A protein kinase C pseudosubstrate peptide inhibits phosphorylation of the CD3 antigen in streptolysin-O-permeabilized human T lymphocytes.

Activation of human T lymphocytes leads to the phosphorylation of the CD3-antigen gamma polypeptide. We have investigated a possible role for protein kinase C (PKC) in mediating this phosphorylation event by using T cells permeabilized with streptolysin-O in the presence of 120 mM-K+ buffers containing Ca2+-EGTA. The gamma-chain was phosphorylated by [gamma-32P]ATP in permeabilized T lymphoblasts in the presence of phorbol 12,13-dibutyrate (Pdbu) or phytohaemagglutinin (PHA). Ca2+ alone in the range 0.5-1.0 microM also induced gamma-chain phosphorylation in some T-lymphoblast preparations; that in Jurkat-6 cells occurred at lower concentrations (50-500 nM). Two experimental approaches were used to investigate the possible involvement of PKC. Firstly, when permeabilization was carried out in buffer lacking free Ca2+, PKC was lost from the cells, and gamma-chain phosphorylation could then no longer be induced on subsequent addition of Pdbu or PHA in 400 nM-Ca2+, or 800 nM-Ca2+ alone, to permeabilized cells. However, when permeabilization was carried out in the presence of these three agents, PKC was translocated to intracellular membranes, and subsequent addition of [gamma-32P]ATP to these cells then resulted in gamma-chain phosphorylation. In the second approach, induction of gamma-chain phosphorylation by Pdbu, 1-oleoyl-2-acetylglycerol, 1,2-diolein, PHA or Ca2+ alone was effectively blocked by permeabilizing T cells in the presence of a PKC pseudosubstrate peptide (50 microM). Pseudosubstrate concentrations in the range 7-20 microM inhibited gamma-chain phosphorylation by 50%. In contrast, addition of four other 'irrelevant' basic peptides (50 microM) did not result in detectable inhibition, and 50 microM-pseudosubstrate did not inhibit the phosphorylation of 17 other polypeptides isolated from permeabilized T cells. These data suggest that Pdbu-, 1,2-diacylglycerol-, PHA- and Ca2+-induced phosphorylation of the CD3-antigen gamma chain in permeabilized T cells is mediated by PKC.     

Tyrosine protein phosphorylation is required for protein kinase C-mediated proliferation in T cells.

We have studied the role of tyrosine kinase in PMA-stimulated T cells. Protein kinase C (PKC)-mediated D10A cell proliferation is inhibited by the specific inhibitor of tyrosine kinase, tyrphostin. This inhibitor selectively blocks the mRNA expression of the proto-oncogene c-myc in response to the phorbol ester, PMA. On the other hand, the same doses of this inhibitor do not affect the mRNA expression of the proto-oncogene c-fos in PMA-stimulated D10A cells. Phorbol esters induce in this T cell line the tyrosine phosphorylation of a unique protein of 42 kDa and the enzyme PKC is required for this activity.     

Cyclic GMP inhibits protein kinase C-mediated secretion in rat pancreatic acini

Dibutyryl cyclic GMP (Bu2cGMP) inhibited agonist-induced secretion of amylase from isolated rat pancreatic acini. In contrast to previous studies, this inhibitory action was not confined to butyryl derivatives of cyclic GMP, since the membrane-permeant cyclic GMP analogues Bu2cGMP and cyclic 8-bromo-GMP (8-Br-cGMP) were equipotent (IC50 2 nM) in their inhibition of amylase secretion stimulated by cholecystokinin-(26-33)-octapeptide (CCK8): at extracellular concentrations up to 1 mM, cyclic GMP itself was devoid of inhibitory activity. Both Bu2cGMP and 8-Br-cGMP also potently inhibited secretion stimulated by 4 beta-phorbol 12-myristate 13-acetate (PMA) (IC50 6 nM), but only partially inhibited responses elicited by bethanechol or bombesin and were without effect on A23187-evoked secretion. Furthermore, agents that are known to raise intracellular cyclic GMP levels (MB22948 (2-o-propoxyphenyl-8-azapurin-6-one) or nitroprusside) or antagonize the actions of protein kinase C (4 alpha-PMA or staurosporine), also inhibited CCK8- or PMA-stimulated secretion but not secretion elicited by bombesin, bethanechol, or A23187. It is concluded from these and other observations reported here that protein kinase C is the major intracellular mediator of amylase secretion stimulated by CCK8 and that this pathway may be regulated by cyclic GMP at a step that follows protein kinase C activation.     

