Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.     

Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism

Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser(1179) in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser(116), Thr(497), and Ser(635). Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser(635), which was consistently slower than that at Ser(1179). Thr(497) was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser(116). Whereas shear-stimulated Ser(635) phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser(635) may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.     

Sphingosine inhibits thyrotropin-releasing hormone binding to pituitary cells by a mechanism independent of protein kinase C

Sphingosine inhibited [3H]methylhistidine-thyrotropin-releasing hormone (MeTRH) binding to intact GH3 cells and to GH3 membranes. This inhibition was dependent on the concentration of sphingosine and on the ratio of sphingosine to cell number (or membrane protein) and was partly reversed by washing. In intact cells, the IC50 was 63 microM (1.8 X 10(6) cells/ml; 2 nM MeTRH), and 100 microM sphingosine was found, by Scatchard analysis, to increase the apparent dissociation constant (Kd) from 1.1 +/- 0.3 to 6.5 +/- 2.3 nM and to decrease the maximal binding capacity (Bmax) to 41 +/- 9.5% of control. Kinetic analysis showed that the major effect of sphingosine on Kd was due to a marked decrease in the apparent association rate constant for MeTRH from 2.5 +/- 0.4 X 10(5) M-1 s-1 to 0.10 +/- 0.015 X 10(5) M-1 s-1. At 100 microM, sterylamine was as effective as sphingosine in inhibiting MeTRH binding, whereas sphinganine was less effective, and psychosine and steroylsphingosine were without effect. The following observations show that sphingosine inhibition of MeTRH binding did not involve protein kinase C. The IC50 for sphingosine inhibition of MeTRH binding was the same in GH3 cells that had been incubated with 1 microM phorbol 12-myristate 13-acetate for 16 h, to "down-regulate" protein kinase C, as in control cells. Sphingosine inhibited MeTRH binding to membranes isolated from GH3 cells that contain very little protein kinase C activity. In GH3 membranes, 100 microM sphingosine increased the Kd for MeTRH from 3.4 +/- 0.1 to 13 +/- 3.1 nM but did not significantly decrease Bmax (12 +/- 5.0% of control, p greater than 0.05). And, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, an inhibitor of protein kinase C, failed to decrease MeTRH binding to intact GH3 cells or to membranes, and did not interfere with the effects of sphingosine. These data show that sphingosine and its analogs have complex actions to inhibit MeTRH binding to GH3 cells, at least some of which are independent of protein kinase C, and thereby demonstrate that sphingolipids cannot be used as specific inhibitors of protein kinase C.     

Regulation of insulin exocytosis by calcium-dependent protein kinase C in beta cells

The control of insulin release from pancreatic beta cells helps ensure proper blood glucose level, which is critical for human health. Protein kinase C has been shown to be one key control mechanism for this process. After glucose stimulation, calcium influx into beta cells triggers exocytosis of insulin-containing dense-core granules and activates protein kinase C via calcium-dependent phospholipase C-mediated generation of diacylglycerol. Activated protein kinase C potentiates insulin release by enhancing the calcium sensitivity of exocytosis, likely by affecting two main pathways that could be linked: (1) the reorganization of the cortical actin network, and (2) the direct phosphorylation of critical exocytotic proteins such as munc18, SNAP25, and synaptotagmin. Here, we review what is currently known about the molecular mechanisms of protein kinase C action on each of these pathways and how these effects relate to the control of insulin release by exocytosis. We identify remaining challenges in the field and suggest how these challenges might be addressed to advance our understanding of the regulation of insulin release in health and disease.     

The involvement of protein kinase C in exocytosis in mast cells.

Diacylglycerol generated from inositolphospholipid hydrolysis and tumor-promoting phorbol esters stimulate protein kinase C. The synthetic diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) have been used in pure rat peritoneal mast cells. Both caused histamine release associated with exocytosis. The release by the stimulation of protein kinase C alone in the absence of secretagogues was slow although up to 50% of the histamine content was released by TPA in 120 min. Remarkable potentiation of histamine release was observed when the mast cells were preincubated with TPA before exposure to the calcium ionophore A23187. The potentiation of histamine release corresponded with an intensification of exocytosis. The potentiation is consistent with a participation of protein kinase C in the secretory process. An inhibitory effect due to protein kinase C activity was also demonstrated using TPA and mast cells from sensitized rats. When sensitized mast cells preincubated with 50 nM TPA for 5 min were exposed to the antigen, the histamine release was substantially reduced compared to the sum of the release by the antigen and TPA or by the antigen alone. There was a corresponding decrease in exocytosis. The inhibition of exocytosis and histamine release seems to reflect a regulatory function of protein kinase C for the termination of the response, as demonstrated in other types of cells apparently acting through an inhibition of inositolphospholipid hydrolysis.     

