Phosphorylation of proteolytically-nicked rat hepatic acetyl-CoA carboxylase

A partially-purified preparation of acetyl-CoA carboxylase was not inactivated by ATP and Mg2+ although it was phosphorylated. SDS gel electrophoresis of the phosphorylated enzyme showed phosphopeptides migrating at 140 and 40 K along with the 250 K native subunit. Phosphorylation by the catalytic subunit of cAMP-dependent protein kinase further phosphorylated an additional 120 K phosphopeptide. Neither cAMP-independent phosphorylation nor the cAMP-dependent phosphorylation of the enzyme resulted in a significant decrease in activity.     

Purification and characterization of a brain-specific protein kinase C substrate, neurogranin (p17). Identification of a consensus amino acid sequence between neurogranin and neuromodulin (GAP43) that corresponds to the protein kinase C phosphorylation site and the calmodulin-binding domain.

Neurogranin, formerly designated p17 (Baudier, J., Bronner, C., Kligman, D., and Cole, R. D.) (1989) J. Biol. Chem. 264, 1824-1828), a brain-specific in vitro substrate for protein kinase C (PKC), has been purified to homogeneity from bovine forebrain. The purified protein has a molecular mass of 7837.1 +/- 0.5 Da, determined by electrospray mass spectrometry. In the absence of reducing agent, dimers and higher oligomers accumulated. On sodium dodecyl sulfate-polyacrylamide gels the protein monomer migrated abnormally with an apparent molecular mass of 15,000-19,000 Da, depending on the percentage of polyacrylamide. The native protein is blocked at its amino terminus. The majority of the primary amino acid sequence was determined following proteolytic and chemical fragmentation. A comparison of the amino acid sequence of neurogranin with that of the brain-specific PKC substrate neuromodulin, revealed a strikingly conserved amino acid sequence AA(X)KIQA-SFRGH(X)(X)RKK(X)K. The two proteins are not related over the rest of their sequences. Neurogranin was shown to be phosphorylated in hippocampal slices incubated with 32Pi and phorbol esters stimulated neurogranin phosphorylation, suggesting that neurogranin is likely to be an in vivo substrate for PKC. In vitro phosphorylation of neurogranin by PKC produced a shift of the isoelectric point of the protein (pI 5.6) to a more acidic value (pI 5.4). Tryptic digestion of the phosphorylated protein yielded a single phosphopeptide having the sequence IQASFR, where the serine residue is the phosphorylated amino acid. This phosphopeptide is part of the conserved sequence shared with neuromodulin and also corresponds to the PKC phosphorylation site on neuromodulin (Apel, E. D., Byford, M. F., Au, D., Walsh, K. A., and Storm, D. R. (1990) Biochemistry 29, 2330-2335). Evidence was obtained suggesting that neurogranin binds to calmodulin in the absence of Ca2+, a feature that also characterizes neuromodulin. We propose that the amino acid sequence shared by neurogranin and neuromodulin reflects a functional relationship between these two proteins and that the consensus sequence represents a conserved PKC phosphorylation site and a calmodulin binding domain that characterizes a class of brain-specific PKC substrates.     

Endotoxin causes phosphorylation of MARCKS in pulmonary vascular endothelial cells

Protein kinase C (PKC) has been implicated in lipopolysaccharide (LPS)-induced endothelial cell (EC) monolayer permeability. Myristoylated alanine-rich C kinase substrate (MARCKS), as a specific PKC substrate, appears to mediate PKC signaling by PKC-dependent phosphorylation of MARCKS and subsequent modification of the association of MARCKS with filamentous actin and calmodulin (CaM). Therefore, in the present study, we investigated LPS-induced MARCKS phosphorylation in bovine pulmonary artery EC (BPAEC). LPS potentiated MARCKS phosphorylation in BPAEC in a time- and dose-dependent manner. The PKC inhibitor, calphostin C, significantly decreased LPS-induced phosphorylation of MARCKS. In addition, downregulation of PKC with phorbol 12-myristate 13-acetate (PMA) did not affect the LPS-induced MARCKS phosphorylation, suggesting that LPS and PMA activate different isoforms of PKC. Pretreatment with SB203580, a specific inhibitor of p38 MAP kinase, or genistein, a tyrosine kinase inhibitor, prevented LPS-induced MARCKS phosphorylation. Phosphorylation at appropriate sites will induce translocation of MARCKS from the cell membrane to the cytosol. However, LPS, in contrast to PMA, did not generate MARCKS translocation in BPAEC, suggesting that MARCKS translocation may not play a role in LPS-induced actin rearrangement and EC permeability. LPS also enhanced both thrombin- and PMA-induced phosphorylation of MARCKS, suggesting that LPS was able to prime these signaling pathways in BPAEC. Because the CaM-dependent phosphorylation of myosin light chains (MLC) results in EC contraction, we studied the effect of LPS on MLC phosphorylation in BPAEC. LPS induced diphosphorylation of MLC in a time-dependent manner, which occurred at lower doses of LPS, than those required to induce MARCKS phosphorylation. In addition, there was no synergism between LPS and thrombin in the induction of MLC phosphorylation. These data indicate that MLC phosphorylation is independent of MARCKS phosphorylation. In conclusion, LPS stimulated MARCKS phosphorylation in BPAEC. This phosphorylation appears to involve activation of PKC, p38 MAP kinase, and tyrosine kinases. Further studies are needed to explore the role of MARCKS phosphorylation in LPS-induced actin rearrangement and EC permeability.     