Oxidative stress induces protein kinase C-mediated activation loop phosphorylation and nuclear redistribution of protein kinase D

Oxidative stress induced by cell treatments with H(2)O(2) activates protein kinase D (PKD) via a protein kinase C (PKC)-dependent signal transduction pathway (Waldron, R. T., and Rozengurt, E. (2000) J. Biol. Chem. 275, 17114-17121). Here we show that oxidative stress induces PKC-dependent activation loop Ser(744) and Ser(748) phosphorylation to mediate dose- and time-dependent activation of PKD, both endogenously expressed in Swiss 3T3 cells and stably overexpressed in Swiss 3T3-GFP.PKD cells. Although oxidative stress induced PKD activation loop phosphorylation and activation with identical kinetics, both were dose-dependently blocked by preincubation of cells with selective inhibitors of PKC (GF109203X and G?6983) or c-Src (PP2). Inhibition of Src tyrosine kinase activity eliminated oxidative stress-induced direct PKD tyrosine phosphorylation, but only partially attenuated activation loop phosphorylation and activation. Mutation of a putative tyrosine phosphorylation site on PKD, Tyr(469) to phenylalanine, had no effect on its activation by oxidative stress in transfected COS-7 cells. Similarly, a mutant with Tyr(469) replaced by aspartic acid had increased basal activity but was also further activated by oxidative stress. Thus, PKD tyrosine phosphorylation at this site neither produced full activation by itself nor was required for oxidative stress-induced activation mediated by activation loop phosphorylation. In addition to PKD activation, activation loop phosphorylation in response to oxidative stress also redistributed activated PKD to cell nuclei, as revealed by PKD indirect immunofluorescence, imaging of a PKD-green fluorescent protein fusion construct (GFP-PKD), and analysis of nuclear pellets. Cell preincubation with G?6983 strongly diminished H(2)O(2)-induced nuclear relocalization of GFP-PKD. Taken together, these results indicate that PKC-mediated PKD Ser(744) and Ser(748) phosphorylation induced by oxidative stress integrates PKD activation with redistribution to the nucleus.     

Role of protein kinase C-mediated protein phosphorylation in mitochondrial translocation of mouse CYP1A1, which contains a non-canonical targeting signal

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH2-terminal-truncated molecular species, +91A1 and +331A1. Mutations Pro-2 --> Leu and Tyr-5 --> Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 --> Asn and Leu-17 --> Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C (PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-35 --> Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, anti-psychotics, and narcotics that are poor substrates for microsomal CYP1A1.     

Chronic ethanol exposure increases levels of protein kinase C delta and epsilon and protein kinase C-mediated phosphorylation in cultured neural cells.

Exposure to ethanol for several days increases the number and function of dihydropyridine-sensitive Ca2+ channels in excitable tissues. In the neural cell line PC12, this process is blocked by inhibitors of protein kinase C (PKC), suggesting that PKC mediates ethanol-induced increases in Ca2+ channels. We report that treatment with 25-200 mM ethanol for 2-8 days increased PKC activity in PC12 cells and NG108-15 neuroblastoma-glioma cells. Detailed studies in PC12 cells showed that ethanol also increased phorbol ester binding and immunoreactivity to PKC delta and PKC epsilon. These changes were associated with increased PKC-mediated phosphorylation. Ethanol did not activate the enzyme directly, nor did ethanol increase levels of diacylglycerol. Ethanol-induced increases in PKC levels may promote up-regulation of Ca2+ channels, and may also regulate the expression and function of other proteins involved in cellular adaptation to ethanol.     

