Attenuation of hen granulosa cell steroidogenesis by a phorbol ester and 1-oleoyl-2-acetylglycerol

A series of studies was conducted to evaluate the effects of phorbol esters and a diacylglycerol analog on basal and hormone-stimulated steroidogenesis in granulosa cells from the largest preovulatory follicle of the domestic hen. Agents that previously have been shown to activate protein kinase C, such as the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the synthetic diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), suppressed luteinizing hormone (LH)-induced progesterone (PMA at levels of 10 and 100 ng/tube; OAG at levels of 10 and 25 micrograms/tube), and androgen (10 and 100 ng PMA; 25 micrograms OAG) production, but had no effect on basal levels of either steroid. Furthermore, PMA decreased the ability of vasoactive intestinal peptide to induce steroidogenesis, suggesting that protein kinase C activation may generally modulate the activity of hormones that act via the adenylyl cyclase/cyclic 3',5'-adenosine monophosphate (cAMP) second messenger system. In further support of this proposal was the finding that PMA and OAG decreased the production of cAMP in response to LH, and attenuated the steroidogenic response in granulosa cells exposed to 10 mM 8-bromo-cAMP. By contrast, the induction of calcium mobilization using a calcium ionophore (A23187; 0.5-2.0 microM) stimulated progesterone and androgen production without increasing intracellular levels of cAMP, and this stimulatory effect on steroidogenesis was not inhibited by the presence of 100 ng PMA/tube. From these data, we suggest that the activation of protein kinase C in granulosa cells of the hen may provide a physiological mechanism by which receptor-mediated steroidogenesis, involving the adenylyl cyclase second messenger system, is modulated.     

Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells.

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.     

Phorbol ester treatment inhibits thrombin but not stable GTP analogue-induced platelet granule secretion despite inhibition of phosphatidate formation with both agonists

Previous work has demonstrated that pre-treatment of platelets with phorbol esters that activate protein kinase C eg phorbol 12-myristate 13-acetate (PMA) results in an inhibition of inositol phospholipid breakdown and granule secretion induced by physiological agonists such as thrombin and collagen. In the present study, the effect of pre-treatment with PMA on granule secretion and [32P]-phosphatidate (PA) formation induced by the stable GTP analogue, guanosine 5'-[gamma thio] triphosphate (GTP gamma S) was examined in saponin-permeabilized platelets. A low concentration of PMA ie 1.6nM, that did not induce significant 5-hydroxytryptamine (5HT) secretion on its own, but inhibited low-dose thrombin-induced 5HT secretion totally and PA formation by 30-40% in intact as well as permeabilised platelets was chosen. Our results demonstrate a lack of inhibition of GTP gamma S (40 microM)-induced 5HT secretion by PMA in permeabilised platelets, despite significant inhibition (70%) of PA formation, suggesting that apart from the diacylglycerol pathway of secretion which may be common to thrombin and GTP analogues, secretion induced by physiological agonists such as thrombin may involve another mechanism that is inhibitable by phorbol esters.     

Phorbol myristate acetate receptors in human polymorphonuclear neutrophils

Resting or phorbol myristate acetate (PMA)-pretreated neutrophils were disrupted by nitrogen cavitation and were fractionated on Percoll density gradients to identify the subcellular location of PMA receptors. Receptors were found in the cytoplasm of resting cells; neither primary nor secondary granules bound [3H]PMA, and the few binding sites located in non-granule membrane fractions appeared to reflect cytosolic contamination. Contrastingly, PMA-pretreated cells lost cytosolic receptors; greater than 80% of PMA-binding sites were associated with non-granule membranes. Protein kinase C activity similarly shifted from cytosol to membranes after PMA treatment. Indeed, protein kinase C and PMA receptors co-sedimented on Percoll gradients, co-eluted from Ultragel AcA 44 columns loaded with neutrophil cytoplasm, and were identically influenced by various phospholipids. Finally, PMA, mezerein, diacylglycerol, and dialkylglycerol activated protein kinase C with potencies that paralleled their respective abilities to stimulate neutrophil aggregation responses and inhibit [3H]PMA binding to whole cells or cytosol. These results fit a model of stimulus-response coupling wherein exogenous PMA or endogenous diacylglycerol solvate in cellular membranes. Cytosolic protein kinase C binds to the intramembranous ligand, forming an active, membrane-associated complex that phosphorylates nearby elements involved in triggering aggregation and other responses.     

Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils

Human neutrophils treated with phorbol 12-myristate 13-acetate (PMA) or dioctanoylglycerol exhibited a large (10-fold), sustained accumulation of the mass of diradylglycerol, beginning 1 min after stimulation and continuing for 30 to 60 min. Phorbol dibutyrate was less potent than PMA in stimulating diradylglycerol accumulation, whereas the 4-alpha analogs of PMA and phorbol dibutyrate were inactive. Submaximal concentrations of PMA (0.5 to 2.5 nM) plus the calcium ionophore, ionomycin (15 to 60 nM), led to synergistic accumulation of diradylglycerols. Chlorpromazine and sphingosine, inhibitors of protein kinase C, blocked PMA-stimulated accumulation of diradylglycerol with IC50 concentrations of 32 and 9 microM, respectively, paralleling their inhibition of PMA-stimulated O2- production. These compounds also inhibited the ionomycin-stimulated accumulation of diradylglycerols. A third protein kinase C inhibitor, H-7, was less effective, inhibiting PMA-stimulated accumulation of diradylglycerol by 25% at 100 microM. Differential sensitivity to alkaline hydrolysis suggests that diradylglycerols that accumulate in response to PMA or ionomycin stimulation are composed of a mixture of two distinct diglyceride species, diacylglycerols and alkylacylglycerols. Whereas diacylglycerol may activate cellular protein kinase C, the importance of the production of alkylacylglycerols is uncertain.     

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.     

Inhibitory effect of the intrauterine infusion of phorbol 12-myristate 13-acetate and 1-oleoyl-2-acetylglycerol on the decidual cell reaction in rats

In rats, prostaglandins (PGs) have an essential role in the decidual cell reaction (DCR), but their mechanism of action at the cellular level within the endometrium is at present uncertain. To test the hypothesis that both protein kinase C activation and calcium mobilization mediate the action of PGs within the endometrium during decidualization, the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the synthetic diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG), activators of protein kinase C in vitro, and the calcium ionophore A23187, which causes calcium mobilization, were infused, alone or combined, into the uterine lumen of rats sensitized for the DCR. The results obtained indicate that both PMA and OAG have an inhibitory effect on the DCR in rats. The calcium ionophore A23187, although having no apparent effect by itself, had a synergistic effect with PMA, but not with OAG, in inhibiting the DCR. The intrauterine infusion of PMA and/or A23187 had no effect on the increase in endometrial vascular permeability (EVP), which precedes the DCR. The inhibitory effect of PMA or PMA plus A23187 on decidualization is probably not mediated by a decrease in uterine PG synthesis, as assessed by the measurement of uterine prostaglandin E concentrations at various times during the intraluminal infusion. These data suggest that activation of protein kinase C can modulate the DCR.     

Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.     

Individual C1 domains of PKD3 in phorbol ester-induced plasma membrane translocation of PKD3 in intact cells

Protein kinase D3 is a novel member of the serine/threonine kinase family PKD. The regulatory region of PKD contains a tandem repeat of C1 domains designated C1a and C1b that bind diacylglycerol and phorbol esters, and are important membrane targeting modules. Here, we investigate the activities of individual C1 domains of PKD3 and their roles in phorbol ester-induced plasma membrane translocation of PKD3. Truncated C1a of PKD3 binds [(3)H]phorbol 12, 13-dibutyrate with high affinity, but no binding activity is detected for C1b. Meanwhile, mutations in C1a of truncated C1ab of PKD3 lead to the loss of binding affinity, while these mutations in C1b have little impact, indicating that C1a is responsible for most of the phorbol ester-binding activities of PKD3. C1a and C1b of the GFP-tagged full length PKD3 are then mutated to assess their roles in phorbol ester-induced plasma membrane translocation in intact cells. At low concentration of phorbol 12-myristate 13-acetate (PMA), the plasma membrane translocations of the C1a and C1ab mutants are significantly impaired, reflecting an important role of C1a in this process. However, at higher PMA concentrations, all C1 mutants exhibit increased rates of translocation as compared to that of wild-type PKD3, which parallel their enhanced activation by PMA, implying that PKD3 kinase activity affects membrane targeting. In line with this, a constitutive active PKD3-GFP translocates similarly as wild-type PKD3, while a kinase-inactive PKD3 shows little translocation up to 2 muM PMA. In addition, RO 31-8220, a potent PKC inhibitor that blocks PMA-induced PKD3 activation in vivo, significantly attenuates the plasma membrane translocation of wild-type PKD3 at different doses of PMA. Taken together, our results indicate that both C1a and the kinase activity of PKD3 are necessary for the phorbol ester-induced plasma membrane translocation of PKD3. PKC, by directly activating PKD3, regulates its plasma membrane localization in intact cells.     

