Opposite Regulation of Adenylyl Cyclase by Protein Kinase C in Astrocyte and Microglia Cultures

Abstract: We studied the regulation of cyclic AMP responses by protein kinase C (PKC) in purified astrocyte and microglia cultures obtained from the neonatal rat brain. In astrocytes, a 10-min treatment with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and 4β-phorbol 12,13-didecanoate (4β-PDD) (but not with 4α-PDD) or with diacylglycerol, which activate PKC, dose-dependently enhanced cyclic AMP accumulation induced by the β-adrenergic agonist isoproterenol and the adenylyl cyclase activator forskolin. Such enhancement was prevented by the PKC inhibitors staurosporine and calphostin-C and by down-regulation of PKC and was not related to activation of membrane receptors or G_s proteins or to inhibition of G_i proteins or phosphodiesterases. Instead, the activity of adenylyl cyclase doubled in PMA-treated astrocytes. In microglia, a 10-min treatment with PMA or PKC inhibitors did not affect cyclic AMP accumulation, whereas longer treatments with PMA or 4β-PDD (but not 4α-PDD) inhibited the cyclic AMP response in a time- and dose-dependent manner. Such inhibition was mimicked by staurosporine and calphostin-C. Also, in the case of microglia, the modulation of cyclic AMP responses appeared to occur at the level of adenylyl cyclase, and not elsewhere in the cyclic AMP cascade. The inhibition of microglial adenylyl cyclase was apparently not due to aspecific cytotoxicity. A differential regulation of adenylyl cyclase by PKC in astrocytes and microglia may help to explain qualitative and quantitative differences in the response of these cells to various physiological and pathological stimuli.     

The role of protein kinase C-delta in PTH stimulation of IGF-binding protein-5 mRNA in UMR-106-01 cells

We have investigated the role of protein kinase C (PKC) signal transduction pathways in parathyroid hormone (PTH) regulation of insulin-like growth factor-binding protein-5 (IGFBP-5) gene expression in the rat osteoblast-like cell line UMR-106-01. Involvement of the PKC pathway was determined by the findings that bisindolylmaleimide I inhibited 40% of the PTH effect, and 1 microM bovine PTH-(3-34) stimulated a 10-fold induction of IGFBP-5 mRNA. PTH-(1-34) and PTH-(3-34) (100 nM) both stimulated PKC-delta translocation from the membrane to the nuclear fraction. Rottlerin, a PKC-delta-specific inhibitor, and a dominant negative mutant of PKC-delta were both able to significantly inhibit PTH-(1-34) and PTH-(3-34) induction of IGFBP-5 mRNA, suggesting a stimulatory role for PKC-delta in the effects of PTH. Phorbol 12-myristate 13-acetate (PMA) stimulated PKC-alpha translocation from the cytosol to the membrane and inhibited approximately 50% of the PTH-(1-34), forskolin, and 8-bromoadenosine 3',5'-cyclic monophosphate-stimulated IGFBP-5 mRNA levels, suggesting that PKC-alpha negatively regulates protein kinase A (PKA)-mediated induction of IGFBP-5 mRNA. These results suggest that the induction of IGFBP-5 by PTH is both PKA and PKC dependent and PKC-delta is the primary mediator of the effects of PTH via the PKC pathway.     

Protein kinase C mediates phosphorylation, desensitization, and trafficking of the D2 dopamine receptor

Previously, D2 dopamine receptors (D2 DARs) have been shown to undergo G-protein-coupled receptor kinase phosphorylation in an agonist-specific fashion. We have now investigated the ability of the second messenger-activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC), to mediate phosphorylation and desensitization of the D2 DAR. HEK293T cells were transiently transfected with the D2 DAR and then treated with intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D2 DAR, suggesting that PKA does not phosphorylate the D2 DAR in HEK293T cells. In contrast, cellular treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in an approximately 3-fold increase in D2 DAR phosphorylation. The phosphorylation was specific for PKC as the PMA effect was mimicked by phorbol 12,13-dibutyrate, but not by 4alpha-phorbol 12,13-didecanoate, active and inactive, phorbol diesters, respectively. The PMA-mediated D2 DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II, and augmented by co-transfection with PKCbetaI. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation, suggesting that PKC is not involved in this response. PKC phosphorylation of the D2 DAR was found to promote receptor desensitization as reflected by a decrease in agonist potency for inhibiting cAMP accumulation. Most interestingly, PKC phosphorylation also promoted internalization of the D2 DAR through a beta-arrestin- and dynamin-dependent pathway, a response not usually associated with PKC phosphorylation of G-protein-coupled receptors. Site-directed mutagenesis experiments resulted in the identification of two domains of PKC phosphorylation sites within the third intracellular loop of the receptor. Both of these domains are involved in regulating sequestration of the D2 DAR, whereas only one domain is involved in receptor desensitization. These results indicate that PKC can mediate phosphorylation of the D2 DAR, resulting in both functional desensitization and receptor internalization.     