A role for protein kinase C-mediated phosphorylation in the mobilization of arachidonic acid in mouse macrophages

Mouse peritoneal macrophages respond to activators of protein kinase C and to zymosan particles and calcium ionophore by rapid enhancement of a phospholipase A pathway and mobilization of arachidonic acid. The pattern of protein phosphorylation induced in these cells by 4 beta-phorbol 12-myristate 13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol, exogenous phospholipase C and by zymosan and ionophore A23187 was found to be virtually identical. The time course of phosphorylation differed among the phosphoprotein bands and in only some of those identified (i.e., those of 45 and 65 kDa) was the phosphorylation sufficiently rapid to be involved in the activation of the phospholipase A pathway. Phosphorylation of lipocortin I or II could not be detected. Down-regulation of kinase C by a 24-h pretreatment with PMA resulted in extensive inhibition of both protein phosphorylation and the mobilization of arachidonic acid in response to PMA or dioctanoylglycerol. The phosphorylation of the 45 kDa protein in response to zymosan and A23187 was also inhibited by pretreatment with PMA, while only arachidonic acid release induced by zymosan was inhibited by this pretreatment. Depletion of intracellular calcium had little effect on kinase C-dependent phosphorylation, although arachidonic acid mobilization is severely inhibited under these conditions. Bacterial lipopolysaccharide and lipid A induced a phosphorylation pattern different from that induced by PMA, and down-regulation of protein kinase C did not affect lipopolysaccharide-induced protein phosphorylation. The results indicate (i) that protein kinase C plays a critical role also in zymosan-induced activation of the phospholipase A pathway mobilizing arachidonic acid; (ii) that such activation requires calcium at some step distal to kinase C-mediated phosphorylation and (iii) that phosphorylation of lipocortins does not explain the kinase C-dependent activation.     

Protamine induces autophosphorylation of protein kinase C: stimulation of protein kinase C-mediated protamine phosphorylation by histone.

Protein kinase C (PKC), a protein phosphorylating enzyme, is characterized by its need for an acidic phospholipid and for activators such as Ca2+ and diacylglycerol. The substrate commonly used in experiments with PKC is a basic protein, histone III-S, which needs the activators mentioned. However, protamine, a natural basic substrate for PKC, does not require the presence of cofactor/activator. We report here that protamine can induce the autophosphorylation of PKC in the absence of any PKC-cofactor or activator; this may represent a possible mechanism of cofactor-independent phosphorylation of this protein. It was investigated if protamine itself can act as a PKC-activator and stimulate histone phosphorylation in the manner of Ca2+ and phospholipids. Experiments however showed that protamine is not a general effector of PKC. On the contrary, histone stimulated PKC-mediated protamine phosphorylation and protamine-induced PKC-autophosphorylation. Histone alone did not induce PKC-autophosphorylation. Kinetic studies suggest that histone increases the maximal velocity (Vmax) of protamine kinase activity of PKC without affecting the affinity (Km). Other polycationic proteins such as polyarginine serine and polyarginine tyrosine were not found to influence PKC-mediated protamine phosphorylation, indicating that the observed effects are specific to histone, and are not general for all polycationic proteins. These results suggest that histone can modulate the protamine kinase activity of PKC by stimulating protamine-induced PKC-autophosphorylation.     

Inhibition of zipper-interacting protein kinase function in smooth muscle by a myosin light chain kinase pseudosubstrate peptide