Phosphorylation of Mnk1 by caspase-activated Pak2/gamma-PAK inhibits phosphorylation and interaction of eIF4G with Mnk

The mitogen-activated protein kinase-interacting kinase 1 (Mnk1) is phosphorylated by caspase-cleaved protein kinase Pak2/gamma-PAK but not by Cdc42-activated Pak2. Phosphorylation of Mnk1 is rapid, reaching 1 mol/mol within 15 min of incubation with Pak2. A kinetic analysis of the phosphorylation of Mnk1 by Pak2 yields a K(m) of 0.6 microm and a V(max) of 14.9 pmol of (32)P/min/microg of Pak2. Two-dimensional tryptic phosphopeptide mapping of Mnk1 phosphorylated by Pak2 yields two distinct phosphopeptides. Analysis of the phosphopeptides by automated microsequencing and manual Edman degradation identified the sites in Mnk1 as Thr(22) and Ser(27). Mnk1, activated by phosphorylation with Erk2, phosphorylates the eukaryotic initiation factor (eIF) 4E and the eIF4G components of eIF4F. Phosphorylation of Mnk1 by Pak2 does not activate Mnk1, as measured with either eIF4E or eIF4F as substrate. Phosphorylation of Erk2-activated Mnk1 by Pak2 has no effect on phosphorylation of eIF4E but reduces phosphorylation of eIF4G by Mnk1 by up to 50%. Phosphorylation of Mnk1 by Pak2 inhibits binding of eIF4G peptides containing the Mnk1 binding site by up to 80%. When 293T cells are subjected to apoptotic induction by hydrogen peroxide, Mnk1 is phosphorylated at both Thr(22) and Ser(27). These results indicate a role for Pak2 in the down-regulation of translation initiation in apoptosis by phosphorylation of Mnk1.     

Myristoylation, phosphorylation, and subcellular distribution of the 80-kDa protein kinase C substrate in BC3H1 myocytes

Numerous reports have described a phosphoprotein with an apparent molecular mass of 68-87 kDa, often referred to as the 80K protein, which serves as a major specific substrate for protein kinase C in a wide variety of cell types. This protein has been shown to be myristoylated in macrophages, apparently in a stimulus-dependent manner. In the present study, we have defined the kinetics for myristoylation of the 80K protein in BC3H1 myocytes and have examined the subcellular distribution of the [3H]myristate and 32P-labeled forms of the protein before and after activation of protein kinase C by phorbol dibutyrate (PDBu). The 80K protein was identified in BC3H1 myocytes by apparent molecular mass of 68 kDa (consistent with the previously reported size of the murine homologue), isoelectric point of 4.6-4.8, PDBu-inducible phosphorylation, peptide mapping, and labeling with [3H]myristate. Incorporation of [3H]myristate by this protein occurred through an amide linkage and was abolished completely by cycloheximide. Pulse labeling of quiescent cells with [3H]myristate revealed no alteration in myristoylation of the 80K protein in either the crude membrane or soluble fractions after PDBu-induced phosphorylation. The subcellular distribution of this protein (approximately 80% membrane, approximately 20% cytosol) also was the same in control and PDBu-stimulated cells. Phosphorylation of both the membrane-bound and soluble forms was increased approximately 6-fold upon stimulation of cultures with PDBu; the soluble form was phosphorylated to a 4-fold higher stoichiometry than its membrane-bound counterpart. Together, these data demonstrate that the 80K protein is myristoylated cotranslationally in BC3H1 cells and that protein kinase C-dependent phosphorylation of the 80K protein does not alter its subcellular distribution or degree of myristoylation. The fact that 20% of total myristoylated 80K protein resides in the cytosol also indicates that myristoylation alone is not sufficient to target this protein to the plasma membrane.     

