Agonist-independent phosphorylation of purified cardiac muscarinic cholinergic receptors by protein kinase C

The results of several studies have suggested that muscarinic cholinergic receptors (mAChR) may be regulated by multiple pathways involving phosphorylation of the receptors. Previous studies have demonstrated that chick heart mAChR are phosphorylated by the beta-adrenergic receptor kinase (beta-AR kinase) in an agonist-dependent manner, and it has been suggested that this process may be linked to receptor desensitization. In this work, we present evidence that protein kinase C can phosphorylate the purified, reconstituted chick heart mAChR and can modify the interaction of the receptors with GTP binding proteins (G-proteins) that couple the receptors to effectors. Phosphorylation of the mAChR with protein kinase C occurred to an extent of approximately 5 mol of P/mol of receptor. Neither the rate nor the extent of the protein kinase C mediated phosphorylation of mAChR was agonist-dependent. Under the conditions tested, the initial rate of phosphorylation of the mAChR by protein kinase C was significantly more rapid than that obtained with the beta-AR kinase. At equilibrium, phosphorylation of mAChR by protein kinase C and beta-AR kinase was partially additive. The functional effects of protein kinase C mediated phosphorylation of the mAChR were assessed by comparing the abilities of purified G-proteins (Gi and Go) to reconstitute high-affinity agonist binding to phosphorylated and nonphosphorylated receptors. A significantly larger percentage of the receptors phosphorylated with protein kinase C exhibited G-protein-dependent high-affinity agonist binding, suggesting that phosphorylation of the receptors by protein kinase C modulates receptor function in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complex role of protein kinase C in mediating the supramaximal inhibition of pancreatic secretion observed with cholecystokinin.

Protein kinase C appears to play an important, yet complex role in the supramaximal inhibition of pancreatic acinar cell secretion observed in response to cholecystokinin (CCK). The addition of protein kinase C activation to the concentration-response curve of a partial agonist acting at the CCK receptor (a phenethyl ester analogue of CCK), transforms a curve without supramaximal inhibition to a full agonist curve typical of CCK. This effect can be elicited by low concentrations of phorbol ester (50pM to 1nM 12-0-tetradecanoyl-phorbol-13-acetate) or by hormonal agonists (0.1 microM carbamylcholine, 10pM bombesin, 1pM CCK-8) which activate protein kinase C, but not by agonists acting via alternate second messengers (VIP). Of interest, this effect is dependent on preincubation of the acinar cells with the protein kinase C activator at 37 degrees C, with the effect rapidly reversed by transient exposure of the cells to lower temperature. This is consistent with mediation by a phosphorylation event. However, the requirement for an extended (greater than 15 min) preincubation period when using minimal kinase activation suggests that this phenomenon is more complicated than a simple bimolecular phosphorylation event and likely includes a series of events such as translocation of substrates and/or enzymes involved.     

Secretagogues differentially activate endoplasmic reticulum stress responses in pancreatic acinar cells

Endoplasmic reticulum (ER) stress leads to the accumulation of misfolded proteins in the ER lumen and initiates the unfolded protein response (UPR). Components of the UPR are important in pancreatic development, and recent studies have indicated that the UPR is activated in the arginine model of acute pancreatitis. However, the effects of secretagogues on UPR components in the pancreas are unknown. The present study aimed to examine the effects of different types and concentrations of secretagogues on acinar cell function and specific components of the UPR. Rat pancreatic acini were stimulated with the CCK analogs CCK8 (10 pM-10 nM) or JMV-180 (10 nM-10 microM) or with bombesin (1-100 nM). Components of the UPR, including chaperone BiP expression, PKR-like ER kinase (PERK) phosphorylation, X box-binding protein 1 (XBP1) splicing, and CCAAT/enhancer binding protein homologous protein (CHOP) expression, were measured, as were effects on amylase secretion and intracellular trypsin activation. CCK8 generated a biphasic secretion dose-response curve, and high concentrations increased intracellular active trypsin levels. In contrast, JMV-180 and bombesin secretion dose-response curves were monophasic, and high concentrations did not increase intracellular trypsin activity. All three secretagogues increased BiP levels and XBP1 splicing. However, only supraphysiological levels of CCK8 associated with inhibited amylase secretion and trypsin activation stimulated PERK phosphorylation and expression of CHOP. The effects of CCK8 on UPR components were rapid, occurring within 5-20 min. In conclusion, ER stress response mechanisms appear to be involved in both pancreatic physiology and pathophysiology, and future efforts should be directed at understanding the roles of these mechanisms in the pancreas.     