As a regulator of smooth muscle contractility, zipper-interacting protein kinase (ZIPK) appears to phosphorylate the regulatory myosin light chain (RLC20), directly or indirectly, at Ser19 and Thr18 in a Ca²⁺-independent manner. The calmodulin-binding and autoinhibitory domain of myosin light chain kinase (MLCK) shares similarity to a sequence found in ZIPK. This similarity in sequence prompted an investigation of the SM1 peptide, which is derived from the autoinhibitory region of MLCK, as a potential inhibitor of ZIPK. In vitro studies showed that SM1 is a competitive inhibitor of a constitutively active 32-kDa form of ZIPK with an apparent K_i value of 3.4 µM. Experiments confirmed that the SM1 peptide is also active against full-length ZIPK. In addition, ZIPK autophosphorylation was reduced by SM1. ZIPK activity is independent of calmodulin; however, calmodulin suppressed the in vitro inhibitory potential of SM1, likely as a result of nonspecific binding of the peptide to calmodulin. Treatment of ileal smooth muscle with exogenous ZIPK was accompanied by an increase in RLC20 diphosphorylation, distinguishing between ZIPK [and integrin-linked kinase (ILK)] and MLCK actions. Administration of SM1 suppressed steady-state muscle tension developed by the addition of exogenous ZIPK to Triton-skinned rat ileal muscle strips with or without calmodulin depletion by trifluoperazine. The decrease in contractile force was associated with decreases in both RLC20 mono- and diphosphorylation. In summary, we present the SM1 peptide as a novel inhibitor of ZIPK. We also conclude that the SM1 peptide, which has no effect on ILK, can be used to distinguish between ZIPK and ILK effects in smooth muscle tissues. inhibitory peptide; calcium sensitization     

Mechanism of regulation of the gap junction protein connexin 43 by protein kinase C-mediated phosphorylation

Phosphorylation of the gap junction protein connexin 43 (Cx43) by protein kinase C (PKC) decreases dye coupling in many cell types. We report an investigation of the regulation by PKC of Cx43 gap junctional hemichannels (GJH) expressed in Xenopus laevis oocytes. The activity of GJH was assessed from the uptake of hydrophilic fluorescent probes. PKC inhibitors increased probe uptake in isolated oocytes expressing recombinant Cx43, indicating that the regulatory effect occurs at the hemichannel level. We identified by mutational analysis the carboxy-terminal (CT) domain sequences involved in this response. We found that 1) Ser368 is responsible for the regulation of Cx43 GJH solute permeability by PKC-mediated phosphorylation, 2) CT domain residues 253-270 and 288-359 are not necessary for the effect of PKC, and 3) the prolinerich CT region is not involved in the effect of phosphorylation by PKC. Our results demonstrate that Ser368 (but not Ser372) is involved in the regulation of Cx43 solute permeability by PKC-mediated phosphorylation, and we conclude that different molecular mechanisms underlie the regulation of Cx43 by intracellular pH and PKC-mediated phosphorylation. protein kinase C blocker; dye loading; hemichannel     

Altered levels and protein kinase C-mediated phosphorylation of substrates in normal and transformed mouse lung epithelial cells.

Protein phosphorylation and protein kinase C (PKC) levels were analyzed in intact cultures of spontaneously transformed, chemically transformed, and untransformed mouse pulmonary epithelial cell lines. It was found that although the transformed cell lines contained about 80% less protein kinase C, measured as total enzyme activity or binding of [3H]phorbol ester, phosphorylation events after phorbol ester treatment could still be easily detected. A commonly described Mr 80-kDa protein kinase C substrate (p80, 80 K, MARKS) was identified using 2D-PAGE, following phosphorylation in intact cells, and found to have reduced availability for phosphorylation in the transformed cell lines C4SE9, C1SA5 and NULB5 in comparison to the untransformed C4E10 and C1C10. Available levels of p80 were further analyzed in heat-denatured extracts from all cell lines using partially purified bovine brain PKC and correlated well with changes seen in intact cells. It was also noted that all transformed cell lines contained large amounts of a family of phosphoproteins of Mr 55-65 kDa, that could not be detected in the untransformed cell lines and whose phosphorylation state was increased by protein kinase C activation. This protein was found to be located in the nucleus. Hence, spontaneously and chemically transformed mouse pulmonary epithelial cells exhibit reduced levels of PKC, along with an altered pattern of PKC-mediated phosphorylation.     