Phorbol Ester- and Retinoic Acid-Induced Regulation of the Protein Kinase C Substrate MARCKS in Immortalized Hippocampal Cells

Abstract: The expression of MARCKS, a major protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 µ M phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to <20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Significant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h, respectively. The reduction in MARCKS protein levels was maximal following 24 h of PMA exposure. MARCKS protein expression was also down-regulated in a dose-dependent manner on exposure of HN33 cells to retinoic acid. In cells exposed to 10 µ M retinoic acid, the MARCKS protein level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (>90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS protein in PMA-treated cultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.     

佛波酯引起蛋白激酶C下降调节的专一性

探讨了佛波酯（ＰＭＡ）对蛋白激酶的下降调节是否有激酶专一性及亚型专一性．用组蛋白Ｈ１作为蛋白激酶Ｃ（ＰＫＣ）和蛋白激酶Ａ（ＰＫＡ）的受体底物,加入ＰＫＣ和ＰＫＡ的特异性激活剂区分ＰＫＣ和ＰＫＡ,用聚谷酪（４１）为酪氨酸蛋白激酶（ＴＰＫ）的专一性受体底物,以３２Ｐ－ＡＴＰ为３２Ｐ共同供体底物测定三种蛋白激酶的活力,并用免疫组化法测定ＰＫＣ亚型．结果发现ＰＭＡ对人７７２１肝癌细胞只引起ＰＫＣ而不引起ＰＫＡ和ＴＰＫ的下降调节,ＰＫＣ的非特异性抑制剂槲皮素和特异性抑制剂Ｄ－鞘氨醇能大部分取消ＰＭＡ对ＰＫＣ的下降调节,但ＴＰＫ抑制剂ｇｅｎｅｓｔｅｉｎ则没有阻断下降调节的作用．用ＨＬ－６０细胞还证明ＰＭＡ只对含量丰富的ＰＫＣα和ＰＫＣβⅡ亚型而不对含量很少的ＰＫＣβⅠ亚型发生下降调节．上述结果说明ＰＭＡ对蛋白激酶的下降调节有激酶和亚型专一性．     

Intracellular regulation of estradiol and progesterone production by cultured bovine granulosa cells.

The objective of the present study was to investigate the implication of protein kinase A (PKA), protein kinase C (PKC), and receptor protein tyrosine kinase (R-PTK) pathways in the regulation of estradiol (E2) and progesterone (P4) production by bovine granulosa cells. Cells were harvested from bovine follicles (8-15 mm diameter) and cultured without serum for an initial 3 days (37 degrees C; 5% CO(2) in air; D1-D3). On the fourth day of culture (D4), E2 and P4 production were stimulated with FSH (1-6 ng/ml) or forskolin (FSK) in the presence or absence of intracellular effectors of PKA, PKC, and R-PTK. Culture medium was collected and replaced each day. Stimulation of granulosa cell adenylate cyclase activity with FSK (0.06-3.75 microM) mimicked FSH, inducing a quadratic increase (P < 0.001) of E2 production and a continuous elevation of P4 (P < 0.01). Inhibition of R-PTK activity with genistein (25-50 microM) increased the sensitivity of cells to FSH as demonstrated by a leftward shift in the dose response curve (P < 0.001). Treatment with transforming growth factor-alpha (TGFalpha; 0. 1 ng/ml) abolished the FSH-induced E2 production (P < 0.001) and this effect was not reversed (P < 0.001) by FSK or by genistein. Furthermore, the inhibitory effect of TGFalpha on FSH-induced E2 production was reproduced by phorbol 12-myristate 13-acetate (PMA; 1. 25-2.5 microM), a PKC activator (P < 0.001). Interestingly, genistein inhibited P4 production (P < 0.05). From these results, we conclude that E2 production by bovine granulosa cells is mediated by intracellular factors and can be stimulated downstream from the FSH receptor. The results also suggest that stimulation of R-PTK and/or PKC activities, as probably occurs with TGFalpha, negatively affects the PKA pathway, thus decreasing E2 production. Furthermore, inhibition of R-PTK leads to an increase production of E2 and may limit luteinization of bovine granulosa cells.     