Contrasting effects of phorbol dibutyrate and phorbol myristate acetate in rabbit aorta

In rat hepatocytes, active phorbol esters inhibited the alpha 1-adrenergic stimulation of phosphatidylinositol labeling with the expected potency order: phorbol myristate acetate (PMA) greater than phorbol dibutyrate (PDB). In contrast, in rabbit aorta the alpha 1-adrenergic action was inhibited dose-dependently by PDB but not by PMA. Similarly PDB (but not PMA) induced a strong contraction in rabbit aorta. The phorbol ester-induced contraction developed slowly, was dose-dependent and independent of extracellular calcium. These effects of PDB in rabbit aorta were neither inhibited by the protein kinase inhibitor H-7 nor mimicked by the synthetic diacylglycerol, OAG. Our results raise some doubts on the mechanism(s) through which the actions of PDB take place in rabbit aorta.     

Identification of an autoinhibitory mechanism that restricts C1 domain-mediated activation of the Rac-GAP alpha2-chimaerin

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in alpha2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type alpha2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize alpha2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the alpha2-chimaerin C1 domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders alpha2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of alpha2-chimaerin in a phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-alpha2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for epidermal growth factor-induced alpha2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of alpha2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, alpha2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.     

Green Fluorescent Protein (GFP)-tagged Cysteine-rich Domains from Protein Kinase C as Fluorescent Indicators for Diacylglycerol Signaling in Living Cells

Cysteine-rich domains (Cys-domains) are ~50–amino acid–long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-γ (Cys1–GFP). Strikingly, stimulation of G-protein or tyrosine kinase–coupled receptors induced a transient translocation of cytosolic Cys1–GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1–GFP in the membrane, whereas DiC8 left Cys1–GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1–GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-γ also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2–GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester–mediated translocation of proteins to selective lipid membranes.     

Endothelin stimulates diacylglycerol accumulation and activates protein kinase C in cultured vascular smooth muscle cells

Endothelin, a novel peptide isolated from the conditioned medium of endothelial cells, causes a slow, sustained contraction of vascular smooth muscle, but its mechanism of action remains unclear. To determine whether the diacylglycerol/protein kinase C signalling pathway is stimulated by endothelin, we exposed cultured rat aortic smooth muscle cells to endothelin and measured diacylglycerol accumulation and protein kinase C-dependent protein phosphorylation. Endothelin stimulated a dose-dependent, biphasic increase in diacylglycerol, which was sustained for at least 20 min. This peptide also induced a prolonged phosphorylation of an acidic protein with a molecular weight of 76,000, which was detectable by 30 s and sustained for at least 20 min. This phosphorylation could be mimicked by phorbol 12-myristate 13-acetate, but not by ionomycin, and was markedly reduced when protein kinase C was down-regulated by a 24-h pretreatment with phorbol 12,13-dibutyrate. These results suggest that endothelin causes a robust stimulation of the diacylglycerol/protein kinase C pathway in cultured vascular smooth muscle cells, and that this mechanism may contribute importantly to the physiologic events stimulated by endothelin in intact blood vessels, including slow, tonic contraction and Ca2+ influx.     