Cholecystokinin inhibits phosphatidylcholine synthesis via a Ca(2+)-calmodulin-dependent pathway in isolated rat pancreatic acini. A possible mechanism for diacylglycerol accumulation.

The effects of cholecystokinin (CCK) and other pancreatic secretagogues on phosphatidylcholine (PC) synthesis were studied in isolated rat pancreatic acini. When acini were incubated with [3H]choline in the presence of 1 nM CCK-octapeptide (CCK8) for 60 min, the incorporations of [3H]choline into both water-soluble choline metabolites and PC in acini were reduced by CCK8 to 74 and 41% of control, respectively. Pulse-chase study revealed that CCK8 reduced both the disappearance of phosphocholine and the synthesis of PC. Other Ca(2+)-mobilizing secretagogues such as carbamylcholine, bombesin, and Ca2+ ionophore A23187 also reduced PC synthesis to the same extent as did CCK8. When combined with 1 nM CCK8, A23187 or carbamylcholine did not further inhibit PC synthesis. Furthermore, W-7 or W-5, a calmodulin antagonist, reversed the inhibition by CCK8 of PC synthesis, suggesting that a Ca(2+)-calmodulin-dependent pathway may be involved in CCK-induced inhibition of PC synthesis in acini. By contrast, neither cAMP-dependent secretagogues such as secretin and dibutyryl cAMP nor a phorbol ester had any effect on PC synthesis in acini. Staurosporine or H-7, a protein kinase C inhibitor, did not affect the inhibition by CCK of PC synthesis. The analysis of enzyme activity involved in PC synthesis via CDP-choline pathway showed that CCK treatment of acini reduced CTP:phosphocholine cytidylyltransferase activity in both cytosolic and particulate fraction, a finding consistent with the delayed disappearance of phosphocholine induced by CCK in pulse-chase study. By contrast, CCK treatment of acini did not alter the activities of choline kinase and phosphocholine transferase in acini. The extent of inhibition by CCK of cytidylyltransferase activity became much larger when subcellular fractions of acini were prepared in the presence of phosphatase inhibitors. In addition, W-7 reversed the inhibitory effect of CCK treatment on cytidylyltransferase activity in acini. When acini were labeled with [3H]myristic acid and chased, CCK8 (1 nM) reduced the synthesis of [3H]myristic acid-labeled PC to 27% of control after a 60-min chase period. This inhibition of PC synthesis induced by CCK was accompanied by a delayed disappearance of [3H]diacylglycerol, the radioactivity of which was 225% of control at 60 min. These results indicate that CCK inhibits PC synthesis by inducing both the reduction of choline uptake into acini and the inhibition of CTP:phosphocholine cytidylyltransferase activity. Furthermore, the results suggest the possibility that the activation of Ca(2+)-calmodulin-dependent kinase in response to CCK may phosphorylate cytidylyltransferase thereby decreasing this enzyme activity in pancreatic acinar cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Residual inhibition of epidermal growth factor binding by pancreatic secretagogues and phorbol ester in rat pancreas

