKC对cdc2和cdc2 5C活性的影响 ,采用免疫印迹和电泳迁移率差异分析的方法 ,观察PKC的激活剂TPA及其抑制剂星形孢子素对小鼠受精卵一细胞期cdc2和cdc2 5C活性的影响 .10nmol L的TPA作用 10min后 ,小鼠受精卵一细胞期卵裂率明显大于对照组 (P <0 0 5 ) ,而星形孢子素作用后卵裂率显著下降 (P <0 0 1) .TPA处理后 ,受精卵中呈去磷酸化状态的活性cdc2明显增加 ,没有活性呈磷酸化状态的cdc2 5C明显减少 ;而星形孢子素处理的受精卵中没有活性的cdc2明显增加 ,有活性的cdc2 5C明显减少 .结果表明 ,TPA短时间作用可以促进小鼠一细胞期受精卵分裂 ,星形孢子素抑制受精卵的分裂 ;TPA可以促进cdc2的去磷酸化以及cdc2 5C的磷酸化 ,从而促进G2 M转换 ,星形孢子素则抑制cdc2和cdc2 5C的活性 ,阻止受精卵由G2 期进入M期     

Inhibition of alpha, betaI, delta, eta, and zeta protein kinase C isoforms by xanthonolignoids

The effect of the xanthonolignoids trans-(+/-)-kielcorin C, cis-(+/-)-kielcorin C, trans-(+/-)-kielcorin D, trans-(+/-)-isokielcorin D and trans-(+/-)-kielcorin E on isoforms alpha, betaI, delta, eta and zeta of protein kinase C (PKC) was studied using the yeast phenotypic assay. All the compounds tested revealed an effect compatible with PKC inhibition, similar to that exhibited by the well established PKC inhibitor chelerythrine, and with differences in their potency towards the distinct isoforms tested, being, in general, potent inhibitors of the atypical PKC isoform (PKC-zeta). PKC inhibition caused by these kielcorins was confirmed using an in vitro kinase assay. The present study constitutes the first attempt to unravel the molecular mechanism of kielcorins activity, and shows that xanthonolignoids are a promising group of compounds to investigate for isoform selective PKC inhibitors.     

Role of protein kinase C in the translational regulation of lipoprotein lipase in adipocytes

The hypertriglyceridemia of diabetes is accompanied by decreased lipoprotein lipase (LPL) activity in adipocytes. Although the mechanism for decreased LPL is not known, elevated glucose is known to increase diacylglycerol, which activates protein kinase C (PKC). To determine whether PKC is involved in the regulation of LPL, we studied the effect of 12-O-tetradecanoyl phorbol 13-acetate (TPA) on adipocytes. LPL activity was inhibited when TPA was added to cultures of 3T3-F442A and rat primary adipocytes. The inhibitory effect of TPA on LPL activity was observed after 6 h of treatment, and was observed at a concentration of 6 nM. 100 nM TPA yielded maximal (80%) inhibition of LPL. No stimulation of LPL occurred after short term addition of TPA to cultures. To determine whether TPA treatment of adipocytes decreased LPL synthesis, cells were labeled with [35S]methionine and LPL protein was immunoprecipitated. LPL synthetic rate decreased after 6 h of TPA treatment. Western blot analysis of cell lysates indicated a decrease in LPL mass after TPA treatment. Despite this decrease in LPL synthesis, there was no change in LPL mRNA in the TPA-treated cells. Long term treatment of cells with TPA is known to down-regulate PKC. To assess the involvement of the different PKC isoforms, Western blotting was performed. TPA treatment of 3T3-F442A adipocytes decreased PKC alpha, beta, delta, and epsilon isoforms, whereas PKC lambda, theta, zeta, micro, iota, and gamma remained unchanged or decreased minimally. To directly assess the effect of PKC inhibition, PKC inhibitors (calphostin C and staurosporine) were added to cultures. The PKC inhibitors inhibited LPL activity rapidly (within 60 min). Thus, activation of PKC did not increase LPL, but inhibition of PKC resulted in decreased LPL synthesis by inhibition of translation, indicating a constitutive role of PKC in LPL gene expression.     

Protein kinase C zeta associates with death inducing signaling complex and regulates Fas ligand-induced apoptosis

Previous studies have shown that Protein kinase C (PKC) stimulation may interfere with Fas signaling pathway and Fas ligand (FasL)-induced apoptosis. In this study, we investigated in Jurkat cells, a FasL-sensitive human T-cell model, whether PKC(zeta) targets apical events of Fas signaling. We describe for the first time that in Jurkat cells, both PKC(zeta) and Prostate apoptosis response-4 (Par-4), one of the major endogenous PKC(zeta) regulators, are components of the death inducing signaling complex (DISC). Using PKC(zeta) overexpressing cells or si-RNA depletion, we demonstrate that PKC(zeta) interferes neither with Fas expression nor Fas clustering in raft microdomains, but negatively regulates FasL-induced apoptosis by interfering with DISC formation and subsequent caspase-8 processing.     

The long-term combined stimulatory effects of ethanol and phorbol ester on phosphatidylethanolamine hydrolysis are mediated by a phospholipase C and prevented by overexpressed alpha-protein kinase C in fibroblasts.