Mechanisms of luteinizing-hormone exocytosis in Staphylococcus aureus-alpha-toxin-permeabilized sheep gonadotropes.

We have used primary gonadotropes permeabilized with the pore-forming protein Staphylococcus aureus alpha-toxin to investigate luteinizing hormone (lutropin, LH) exocytosis. The diameter of the alpha-toxin pores (2-3 nm) allows the exchange of small molecules, whereas larger cytosolic proteins are retained. Because of the slow exchange of small molecules through the pores, we have developed a protocol which combines prolonged pre-equilibration of the permeabilized cells at 0 degrees C before stimulation with strong Ca2+ buffering. Under these conditions, increasing the free Ca2+ concentration from 0.1 microM to 10 microM [EC50 (concentration effecting half-maximal response) 2-3 microM] resulted in a 15-20-fold increase in LH exocytosis. LH exocytosis was maximal in the first 3 min and completed by 12 min. When permeabilized cells were equilibrated for prolonged periods in the absence of MgATP, Ca2(+)-stimulated LH secretion gradually declined (greater than 90% decrease by 60 min). Addition of MgATP (5 mM) rapidly restored full Ca2(+)-stimulated LH secretion. MgATP supported Ca2(+)-stimulated LH secretion at a half-maximal concentration of 1.5 mM. UTP and adenosine 5'-[gamma-thio]triphosphate were 40 and 31% as effective as MgATP, whereas other nucleotide triphosphates were ineffective. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 50 nM) stimulated LH exocytosis at free Ca2+ concentrations as low as 1 nM and was additive with Ca2+ at higher free Ca2+ concentrations. PMA-stimulated exocytosis required MgATP at concentrations similar to those required for Ca2(+)-stimulated LH exocytosis. These results demonstrate that LH exocytosis can be triggered both by micromolar Ca2+ concentrations or, in the virtual absence of Ca2+, by PKC activation. Both mechanisms of stimulated exocytosis have an absolute requirement for millimolar ATP. Because they retain cytosolic proteins, alpha-toxin-permeabilized cells may have advantages over alternative permeabilization methods provided that conditions are used that compensate for slow diffusion through alpha-toxin pores.     

The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.     

Calcium free inositol (1,4,5)-trisphosphate stimulates protein kinase C dependent protein phosphorylation in nuclei isolated from mitogen-treated Swiss 3T3 cells

As a step towards the elucidation of the role played by nuclear polyphosphoinositides, we have investigated the effect of exogenous calcium free inositol (1,4,5)-trisphosphate on the in vitro phosphorylation of proteins in nuclei prepared from Swiss 3T3 cells treated with bombesin and insulin-like growth factor I. When present in combination with phosphatidylserine, inositol (1,4,5)-trisphosphate enhanced the phosphorylation of two nuclear proteins, Mr 21,000 and 31,000, as well as of exogenous histone H1, to the same extent as a combination of phosphatidylserine and diacylglycerol. Inositol (1,4,5)-trisphosphate alone had no effect. This stimulation could be abolished by the protein kinase C inhibitor sphingosine and by EGTA, while could be restored by a combination of phosphatidylserine and exogenous Ca+(+) ions. These results raise the possibility that inositol (1,4,5)-trisphosphate is capable of liberating Ca+(+) ions from a nuclear store thus stimulating protein kinase C activity.     

Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism.

Intrahepatic bile duct epithelial cells, or cholangiocytes, contribute to bile secretion in response to hormones, including secretin. However, the mechanism by which secretin stimulates ductular bile flow is unknown. Since recent data in nonhepatic epithelia have suggested a role for exocytosis in fluid secretion, we tested the hypothesis that secretin stimulates exocytosis by isolated cholangiocytes. Cholangiocytes were isolated from normal rat liver by a newly described method employing enzymatic digestion and mechanical disruption followed by immunomagnetic separation using specific monoclonal antibodies, and exocytosis was measured using a fluorescence unquenching assay employing acridine orange. Secretin caused a dose-dependent (10(-12)-10(-7) M) increase in acridine orange fluorescence by acridine orange-loaded cholangiocytes with a peak response at 10 min; the half-maximal concentration of secretin was 7 x 10(-9) M. The secretin effect was inhibited by preincubation of cholangiocytes with colchicine (30% inhibition, p less than 0.05) or trypsin (90% inhibition, p less than 0.001); no inhibition was seen with lumicolchicine and heat-inactivated trypsin. Cholecystokinin, insulin, and somatostatin had no effect on fluorescence of acridine orange-loaded cholangiocytes; secretin had no effect on fluorescence of acridine orange-loaded hepatocytes or hepatic endothelial cells. Exposure of isolated cholangiocytes to secretin at doses that stimulated exocytosis caused a dose-dependent increase in cyclic AMP levels (218% maximal increase, p less than 0.05); moreover, an analogue of cyclic AMP stimulated exocytosis by cholangiocytes. Secretin had no effect on intracellular calcium concentration using Fura-2-loaded cholangiocytes assessed by digitized video microscopy. Our results demonstrate, for the first time, that secretin stimulates exocytosis by rat cholangiocytes. The effect is cell- and hormone-specific, dependent on intact microtubules, on a protein(s) on the external surface of cholangiocytes, and on changes in cellular levels of cyclic AMP. The results are consistent with the hypothesis that secretin-induced changes in bile flow may involve an exocytic process.     