Tumor promoter phorbol myristate acetate inhibits Ca2+ influx through voltage-gated Ca2+ channels in two secretory cell lines, PC12 and RINm5F

Protein kinase C is known to be involved both in initiation and termination of cellular responses due to phosphoinositide breakdown. Here we report that in PC12 cells (a line of neurosecretory cells derived from a rat pheochromocytoma), pretreatment with nanomolar concentrations of phorbol myristate acetate, PMA, which is believed to specifically activate protein kinase C, inhibits the cytosolic-free Ca2+ concentration rise induced by depolarizing agents. In contrast, plasma membrane potential and 45Ca efflux from preloaded cells were unaffected by PMA pretreatment. Inhibition by PMA and diacylglycerol of the cytosolic-free Ca2+ concentration rise induced by depolarization was observed also in another cell line, the insulin secreting line RINm5F. These results raise the possibility that the voltage-gated Ca2+ channel is under inhibitory control by protein kinase C.     

Differential effects of phorbol ester on phenylephrine and vasopressin-induced Ca2+ mobilization in isolated hepatocytes

Receptor-mediated breakdown of PtdIns(4,5)P2 produces two cellular signals, Ins(1,4,5)P3, which can release intracellular Ca2+, and diacylglycerol, which activates a Ca2+- and phospholipid-dependent protein kinase (protein kinase C). This study assesses the significance of protein kinase C in relation to phenylephrine- and vasopressin-induced Ca2+ mobilization in hepatocytes. Phorbol ester (4 beta-phorbol-12-myristate-13-acetate), which can directly activate protein kinase C, had no effect either on Ca2+ efflux from the cell (measured with arsenazo III) or on Ca2+ influx (measured with Quin-2), processes which are inhibited and stimulated, respectively, by both phenylephrine and vasopressin. No evidence of synergism between phorbol ester pretreatment of hepatocytes and the Ca2+ ionophore (ionomycin)-mediated effects on the increase of cytosolic free Ca2+ and phosphorylase activation could be obtained. These findings suggest that protein kinase C is not obligatorily involved in the regulation of hepatocyte Ca2+ fluxes. Pretreatment of hepatocytes with phorbol ester (PMA) or 1-oleoyl-2-acetylglycerol totally inhibited the effects of phenylephrine in elevating the cytosolic free Ca2+; half-maximal inhibitory effects occurred at PMA and 1-oleoyl-2-acetylglycerol concentrations of 1 ng/ml and 12 micrograms/ml, respectively. In contrast, pretreatment with PMA had a much smaller effect on Ca2+ mobilization induced by vasopressin. These observations suggest that protein kinase C may be involved in "down-regulation" of the alpha 1-receptor in hepatocytes and may thus exert a negative influence on the Ca2+-signalling pathway.     

Contribution of the C1A and C1B domains to the membrane interaction of protein kinase C

The hallmark for protein kinase C activation is its "translocation" to membranes following generation of lipid second messengers. This translocation is mediated by the C1 and C2 domains, two membrane-targeting modules, whose engagement on membranes provides the energy for an activating conformational change in which an autoinhibitory pseudosubstrate sequence is released from the active site. Novel and conventional protein kinase C isozymes contain a tandem repeat of C1 domains, the C1A and C1B, which each contain a binding pocket for phorbol esters/diacylglycerol. This study addresses the contribution of the C1A and C1B domains in the regulation of protein kinase C's membrane interaction using bisfunctional (dimeric) phorbol myristate acetate (PMA) molecules. We show that dimeric bisphorbols are an order of magnitude more effective at recruiting full-length PKC betaII to membranes compared with monomeric PMA and that the effectiveness of the interaction depends on the nature and length of the cross-link between the PMA moieties. Most effective were dimeric phorbol 12-acetate 13-esters linked at the 13 position with a 14 carbon spacer. The increased potency of dimeric phorbol esters is reduced if either the C1A or C1B domains are mutated so that they are unable to bind PMA, if one moiety of the dimer contains a nonfunctional phorbol, or if the binding to the isolated C1B domain is measured. Thus, the increased potency of the dimeric phorbol esters results primarily from their ability to engage, to a limited extent, both C1 modules on the same molecule. Although dimeric phorbols were more potent than monomeric phorbol esters in recruiting protein kinase C to membranes, the magnitude of the increase was still several orders of magnitude lower than what would be predicted on the basis of the reduction in dimensionality that occurs when the first C1 domain is engaged on the membrane. Thus, engaging both domains can be forced but is highly unfavored. In summary, our data reveal that both C1 domains are oriented for potential membrane interaction but only one C1 domain binds ligand in a physiological context.     