Cholecystokinin-octapeptide (CCK8) inhibits 125I-labeled epidermal growth factor (EGF) cell-associated radioactivity in pancreatic acini, ostensibly as a result of its ability to mobilize cellular Ca2+. The phorbol ester tetradecanoyl phorbol acetate (TPA), a compound that activates protein kinase C, mimics the inhibitory action of CCK8. In the present study we examined the relationship between occupancy of the cholecystokinin (CCK) receptor, the subsequent inhibition of EGF binding, and the potential role of C-kinase activation in mediating this inhibition. Proglumide and dibutyryl cyclic GMP (dbGMP), two distinct competitive antagonists of CCK8, reversed the inhibitory actions of CCK8. Analysis of steady-state saturation kinetics of 125I-EGF binding indicated that CCK8 decreased the apparent affinity of the EGF receptor, mainly as a result of a marked decrease in the amount of internalized ligand. TPA also inhibited 125I-EGF internalization. Removal of CCK8 and TPA from incubation medium did not abolish their inhibitory actions. Carbachol, but not bombesin, exerted a similar residual inhibitory effect. It is suggested that in addition to acting via Ca2+, certain pancreatic secretagogues may also act through C-kinase to regulate EGF binding.     

Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.     

Receptor-operated Ca2+ influx and its association with the Src family in secretagogue-stimulated pancreatic acini

We investigated signal transduction between receptor-operated Ca(2+) influx (ROCI) and Src-related nonreceptor protein tyrosine kinase (PTK) in rat pancreatic acini. CCK and the Ca(2+) ionophore enhanced the Src-related PTK activity, whereas the high-affinity CCK-A receptor agonists, fibroblast growth factor (FGF), and the protein kinase C (PKC) activator had no or little effect. This increase was abolished by eliminating [Ca(2+)](o), loading of the intracellular Ca(2+) chelator, and administering the PTK inhibitor genistein. While genistein inhibited extracellular Ca(2+) or Mn(2+) entry induced by CCK and carbachol, it did not affect intracellular Ca(2+) release and oscillations. CCK dose-dependently increased the Src phosphotransferase activity, which was abolished by inhibitors of G(q) protein, phospholipase C (PLC), and Src, but not by the calmodulin kinase (CaMK) inhibitor. Intensities of the Src band and amounts of tyrosine phosphorylated Src were enhanced by CCK stimulation. Thus, Src cascades appear to be coupled to the low-affinity CCK-A receptor and utilize G(q)-PLC pathways for their activation, independent of PKC and CaMK cascades. The low-affinity CCK-A receptor regulates ROCI via mediation of Src-related PTK and activates Src pathways to cause [Ca(2+)](o)-dependent pancreatic exocytosis.     

Coincident regulation of PKCdelta in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling

PKC (protein kinase C)d plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCd. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCd with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCd is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCd. Both thrombin and PMA induce recruitment of PKCd to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCd, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCd is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity.     

Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C

We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.     

Activation of protein kinase C accelerates internalization of transferrin receptor but not of major histocompatibility complex class I, independent of their phosphorylation status.

Phosphorylation of membrane glycoproteins has often been invoked as a determinant of receptor internalization and receptor trafficking in a more general sense. Here we have studied the trafficking of major histocompatibility complex (MHC) Class I molecules and transferrin receptor (Tfr) related to their phosphorylation status in the human lymphoblastoid cell line JY. High resolution isoelectric focusing (IEF) allows the visualization of phosphorylated and non-phosphorylated protein species simultaneously, using protein backbone-labeling. Analysis on IEF was combined with a neuraminidase protection assay, in which sialic acid modification of the N-linked glycans present on Tfr and Class I molecules is used as a reporter group for cell surface expression. Phosphorylation of Class I heavy chains and Tfr was induced by exposure of cells to the phorbol ester tetradecanoyl phorbol acetate. We show that 1) phosphorylation of MHC Class I molecules is restricted to the cell surface fraction, 2) phosphorylation of MHC Class I molecules by protein kinase C (PKC) is not correlated with their internalization, as no internalization of Class I molecules, phosphorylated or non-phosphorylated, could be detected, 3) the initial rate, but not the final extent of the internalization of Tfr is affected by activation of PKC, and 4) phosphorylated Tfr behaves in a manner identical to non-phosphorylated Tfr in terms of internalization. The effect of activation of PKC on internalization of Tfr therefore most likely takes place at the level of the internalization machinery. Our data concerning the internalization of MHC Class I molecules contrast with earlier studies describing constitutive internalization in the B lymphoblastoid cell line A 46 and in HPB-ALL cells.     