The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) has been shown to potentiate the stimulatory effect of ethanol on the hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Following an initial 20-min period, the main product of PtdEtn degradation in cells treated with TPA plus ethanol was ethanolamine phosphate. Here, we have examined the regulatory role of PKC and the possible catalytic role of phospholipase C in the formation of ethanolamine phosphate. TPA, bryostatin, and bombesin, direct or indirect activators of PKC, had similar potentiating effects on ethanol-induced formation of [14C]ethanolamine phosphate from [14C]PtdEtn in [14C]ethanolamine-prelabelled NIH 3T3 fibroblasts. At lower concentrations of ethanol (40-80 mM), significant stimulation of ethanolamine phosphate formation required longer treatments (2 h or longer). The combined effects of TPA (100 nM) and ethanol (50-200 mM) on ethanolamine phosphate formation were not inhibited by the PKC inhibitors staurosporine or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7). In contrast, these inhibitors significantly inhibited TPA-induced formation of ethanolamine, catalyzed by a phospholipase-D-type enzyme. In membranes isolated from TPA+ethanol-treated cells, enhanced formation of ethanolamine phosphate was maintained for at least 20 min. Down-regulation of PKC by prolonged (24-h) treatment of NIH 3T3 fibroblasts by 300 nM TPA enhanced, while overexpression of alpha-PKC in Balb/c fibroblasts diminished, the stimulatory effect of ethanol on the formation of ethanolamine phosphate. Finally, addition of the protein phosphatase inhibitor okadaic acid (2 microM) to fibroblasts inhibited TPA+ethanol-induced formation of ethanolamine phosphate. These results suggest that alpha-PKC-mediated protein phosphorylation may negatively regulate PtdEtn hydrolysis and that the potentiating effect of TPA may result, at least partly, from increased degradation of this PKC isoform.     

Regulation of human involucrin promoter activity by novel protein kinase C isoforms

Human involucrin (hINV) mRNA level and promoter activity increase when keratinocytes are treated with the differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). This response is mediated via a p38 mitogen-activated protein kinase-dependent pathway that targets activator protein 1 (Efimova, T., LaCelle, P. T. , Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395). In the present study we examine the role of various PKC isoforms in this regulation. Transfection of expression plasmids encoding the novel PKC isoforms delta, epsilon, and eta increase hINV promoter activity. In contrast, neither conventional PKC isoforms (alpha, beta, and gamma) nor the atypical isoform (zeta) regulate promoter activity. Consistent with these observations, promoter activity is inhibited by the PKCdelta-selective inhibitor, rottlerin, but not by Go-6976, an inhibitor of conventional PKC isoforms, and novel PKC isoform-dependent promoter activation is inhibited by dominant-negative PKCdelta. This regulation appears to be physiologically important, as transfection of keratinocytes with PKCdelta, -epsilon, or -eta increases expression of the endogenous hINV gene. Synergistic promoter activation (>/=100-fold) is observed when PKCepsilon- or -eta-transfected cells are treated with TPA. In contrast, the PKCdelta-dependent response is more complex as either activation or inhibition is observed, depending upon PKCdelta concentration.     

Regulation of c-Myc and Max in megakaryocytic and monocytic-macrophagic differentiation of K562 cells induced by protein kinase C modifiers: c-Myc is down-regulated but does not inhibit differentiation.

We have studied the regulation and role of c-Myc and Max in the differentiation pathways induced in K562 cells by 12-O-tetradecanoyl phorbol-13 acetate (TPA) and staurosporine, an activator and inhibitor, respectively, of protein kinase C (PKC). We found that staurosporine induced megakaryocytic differentiation, as revealed by the cellular ultrastructure, platelet formation, and DNA endoreduplication. In contrast, TPA induced a differentiated phenotype that more closely resembled that of the monocyte-macrophage lineage. c-myc expression was down-regulated in K562 differentiated by both TPA and staurosporine, whereas max expression did not change in either case. Although PKC enzymatic activity was low in cells terminally differentiated with TPA and staurosporine, inhibition of PKC activity by itself did not induce c-myc down-regulation. We conclude that the c-myc gene is switched off as a consequence of the differentiation process triggered by these drugs in a manner independent from PKC. Ectopic overexpression of c-Myc in K562 cells did not affect the monocytic-macrophagic and megakaryocytic differentiation, indicating that c-Myc suppression is not required for these processes in K562. Similarly, both differentiation pathways were not affected by Max overexpression or by concomitant overexpression of c-Myc and Max. This result is in contrast with the inhibition of erythroid differentiation of K562 exerted by c-Myc, suggesting divergent roles for c-Myc/Max, depending on the differentiation pathway.     

PKC zeta is a molecular switch in signal transduction of TNF-alpha, bifunctionally regulated by ceramide and arachidonic acid.

Tumor necrosis factor (TNF-alpha) stimulates a number of signal transduction pathways in which phospholipases produce lipid second messengers. However, the immediate molecular targets of these messengers, in particular those of ceramide and arachidonic acid (AA) and their role in TNF signaling are not well defined. In this study we investigated the relationship of ceramide and AA in regulating an atypical PKC isozyme, PKC zeta. U937 cells responding to TNF-alpha treatment with NF kappa B activation displayed enhanced phosphorylation of PKC zeta, which is already detectable 30 s after stimulation. [14C]ceramide specifically binds to and regulates kinase activity of PKC zeta in a biphasic manner. Binding studies indicate high and low affinity binding with bmax values of 60 and 600 nM and Kd values of 7.5 and 320 nM respectively. At ceramide concentrations as low as 0.5 nM an up to 4-fold increase in autophosphorylation is obtained, which, at concentrations > 60 nM, again declines to basal levels. Interestingly, AA competes for ceramide binding and inhibits basal and ceramide-stimulated PKC zeta kinase activity at < 100 nM. Metabolism of [14C]ceramide in cells is slow and is inhibited in the presence of equimolar concentrations of lyso-phosphatidylcholine. Based on the bifunctional modulation of PKC zeta by the lipid messengers ceramide and AA, a model of TNF signal pathways is suggested in which PKC zeta takes a central position, acting as a molecular switch between mitogenic and growth inhibitory signals of TNF-alpha.