Phosphatidylcholine hydrolysis stimulated by phorbol myristate acetate is mediated principally by phospholipase D in endothelial cells

The mechanism of phosphatidylcholine (PC) degradation stimulated by phorbol myristate acetate (PMA) was investigated in bovine pulmonary artery endothelial cells prelabeled with [methyl-3H]choline ([3H]choline) or [9,10-3H]myristic acid ([3H]myristic acid). Both labels were selectively incorporated into PC, and addition of PMA stimulated comparable losses of 3H from PC in cells prelabeled with [3H]choline or [3H]myristate. In cells prelabeled with [3H]choline, the loss of 3H from PC correlated with a rapid increase in intracellular free [3H]choline. The increase in intracellular [3H]choline stimulated by PMA was not preceded by an increase in any other 3H-labeled PC degradation product. PMA did not stimulate the formation of PC deacylation products in cells prelabeled with [3H]choline. In permeabilized cells prelabeled with [3H]choline, PMA stimulated the formation of [3H]choline but not [3H]phosphocholine. In intact cells prelabeled with [3H]myristate, the loss of 3H from PC induced by PMA correlated with the formation of [3H]phosphatidic acid ([3H]PA) and [3H]diacylglycerol. In the presence of ethanol, PMA stimulated the formation of [3H]phosphatidylethanol ([3H]PEt) at the expense of [3H]PA. The time-course of [3H]PEt formation was similar to the time-course of intracellular [3H]choline formation in cells stimulated with PMA. These data taken together support the notion that PC degradation in endothelial cells stimulated with PMA is mediated principally by phospholipase D. PC breakdown via phospholipase D was not observed in cells treated with phorbol esters incapable of interacting with protein kinase C. Activation of phospholipase D by phorbol esters was inhibited by long-term pretreatment of cells with PMA to down-regulate protein kinase C and by pretreatment of the cells with staurosporine. These data support the notion that activation of phospholipase D by phorbol esters is dependent upon protein kinase C.     

Conserved domains subserve novel mechanisms and functions in DKF-1, a Caenorhabditis elegans protein kinase D

Protein kinase D (PKD) isoforms are effectors in signaling pathways controlled by diacylglycerol. PKDs contain conserved diacylglycerol binding (C1a, C1b), pleckstrin homology (PH), and Ser/Thr kinase domains. However, the properties of conserved domains may vary within the context of distinct PKD polypeptides. Such functional/structural malleability (plasticity) was explored by studying Caenorhabditis elegans D kinase family-1 (DKF-1), a PKD that governs locomotion in vivo. Phorbol ester binding with C1b alone activates classical PKDs by relieving C1-mediated inhibition. In contrast, C1a avidly ligated phorbol 12-myristate 13-acetate (PMA) and anchored DKF-1 at the plasma membrane. C1b bound PMA (moderate affinity) and cooperated with C1a in targeting DKF-1 to membranes. Mutations at a "Pro(11)" position in C1 domains were inactivating; kinase activity was minimal at PMA concentrations that stimulated wild type DKF-1 approximately 10-fold. DKF-1 mutants exhibited unchanged, maximum kinase activity after cells were incubated with high PMA concentrations. Titration in situ revealed that translocation and activation of wild type and mutant DKF-1 were tightly and quantitatively linked at all PMA concentrations. Thus, C1 domains positively regulated phosphotransferase activity by docking DKF-1 with pools of activating lipid. A PH domain inhibits kinase activity in classical PKDs. The DKF-1 PH module neither inhibited catalytic activity nor bound phosphoinositides. Consequently, the PH module is an obligatory, positive regulator of DKF-1 activity that is compromised by mutation of Lys(298) or Trp(396). Phosphorylation of Thr(588) switched on DKF-1 kinase activity. Persistent phosphorylation of Thr(588) (activation loop) promoted ubiquitinylation and proteasome-mediated degradation of DKF-1. Each DKF-1 domain displayed novel properties indicative of functional malleability (plasticity).