Cholecystokinin and EGF activate a MAPK cascade by different mechanisms in rat pancreatic acinar cells

The effects of activating the G_qprotein-coupled cholecystokinin (CCK) receptor on differentproteins/signaling molecules in the mitogen-activated protein kinase(MAPK) cascade in pancreatic acinar cells were analyzed and comparedwith the effects of activating the tyrosine kinase-coupled epidermalgrowth factor (EGF) receptor. Both EGF and CCK octapeptide rapidlyincreased the activity of the MAPKs [extracellular signal-regulatedkinase (ERK) 1 and ERK2], reaching a maximum within 2.5 min when 3.9- and 8.5-fold increases, respectively, were observed. The EGF-inducedincrease of MAPK activity was transient, with only a slight elevationafter 30 min, whereas CCK-stimulated MAPK remained at a high level ofactivation to 60 min. The protein kinase C inhibitor GF-109203Xabolished the activation by phorbol ester and inhibited the effect ofCCK by 78% but had no effect on EGF-activated MAPK activity. EGF and CCK activated both forms of MAPK kinase (MEK), with CCK having a muchlarger effect, activating MEK1 by 6-fold and MEK2 by 10-fold, whereasEGF activated both MEKs by only 2-fold. Immunoblotting revealed threedifferent forms of Raf in pancreatic acinar cells. Of the total basalRaf kinase activity, 3.7% was Raf-A, 89.0% was Raf-B, and 7.3% wasc-Raf-1. All three forms of Raf were stimulated to a greater extent byCCK than by EGF, which was especially evident for Raf-A and c-Raf-1.The effect of CCK in activating Rafs was at least partially mimicked bystimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. EGF significantlyincreased GTP-bound Ras by 183 and 164% at 2.5 and 10 min,respectively; CCK and TPA had no measurable effect. Our study suggeststhat CCK and EGF activate the MAPK cascade by distinct mechanisms in pancreatic acinar cells.

     

Phosphorylation of nerve growth factor receptor proteins in sympathetic neurons and PC12 cells. In vitro phosphorylation by the cAMP-independent protein kinase FA/GSK-3

We have examined phosphorylation of nerve growth factor (NGF) receptor in cultured sympathetic neurons and PC12 cells. Dissociated rat superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and NGF receptor proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that NGF receptor components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or phorbol 12-myristate 13-acetate. When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only phosphoserine; the other peptide contained phosphoserine and phosphothreonine. No phosphotyrosine was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P]ATP and the cAMP-independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on serine and to a lesser extent on threonine. However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by cAMP-dependent protein kinase, casein kinase II, or protein kinase C (the Ca2+/phospholipid-dependent enzyme). We conclude that binding units of the NGF receptor are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro.     

MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase.

A 'MAP kinase activator' was purified several thousand-fold from insulin-stimulated rabbit skeletal muscle, which resembled the 'activator' from nerve growth factor-stimulated PC12 cells in that it could be inactivated by incubation with protein phosphatase 2A, but not by protein tyrosine phosphatases and its apparent molecular mass was 45-50 kDa. In the presence of MgATP, 'MAP kinase activator' converted the normal 'wild-type' 42 kDa MAP kinase from an inactive dephosphorylated form to the fully active diphosphorylated species. Phosphorylation occurred on the same threonine and tyrosine residues which are phosphorylated in vivo in response to growth factors or phorbol esters. A mutant MAP kinase produced by changing a lysine at the active centre to arginine was phosphorylated in an identical manner by the 'MAP kinase activator', but no activity was generated. The results demonstrate that 'MAP kinase activator' is a protein kinase (MAP kinase kinase) and not a protein that stimulates the autophosphorylation of MAP kinase. MAP kinase kinase is the first established example of a protein kinase that can phosphorylate an exogenous protein on threonine as well as tyrosine residues.     