Regulation of mRNA expression of 3 beta-hydroxy-5-ene steroid dehydrogenase in porcine granulosa cells in culture: a role for the protein kinase-C pathway

We studied the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase-C, on porcine granulosa cells in culture. PMA as well as cholera toxin, forskolin, and hCG increased cAMP accumulation. PMA further augmented the elevation in cAMP accumulation induced by cholera toxin, forskolin, and hCG. In the same cell culture model, hCG induced a time-dependent increase in the 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) mRNA levels with a maximal 3-fold stimulation obtained at 8-16 h of incubation with 1 IU hCG/ml. PMA inhibited the increase in 3 beta HSD mRNA levels induced by hCG in a dose-dependent manner. The phorbol ester also inhibited the increase in 3 beta HSD mRNA levels stimulated by LH as well as cholera toxin and forskolin and the cAMP analogs (Bu)2cAMP and 8-bromo-cAMP. Activation of protein kinase-C by mezerein similarly inhibited hCG stimulation of 3 beta HSD mRNA levels. The present data indicate that activation of the protein kinase-C pathway induces generation of cAMP, but causes a near-complete inhibition of the stimulatory effects of hCG, LH, forskolin, cholera toxin, and cAMP analogs on 3 beta HSD mRNA levels in porcine granulosa cells in culture.     

Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells

Gonadotropin-stimulated steroidogenesis in the differentiating ovarian granulosa cell is mediated through the activation of cAMP-dependent protein kinase, and is also modulated by calcium-dependent mechanisms. Granulosa cells contain calcium-activated, phospholipid-dependent protein kinase (C kinase), and show an increase in phosphatidylinositol turnover in response to GnRH agonist analogs. To evaluate the role of C kinase in ovarian steroidogenesis, the potent phorbol ester, TPA, and the permeant diacylglycerol, OAG, were used to activate C kinase in granulosa cells from PMSG-treated immature rats. Both TPA and OAG caused dose-dependent stimulation of progesterone production without affecting intra- or extracellular cAMP levels. However, the maximum steroid responses to these compounds were less than those stimulated by cAMP. The ED50 for TPA-stimulated progesterone production was 3 nM, which is close to the known Km for activation of C kinase. Stimulation of steroidogenesis was only observed with biologically-active phorbol esters and permeant diacylglycerols such as OAG and DOG. Exposure of granulosa cells to phospholipase C also increased progesterone production in a dose-dependent manner without changing the cAMP content. Although TPA and OAG did not increase basal cAMP production, both agents enhanced the cAMP responses stimulated by hCG and forskolin; likewise, phospholipase C alone did not change cAMP production but caused a dose-dependent increase in the cAMP responses to hCG and forskolin. These results demonstrate that activation of C kinase promotes steroidogenesis in ovarian granulosa cells, and potentiates the activation of adenylate cyclase by hCG and forskolin. Such findings support the possibility that the calcium, phospholipid-dependent enzyme could be involved in the regulation of progesterone production by hormonal ligands such as gonadotropins and GnRH.     

Ca2+ Regulates Hormone Secretion and Proopiomelanocortin Gene Expression in Melanotrope Cells via the Calmodulin and the Protein Kinase C Pathways