Calcium- and guanine-nucleotide-dependent exocytosis in permeabilized rat mast cells. Modulation by protein kinase C.

We have used a digitonin-permeabilized cell system to study the signal transduction pathways responsible for stimulus-secretion coupling in the rat peritoneal mast cell. Conditions were established for permeabilizing the mast cell plasma membrane without disrupting secretory vesicles. Exocytotic release of histamine from digitonin-permeabilized cells required a combination of micromolar concentrations of Ca2+ and the stable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but was independent of exogenous ATP. In the presence of 40 microM-GTP[S], exocytosis was half-maximal at 1.3 microM-Ca2+ and maximal at 10 microM-Ca2+; GTP[S] alone (100 microM) had no effect on histamine release in the absence of added Ca2+. In the presence of 10 microM free Ca2+, 5 microM-GTP[S] was required for half-maximal exocytosis. To examine the possible role of protein kinase C (PKC) in exocytosis, we utilized 12-O-tetradecanoylphorbol 13-acetate (TPA) to activate PKC and studied its effect on histamine release from permeabilized mast cells. Cells that had been incubated with TPA (25 nM for 5 min) exhibited increased sensitivity to both GTP[S] and Ca2+. The PKC inhibitor staurosporine blocked the effect of TPA without inhibiting normal exocytosis in response to the combination of GTP[S] and Ca2+. In addition, down-regulation of mast-cell PKC by long-term TPA treatment (25 nM for 20 h) blocked the ability of the cells to respond to TPA and inhibited exocytosis in response to Ca2+ and GTP[S] by 40-50%. These results suggest that the sensitivity of the exocytotic machinery of the mast cell can be altered by PKC-catalysed phosphorylation events, but that activation of PKC is not required for exocytosis to occur.     

Phosphorylation of Munc18 by protein kinase C regulates the kinetics of exocytosis

Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.     

C-fos gene activation in murine thymocytes by a mechanism independent of protein kinase C or a Ca2+ signal

The accumulation of c-fos mRNA in mouse thymocytes was compared when the cells were stimulated by concanavalin A (Con A), the Ca2+ ionophore A23187 or the phorbol ester, TPA, either separately or by combinations of these mitogens. The c-fos response to mitogenic concentrations of Con A could not be attributed either to the rise in [Ca2+]i it induces or to activation of protein kinase C. Thus, although Con A causes the breakdown of phosphatidylinositol 4,5-bisphosphate in these cells, neither of the signals which can be generated by this response was responsible for the activation of the c-fos gene by Con A. This implies that some other unidentified signal generated by Con A activates the c-fos gene.     

Activation of phospholipase D by protein kinase C. Evidence for a phosphorylation-independent mechanism.

The role of protein kinase C (PKC) in the regulation of phosphatidylcholine-hydrolyzing phospholipase D (PLD) was investigated. In membranes from Chinese hamster lung fibroblasts that had been incubated with [14C]choline to label endogenous phosphatidylcholine, phorbol 12-myristate 13-acetate (PMA) failed to stimulate production of [14C]choline. However, stimulation was observed if fibroblast cytosolic fraction or PKC partially purified from this fraction was added. When incubated with membranes in the presence of PMA, pure PKC from rat brain stimulated [14C]choline production in a concentration-dependent manner, with a maximal 2-3-fold effect. PMA similarly stimulated [14C]phosphatidylpropanol formation from propanol using membranes from [14C]myristic acid-prelabeled cells, confirming the activation of PLD. None of the effects described required exogenous ATP. To probe the role of phosphorylation in the PKC effect, we included high concentrations of apyrase in the assay. This ATPase had no effect on the ability of PKC to activate PLD, but under exactly the same conditions, it eliminated autophosphorylation of PKC. The results provide conclusive evidence for the involvement of PKC in the activation of PLD and suggest that ATP-dependent phosphorylation is not required.     

Phosphorylation of type II (beta) protein kinase C by casein kinase II.