Use of pseudosubstrate affinity to measure active protein kinase A

Traditional cAMP-dependent protein kinase (also known as protein kinase A [PKA]) assays, which are based on substrate phosphorylation, often have high background activity from other kinases, thereby limiting sensitivity and making it difficult to detect low levels of active PKA in cell lysates. Therefore, a better technique that measures active PKA in crude cell lysates undoubtedly is necessary. We developed an efficient and sensitive assay to compare active PKA levels based on binding of the active PKA catalytic subunit to its pseudosubstrate domain inhibitor (PKI) fused with glutathione S-transferase (GST-PKI). This pseudosubstrate affinity assay can detect variations in the active PKA levels in the presence of common inducers of PKA activity such as forskolin and prostaglandins. It has resolution to detect a concentration-dependent curve of active PKA in a linear range, and it also has sensitivity to detect up to 2.5 ng of active enzyme. An observed change in the binding affinity between PKA and PKI in the presence of the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) shows that this assay can be successfully used to measure how active PKA is affected by specific inhibitors. We conclude that this method is a simple, inexpensive, and nonhazardous method to compare active PKA levels with high sensitivity and specificity with negligible background.     

Induction of apoptosis by protein kinase C pseudosubstrate lipopeptides in several human cells

Intracellular enzymes or receptors are interesting targets for thepharmacomodulation of cellular metabolism. We have previously shown thatmodification of relatively long peptides by a palmitoyl-lysine residue couldfacilitate their delivery into the cytoplasm of living cells. Severalpeptides containing pseudosubstrate sequences of protein kinase C (PKC) havebeen evaluated for their ability to modulate phosphorylation of modelsubstrate, neuronal morphology or tumor necrosis factor secretion. In thiswork we have evaluated the effect of palmitoyl-modified PKC-pseudosubstratepeptides on induction of apoptosis. We have established that these peptidesare able to induce apoptosis in different human cell types (primaryfibroblasts, T- and B-lymphocyte cell lines) as assessed by (terminal deoxynucleotidyl transferase dUTP nick-end labelling) and DNAfragmentation. In contrast, control peptides (non-lipidicPKC-pseudosubstrate peptides and irrelevant lipopeptides) had no or littleeffect on programmed cell death. This work highlights the pharmacologicalinterest of lipopeptides and argues in favor of the potential role of PKC(s)in the cell death machinery.     

Induction of apoptosis by protein kinase C pseudosubstrate lipopeptides in several human cells

Summary Intracellular enzymes or receptors are interesting targets for the pharmacomodulation of cellular metabolism. We have previously shown that modification of relatively long peptides by a palmitoyl-lysine residue could facilitate their delivery into the cytoplasm of living cells. Several peptides containing pseudosubstrate sequences of protein kinase C (PKC) have been evaluated for their ability to modulate phosphorylation of model substrate, neuronal morphology or tumor necrosis factor secretion. In this work we have evaluated the effect of palmitoyl-modified PKC-pseudosubstrate peptides on induction of apoptosis. We have established that these peptides are able to induce apoptosis in different human cell types (primary fibroblasts, T- and B-lymphocyte cell lines) as assessed by (terminal deoxynucleotidyl transferase dUTP nick-end labelling) and DNA fragmentation. In contrast, control peptides (non-lipidic PKC-pseudosubstrate peptides and irrelevant lipopeptides) had no or little effect on programmed cell death. This work highlights the pharmacological interest of lipopeptides and argues in favor of the potential role of PKC(s) in the cell death machinery. K. Thiam and E. Loing have contributed equally to this work.     

Role of troponin I phosphorylation in protein kinase C-mediated enhanced contractile performance of rat myocytes

Our goal was to define the role of phosphorylated cardiac troponin-I in the adult myocyte contractile performance response to activated protein kinase C. In agreement with earlier work, endothelin enhanced both adult rat myocyte contractile performance and cardiac troponin-I phosphorylation. Protein kinase C participated in both responses. The role of cardiac troponin-I phosphorylation in the contractile function response to protein kinase C was further investigated using gene transfer into myocytes of troponin-I isoforms/mutants lacking one or more phosphorylation sites previously identified in purified cardiac troponin-I. Sarcomeric replacement with slow skeletal troponin-I-abrogated protein kinase C-mediated troponin-I phosphorylation. In functional studies, endothelin slowed relaxation in myocytes expressing slow skeletal troponin-I, while the relaxation rate increased in myocytes expressing cardiac troponin-I. Based on these results, acceleration of myocyte relaxation during protein kinase C activation largely depended on cardiac troponin-I phosphorylation. Experiments with troponin-I isoform chimeras provided evidence that phosphorylation sites in the amino portion of cardiac troponin I-mediated the protein kinase C acceleration of relaxation. The cardiac troponin-I Thr-144 phosphorylation site identified in earlier biochemical studies was not significantly phosphorylated during the acute contractile response. Thus, amino-terminal protein kinase C-dependent phosphorylation sites in cardiac troponin-I are likely responsible for the accelerated relaxation observed in adult myocytes.     