The mechanism by which Ca2+ regulates proopiomelanocortin (POMC)-derived peptide secretion and POMC mRNA levels was investigated in primary cultures of porcine intermediate lobe (IL) cells maintained in serum-free medium. POMC gene expression was evaluated by the dot blot hybridization assay with a 32P-labeled DNA probe complementary to the full-length sequence of porcine POMC mRNA. Treatment of IL cells for 24 h with the calmodulin (CAM) antagonists W7 and W13 reduced POMC mRNA levels by a maximum of 50% in a dose-dependent manner (ED50 approximately 10(-8) M). Accumulation of alpha-melanocyte-stimulating hormone (alpha-MSH) in the medium was also depressed by 50% after 8 h of treatment. The role of protein kinase C (PKC) was investigated by depleting the IL cell PKC content with phorbol ester treatment. Phorbol 12-myristate 13-acetate (PMA) at 5 X 10(-8) M induced a rapid translocation of cytoplasmic PKC activity toward the membrane. After 12 h of PMA treatment, PKC activity was undetectable in either the cytoplasmic or the particulate fractions. The same dose of PMA induced a time-dependent decrease in POMC mRNA levels (50% inhibition after 24 h). The same effect was seen with the phorbol ester phorbol 12,13-dibutyrate at 5 X 10(-8) M, whereas the inactive phorbol ester 4 alpha-phorbol at 5 X 10(-8) M was without effect after 24 h of treatment. PMA treatment had a biphasic effect on alpha-MSH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual intracellular pathways in gonadotropin releasing hormone (GNRH) induced desensitization of luteinizing hormone (LH) secretion.

The mechanisms of GnRH-induced desensitization of LH secretion are poorly understood. Protein kinase C (PKC) and protein kinase A (PKA) desensitize some receptors of the 7-membrane type, and the GnRH receptor has consensus phosphorylation sites for PKC in the first and third intracellular loops, and a site for PKA in the first intracellular loop. In the first set of experiments we determined whether synthetic peptides representing the three intracellular loops of the receptor could be phosphorylated in vitro by purified PKC and PKA. As compared with a model substrate peptide for PKC, the third intracellular loop was phosphorylated 74% and the first intracellular loop 21%; PKA-phosphorylated the first intracellular loop peptide 17% as well as a model peptide substrate. In the second set of experiments, we used phorbol 12-myristate 13 acetate (PMA), an established PKC stimulator, and cholera toxin (CTX), established to activate the Gs protein and presumed to activate PKA, to treat cultured rat pituitary cells followed by LH measurements. Treatment with both drugs severely impaired GnRH-stimulated LH secretion whereas neither drug alone reduced LH secretion. Dibutyryl cAMP did not duplicate the effects of cholera toxin suggesting that the CTX action could not be explained by an increase in cAMP. These results suggest that more than one intracellular signaling pathway requires activation in order to induce desensitization; one pathway involves PKC and the other involves a pathway stimulated by cholera toxin, presumably Gs protein, which does not involve PKA.     

Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.     

Protein kinase C induces phosphorylation and desensitization of the human 5-HT1A receptor

The effects of short-term phorbol ester treatment of CHO cells that stably express 900 fmol of recombinant human serotonin 5-HT1A receptor/mg of protein on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the receptor were studied. Pretreatment of cell monolayers with phorbol 12-myristate 13-acetate (PMA) caused a dose- and time-dependent shift of the half-maximal dose of serotonin (5-HT) required to inhibit membrane adenylyl cyclase (from IC50 approximately 100 nM to approximately 400 nM). This desensitization (shift in IC50) was rapid, occurring with 5 min of pretreatment and being maximal by 10-15 min; it was also dose-dependent, being half-maximal at approximately 300 nM PMA. Desensitization was also induced by sn-dioctanoylglycerol (DiC8) and blocked by the protein kinase C (PKC) inhibitors sphingosine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). In detached permeabilized cells, PMA pretreatment caused a rapid phosphorylation of immunoprecipitated 5-HT1A receptors, with an approximately 3-4-fold increase that was maximal after 15 min and persisted for 90 min. The phosphorylation occurred at a similar dose of PMA as that which induced desensitization (half-maximal at approximately 300 nM, maximal at 500 nM to 1 microM), could be reproduced by pretreatment with the PKC activators DiC8 or phorbol 12,13-dibutyrate (PDBu), and could be blocked by the PKC inhibitors sphingosine or H-7. The stoichiometry of the phosphorylation was approximately 2 mol of [32P]ATP/mol of receptor, suggesting the involvement at least two of three putative PKC sites within the 5-HT1A receptor. The close concordance between the PKC-induced desensitization and phosphorylation suggests a potential causative link between these two effects of PKC on the human 5-HT1A receptor.     