Ca2+-dependent and cAMP-dependent control of nicotinic acetylcholine receptor phosphorylation in muscle cells

Mouse BC3H1 myocytes were incubated with 32Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the 32P found in the receptor was bound to the delta subunit. Two phosphorylation sites in this subunit were resolved by reverse phase high performance liquid chromatography after exhaustive proteolysis of the protein with trypsin. Sites 1 and 2 were phosphorylated to approximately the same level in control cells. The divalent cation ionophore, A23187, increased 32P in site 1 by 40%, but did not affect the 32P content of site 2. In contrast, isoproterenol increased 32P in site 2 by more than 60%, while increasing 32P in site 1 by only 20%. When dephosphorylated receptor was incubated with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase, the delta subunit was phosphorylated to a maximal level of 1.6 phosphates/subunit. Approximately half of the phosphate went into site 2, with the remainder going into a site not phosphorylated in cells. The alpha subunit was phosphorylated more slowly, but phosphorylation of both alpha and delta subunits was blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. Phosphorylation of the receptor was also observed with preparations of phosphorylase kinase. In this case phosphorylation occurred in the beta subunit and site 1 of the delta subunit, neither of which were phosphorylated by cAMP-dependent protein kinase. The rate of receptor phosphorylation by phosphorylase kinase was slow relative to that catalyzed by cAMP-dependent protein kinase. Therefore, it can not yet be concluded that phosphorylase kinase phosphorylates the beta subunit and the delta subunit site 1 in cells. However, the results strongly support the hypothesis that phosphorylation by cAMP-dependent protein kinase accounts for phosphorylation of the alpha subunit and the delta subunit site 2 in response to elevations in cAMP.     

Mechanism of phorbol diester-induced regulation of surface transferrin receptor involves the action of activated protein kinase C and an intact cytoskeleton

Phorbol diesters are tumor-promoting agents that cause differentiation of HL60 human leukemic cells and concomitantly regulate surface transferrin receptors. Regulation of transferrin receptors by phorbol diesters involves receptor internalization in association with increased receptor phosphorylation (hyperphosphorylation). The intracellular mechanism of action of phorbol diester involves binding to and activation of the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Present studies comparing results obtained with whole cells and those from a cell-free system reconstituted from purified protein kinase C and transferrin receptor components have revealed that the transferrin receptor is phosphorylated by protein kinase C activated by phorbol esters. Following tryptic digestion and two-dimensional separation of phosphopeptides of phosphorylated transferrin receptors, two major and several minor phosphoserine-containing fragments are resolved. These fragments are identical whether transferrin receptor is phosphorylated in whole cells incubated with phorbol diesters or following phosphorylation of affinity immobilized transferrin receptor in the in vitro reconstitution system. Phosphoamino acid analysis of these fragments indicates that serine is the only amino acid phosphorylated in whole cells or in the cell-free system. In addition, colchicine is shown to inhibit in a dose-dependent manner phorbol diester-induced internalization but not hyperphosphorylation of the surface transferrin receptor in whole cells. This inhibition is specific for colchicine since inactive beta- and gamma-Lumicolchicine have no such effect, while taxol reverses the inhibition. These results indicate that the phorbol diester-mediated process of down-regulation of the surface transferrin receptor is associated with phosphorylation of the receptor by activated protein kinase C and requires an intact cytoskeleton to affect receptor internalization.     