Rat brain type II (beta) protein kinase C (PKC) was phosphorylated by rat lung casein kinase II (CK-II). Neither type I (gamma) nor type III (alpha) PKC was significantly phosphorylated by CK-II. CK-II incorporated 0.2-0.3 mol of phosphate into 1 mol of type II PKC. This phosphate was located at the single seryl residue (Ser-11) in the V1-variable region of the regulatory domain of the PKC molecule. A glutamic acid cluster was located at the carboxyl-terminal side of Ser-11, showing the consensus sequence for phosphorylation by CK-II. The velocity of this phosphorylation was enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which are all required for the activation of PKC. Phosphorylation of casein or synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or phosphatidylserine. Available evidence suggests that CK-II phosphorylates preferentially the activated form of type II PKC. It remains unknown, however, whether this reaction has a physiological significance.     

The phosphoinositide-dependent kinase, PDK-1, phosphorylates conventional protein kinase C isozymes by a mechanism that is independent of phosphoinositide 3-kinase

Phosphorylation by the phosphoinositide-dependent kinase, PDK-1, is required for the activation of diverse members of the AGC family of protein kinases, including the protein kinase C (PKC) isozymes. Here we explore the subcellular location of the PDK-1-mediated phosphorylation of conventional PKCs, and we address whether this phosphorylation is regulated by phosphoinositide 3-kinase. Pulse-chase experiments reveal that newly synthesized endogenous PKC alpha is primarily phosphorylated in the membrane fraction of COS-7 cells, where it is processed to a species that is phosphorylated at the activation loop and at two carboxyl-terminal positions. This "mature" species is then released into the cytosol. Deletion of the plekstrin homology domain of PDK-1 results in a 4-fold increase in the rate of processing of PKC indicating an autoinhibitory role for this domain. Autoinhibition by the plekstrin homology domain is not relieved by binding 3'-phosphoinositides; PKC is phosphorylated at a similar rate in serum-treated cells and serum-starved cells treated with the phosphoinositide 3-kinase inhibitors, LY294002 and wortmannin. Under the same conditions, the PDK-1-catalyzed phosphorylation of another substrate, Akt/protein kinase B, is abolished by these inhibitors. Our data are consistent with a model in which PDK-1 phosphorylates newly synthesized PKC by a mechanism that is independent of 3'-phosphoinositides.     

Diacylglycerol mediates the T-cell receptor-driven Ca(2+) influx in T cells by a novel mechanism independent of protein kinase C activation

The mechanism of Ca(2+) influx in nonexcitable cells is not known yet. According to the capacitative hypothesis, Ca(2+) influx is triggered by IP(3)-mediated Ca(2+) release from the intracellular Ca(2+) stores. Conversely, many workers have reported a lack of association between release and influx. In this work, the role of diacylglycerol (DAG) as the mediator of T-cell receptor (TCR)-driven Ca(2+) influx in T cells was investigated. Stimulation of mouse splenic T cells with naturally occurring DAG caused Ca(2+) entry in a dose- and time-dependent manner. Such stimulation was blocked by Ni(2+), a divalent cation known to block Ca(2+) channels. Inhibition of protein kinase C (PKC) by calphostin C did not inhibit, but slightly enhanced, the DAG-stimulated Ca(2+) entry. However, inhibition of DAG metabolism by DAG kinase and lipase inhibitors enhanced the DAG-stimulated Ca(2+) entry. DAG lipase and kinase inhibitors also enhanced the Ca(2+) entry in T cells stimulated through TCR/CD3 complex with anti-CD3 antibody. Calphostin C did not affect the anti-CD3-stimulated Ca(2+) entry. These results showed that TCR-driven Ca(2+) influx in T cells is mediated by DAG through a novel mechanism(s) independent of PKC activation.     

Induction of apoptosis by protein kinase C delta is independent of its kinase activity

Protein kinase C, a multigene family of phospholipid-dependent and diacylglycerol-activated Ser/Thr protein kinases, is a key component in many signal transduction pathways. The kinase activity was thought to be essential for a plethora of biological processes attributed to these enzymes. Here we show that at least one protein kinase C function, the induction of apoptosis by protein kinase C delta, is independent of the kinase activity. Stimulation of green fluorescent protein-protein kinase C delta fusion protein with phorbol ester or diacylglycerol led to its redistribution within seconds after the stimulus. Membrane blebbing, an early hallmark of apoptosis, was visible as early as 20 min after stimulation, and nuclear condensation was visible after 3-5 h. Apoptosis could be inhibited by expression of Bcl-2 but not by specific protein kinase C inhibitors. In addition, a kinase-negative mutant of protein kinase C delta also induced apoptosis to the same extent as the wild type enzyme. Apoptosis was confined to the protein kinase C delta-overexpressing cells. Stimulation of overexpressed protein kinase C epsilon did not result in increased apoptosis. Our results indicate that distinct protein kinase C isozymes induce apoptosis in vascular smooth muscle cells. More importantly, they show that some protein kinase C effector functions are independent of the catalytic activity.