Atrial natriuretic peptide inhibits protein phosphorylation stimulated by angiotensin II in bovine adrenal glomerulosa cells

Bovine adrenal glomerulosa cells were incubated with 32PO4 and either angiotensin II, atrial natriuretic peptide, or both. Solubilized cells were subjected to one-dimensional gel electrophoresis. Autoradiography and scintillation counting of gels showed that angiotensin increased labeling of one band, with an apparent molecular weight of 17,600. Atrial natriuretic peptide inhibited the angiotensin effect. Together with earlier results, this observation suggests that atrial natriuretic peptide affects aldosteronogenesis at the level of protein phosphorylation, but not by altering angiotensin receptors, calcium fluxes or phosphoinositide metabolism.     

Atrial natriuretic peptide inhibits cardiomyocyte hypertrophy through mitogen-activated protein kinase phosphatase-1

Cardiac hypertrophy is formed in response to hemodynamic overload. Although a variety of factors such as catecholamines, angiotensin II (AngII), and endothelin-1 (ET-1) have been reported to induce cardiac hypertrophy, little is known regarding the factors that inhibit the development of cardiac hypertrophy. Production of atrial natriuretic peptide (ANP) is increased in the hypertrophied heart and ANP has recently been reported to inhibit the growth of various cell types. We therefore examined whether ANP inhibits the development of cardiac hypertrophy. Pretreatment of cultured cardiomyocytes with ANP inhibited the AngII- or ET-1-induced increase in the cell size and the protein synthesis. ANP also inhibited the AngII- or ET-1-induced hypertrophic responses such as activation of mitogen-activated protein kinase (MAPK) and induction of immediate early response genes and fetal type genes. To determine how ANP inhibits cardiomyocyte hypertrophy, we examined the mechanism of ANP-induced suppression of the MAPK activation. ANP strongly induced expression of MAPK phosphatase-1 (MKP-1) and overexpression of MKP-1 inhibited AngII- or ET-1-induced hypertrophic responses. These growth-inhibitory actions of ANP were mimicked by a cyclic GMP analog 8-bromo-cyclic GMP. Taken together, ANP directly inhibits the growth factor-induced cardiomyocyte hypertrophy at least partly via induction of MKP-1. Our present study suggests that the formation of cardiac hypertrophy is regulated not only by positive but by negative factors in response to hemodynamic load.     

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.     

Functional effects of protein kinase C-mediated phosphorylation of chick heart muscarinic cholinergic receptors.

Muscarinic cholinergic receptors (mAChR) purified from chick heart were phosphorylated by protein kinase C (PKC) and reconstituted with the purified GTP-binding regulatory protein Go. The effects of PKC phosphorylation on the interaction of mAChR with Go were assessed by monitoring for agonist-stimulated guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) binding to Go, agonist-stimulated GTPase activity of Go, and the capability of Go to induce high affinity agonist binding to mAChR. Both the receptor-stimulated GTP gamma S binding and GTPase activity of Go were markedly diminished as a result of PKC-mediated phosphorylation of the mAChR, whereas the ability of Go to induce high affinity agonist binding to the receptors was unaffected. When mAChR were first reconstituted with Go and then subjected to phosphorylation with PKC, a complete inhibition of the phosphorylation of mAChR by PKC was observed. The inhibitory effect of Go on mAChR phosphorylation was concentration-dependent and was prevented by the presence of GTP gamma S in the reaction mixtures. Taken together, these results indicate that the phosphorylation of mAChR by PKC modulates receptor/G-protein interactions and that the ability of the receptors to act as substrates for PKC may be regulated by receptor/G-protein interactions.