Mechanism of activation of protein kinase D2(PKD2) by the CCK(B)/gastrin receptor

Recently, we cloned a novel serine/threonine kinase termed protein kinase D2 (PKD2). PKD2 can be activated by phorbol esters both in vivo and in vitro but also by gastrin via the cholecystokinin/CCK(B) receptor in human gastric cancer cells stably transfected with the CCK(B)/gastrin receptor (AGS-B cells). Here we identify the mechanisms of gastrin-induced PKD2 activation in AGS-B cells. PKD2 phosphorylation in response to gastrin was rapid, reaching a maximum after 10 min of incubation. Our data demonstrate that gastrin-stimulated PKD2 activation involves a heterotrimeric G alpha(q) protein as well as the activation of phospholipase C. Furthermore, we show that PKD2 can be activated by classical and novel members of the protein kinase C (PKC) family such as PKC alpha, PKC epsilon, and PKC eta. These PKCs are activated by gastrin in AGS-B cells. Thus, PKD2 is likely to be a novel downstream target of specific PKCs upon the stimulation of AGS-B cells with gastrin. Our data suggest a two-step mechanism of activation of PKD2 via endogenously produced diacylglycerol and the activation of PKCs.     

Phorbol ester- and protein kinase C-mediated phosphorylation of the cellular Kirsten ras gene product

The effect of phorbol 12-myristate 13-acetate on the phosphorylation of the ras p21 protein was studied by metabolically labeling cultured cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. Phorbol 12-myristate 13-acetate (100 nM) induced phosphorylation of cKi-ras p21 in a mouse adrenocortical cell line (Yl) expressing high levels of cKi-ras with exon 4B. Phosphorylation was detected at 10 min and was maximal at 2 h. The ras protein was not phosphorylated in response to phorbol 12-myristate 13-acetate in NIH 3T3 cells expressing activated cHa-ras or vHa-ras. In vitro, protein kinase C phosphorylated cKi-ras in a phosphatidylserine and diolein-dependent manner. Both in intact cells and in vitro the amino acid phosphorylated was serine. Analysis of p21 from NIH 3T3 cells expressing a variety of ras proteins indicated that phosphorylation occurs within a domain encoded by exon 4B of cKi-ras. Phosphorylation affected neither the binding nor the GTPase activity of the ras protein. We conclude that cKi-ras is a substrate for protein kinase C and that the site of phosphorylation is likely to be serine 181 encoded by exon 4B.     

Phosphorylation of low molecular mass cytosolic proteins by protein kinase C and protein kinase A in the rabbit exocrine pancreas

Subcellular fractionation of rabbit pancreatic acini was performed to study the distribution of endogenous substrates for protein kinase C. Substrates for protein kinase C were found to be predominantly low molecular mass proteins of cytosolic origin. At least three of these soluble substrates, with molecular masses of 17-19 kDa, were relatively heavily phosphorylated by endogenous as well as purified pancreatic protein kinase C. In the same molecular mass range, 16-18 kDa, soluble proteins were also phosphorylated by protein kinase A. Moreover, addition of cyclic AMP under conditions that activated protein kinase C gave a more than additive labelling of these low molecular mass proteins. The latter observation may be of interest in view of the potentiating effect cyclic-AMP-activated protein kinase A has on amylase secretion stimulated by secretagogues which increase free cytosolic Ca2+ and activate protein kinase C.     

Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.     

Epidermal growth factor stimulates phosphorylation of RAF-1 independently of receptor autophosphorylation and internalization.

Phosphorylation of the RAF-1 protooncogene product and activation of its associated serine/threonine kinase are common features of the response of cells to peptide growth factors. We have used wild-type and mutant epidermal growth factor (EGF) receptors to investigate mechanisms of RAF-1 phosphorylation. In vivo EGF treatment rapidly stimulated phosphorylation of RAF-1 exclusively on serine residues. Stimulation of RAF-1 phosphorylation occurred at 37 degrees C but not at 4 degrees C and persisted after dissociation of EGF from its receptor. EGF-induced RAF-1 serine phosphorylation required the intrinsic tyrosine kinase activity of the EGF receptor but was independent of EGF receptor self-phosphorylation and of ligand-induced receptor internalization. Down-regulation of protein kinase C did not affect the EGF-induced increase in RAF-1 phosphorylation. These data suggest that the activated tyrosine kinase activity of the EGF receptor enhances serine phosphorylation of RAF-1 via an intermediary molecule(s).