Dependence of gonadotropin-releasing hormone-induced neuronal MAPK signaling on epidermal growth factor receptor transactivation

The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), utilizes multiple signaling pathways to activate extracellularly regulated mitogen-activated protein kinases (ERK1/2) in normal and immortalized pituitary gonadotrophs and transfected cells expressing the GnRH receptor. In immortalized hypothalamic GnRH neurons (GT1-7 cells), which also express GnRH receptors, GnRH, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) caused marked phosphorylation of ERK1/2. This action of GnRH and PMA, but not that of EGF, was primarily dependent on activation of protein kinase C (PKC), and the ERK1/2 responses to all three agents were abolished by the selective EGF receptor kinase inhibitor, AG1478. Consistent with this, both GnRH and EGF increased tyrosine phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase, Pyk2, at Tyr(402). This was reduced by Ca(2+) chelation and inhibition of PKC, but not by AG1478. GnRH stimulation caused translocation of PKC alpha and -epsilon to the cell membrane and enhanced the association of Src with PKC alpha and PKC epsilon, Pyk2, and the EGF receptor. The Src inhibitor, PP2, the C-terminal Src kinase (Csk), and dominant-negative Pyk2 attenuated ERK1/2 activation by GnRH and PMA but not by EGF. These findings indicate that Src and Pyk2 act upstream of the EGF receptor to mediate its transactivation, which is essential for GnRH-induced ERK1/2 phosphorylation in hypothalamic GnRH neurons.     

Phorbol esters reduce gonadotrope responsiveness to protein kinase C activators but not to Ca2+-mobilizing secretagogues. Does protein kinase C mediate gonadotropin-releasing hormone action?

The demonstration that activators of the Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), such as phorbol esters and diacylglycerols, can provoke luteinizing hormone (LH) release from pituitary gonadotropes, suggests a possible role for protein kinase C in stimulus-release coupling. We now report that administration of phorbol myristate acetate (PMA) to pituitary cell cultures causes a sustained reduction in Triton X-100-extracted protein kinase C activity. Further, phorbol ester- and diacylglycerol-stimulated LH release, as well as inhibition by PMA of gonadotropin-releasing hormone (GnRH)-stimulated inositol phosphate production, were reduced by pretreatment with PMA. The effects of phorbol ester pretreatment on PMA-stimulated LH release and protein kinase C activity were dose-dependent, sustained (greater than or equal to 24 h) and specific (no measurable effect with 4 alpha-phorbol didecanoate). The effect on PMA-stimulated LH release was apparently Ca2+-independent. In pituitary cell cultures with reduced protein kinase C activity, the gonadotropes have reduced responsiveness to PMA but release a similar proportion of cellular LH in response to Ca2+-mobilizing secretagogues (GnRH and A23187) as do control cells. The normal responsiveness to GnRH of cells with reduced responsiveness to protein kinase C activators calls into question the requirement for this enzyme for GnRH-stimulated LH release.     

PKC-Dependent Long-Term Effect of PMA on Protein Cell Surface Expression in Caco-2 Cells

Several recent data indicate that protein traffic is under the control of different phosphorylation pathways. In previous works, we have shown that cell surface expression of apical hydrolases and of a basolateral protein, “525” antigen, was impaired in Caco-2 cells treated with forskolin, a potent PKA activator (L. Baricaultet al.,1995,J. Cell Sci.,108, 2109–2121). Surprisingly, in these experiments forskolin did not seem to act through PKA activation. These cAMP-independent effects of FK may rely on cross-talk between intracellular phosphorylation pathways as described recently for PKA and PKC pathways. Therefore, we tested the hypothesis that PKC activation may induce effects comparable to those of FK on three brush border hydrolases as well as on 525 antigen cell surface expression in Caco-2 cells. Using enzymatic activity measurements and pulse–chase experiments combined with cell surface biotinylation assays, we show that long-term treatment with phorbol 12-myristate 13-acetate (PMA) impairs the overall expression of neither brush border hydrolases nor that of the 525 antigen but decreases total cell surface expression of these proteins. The apical and basolateral delivery pathways are equally affected. Using confocal laser scanning microscopy we show that the DPP IV and the 525 antigen that were not recovered from the cell surface were sequestrated in Lamp-1-positive lysosomal-related vesicles. PMA stimulates PKC translocation even after a 3-week treatment and induces PKC? redistribution to a vesicular- and membrane-associated compartment also labeled with cytokeratins. These results demonstrate that PMA-dependent PKC activation strongly impairs protein cell surface targeting. They also suggest that these PKC-dependent effects which are similar to those previously obtained with FK are relevant to the described cross-talk between PKA- and PKC-dependent phosphorylation pathways.