Phorbol esters stimulate phosphorylation of eukaryotic initiation factors 3, 4B, and 4F

Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, was isolated by m7 GTP-Sepharose affinity chromatography from rabbit reticulocytes incubated with [32P]orthophosphate. Following treatment of reticulocytes with phorbol 12-myristate 13-acetate (PMA) for 30 min, stimulation of phosphorylation of both the p25 and p220 subunits was observed (2.5-5-fold). Two variants were observed for p25 in the absence and presence of PMA when analyzed by two-dimensional gel electrophoresis. Only the more acidic of these was phosphorylated, with the level of phosphorylation increased upon PMA treatment. One main variant was observed for p220; following PMA stimulation, in addition to increased labeling of this variant, two more acidic phosphorylated variants were observed. Low levels of eIF-3 and -4B were associated with purified eIF-4F, and PMA treatment stimulated phosphorylation of eIF-3 (p170) by 2-4-fold and eIF-4B by 1.5-2.5 fold. Two-dimensional phosphopeptide mapping of p25 phosphorylated in the absence or presence of PMA generated a single tryptic phosphopeptide, suggesting a single phosphorylation site. A more complex phosphopeptide map was observed with p220 subunit. The maps for both subunits contained the same phosphopeptides as those obtained when eIF-4F was phosphorylated in vitro by the Ca2+/phospholipid-dependent protein kinase, indicating this protein kinase directly modulated eIF-4F in response to PMA.     

Hormone-induced phosphorylation of the plasma membrane calcium pump in cultured aortic endothelial cells.

The regulation of the plasma membrane Ca2+ pump by hormones via phosphorylation in intact cells has not been clearly established. We now present evidence that the Ca2+ pump is phosphorylated on both serine and threonine residues in unstimulated and stimulated cultured rat aortic endothelial cells. Among the stimuli tested, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was most potent and increased the level of phosphorylation threefold, while the cAMP-dependent protein kinase activator 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) stimulated the phosphorylation 1.6-fold. Two-dimensional tryptic phosphopeptide maps of the Ca2+ pump from unstimulated and CPT-cAMP-stimulated cells have identical patterns (five phosphopeptides) while PMA-stimulated cells have three additional phosphopeptides. Isoproterenol-, ATP-, angiotensin II-, and bradykinin-stimulated cells also have increased levels of Ca2+ pump phosphorylation. Stimuli-induced phosphorylation of the Ca2+ pump was rapid (5-10 min) and was concomitant with stimulated calcium efflux from the same cells. This is the first direct evidence that the plasma membrane Ca2+ pump in intact cells is regulated by various hormones or agonists via cAMP-dependent protein kinase or protein kinase C phosphorylation.     

Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes.

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.     

Protein kinase C activation stimulates the phosphorylation and internalization of the sst2A somatostatin receptor

The sst2A receptor is expressed in the endocrine, gastrointestinal, and neuronal systems as well as in many hormone-sensitive tumors. This receptor is rapidly internalized and phosphorylated in growth hormone-R2 pituitary cells following somatostatin binding (Hipkin, R. W., Friedman, J., Clark, R. B., Eppler, C. M., and Schonbrunn, A. (1997) J. Biol. Chem. 272, 13869-13876). The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), also stimulates sst2A phosphorylation. Here we examine the mechanisms and consequences of PMA and agonist-induced sst2A phosphorylation. Like somatostatin, both PMA and bombesin increased sst2A receptor phosphorylation within 2 min. The PKC inhibitor GF109203X blocked PMA- and bombesin- stimulated sst2A phosphorylation, whereas stimulation by the somatostatin analog SMS 201-995 was unaffected. Agonist and PMA each stimulated phosphorylation in two receptor domains, the third intracellular loop and the C-terminal tail. Functionally, PMA dramatically increased the internalization of the sst2A receptor-ligand complex. This PMA stimulation was blocked by GF109203X, whereas basal internalization was unaffected. However, neither basal nor PMA-stimulated internalization was altered by pertussis toxin, whereas both were blocked by hypertonic sucrose. Therefore PKC activation and agonist binding stimulate sst2A phosphorylation by distinct mechanisms, and PKC potentiates internalization of the sst2A receptor via clathrin-coated pits. Thus, hormonal stimulation of PKC-coupled receptors may provide a mechanism for regulating the inhibitory actions of somatostatin in target tissue.     

Differing signal requirements for the activation of macrophages from C3H/HeJ and C3H/OuJ mice

Abstract Endotoxin-associated protein (EP) from Salmonella typhi stimulated the release of prostaglandin E2 (PGE2), interleukin-1 (IL-1), and interferon (IFN) activity in macrophages from the lipopolysaccharide (LPS) responder C3H/OuJ mouse strain. However, only PGE2 and IL-1 were stimulated by EP in macrophages from the LPS nonresponder C3H/HeJ mouse strain. LPS stimulated the release of PGE2, IL-1 and IFN activity in C3H/OuJ macrophages, but not in C3H/HeJ macrophages. The protein kinase C (PKC) activator phorbol myristic acid (PMA) stimulated PGE2 production in both strains but not IL-1 production, suggesting that signalling pathways other than PKC may be involved in IL-1 production. The calcium ionophore ionomycin stimulated PGE2 production in C3H/OuJ but not C3H/HeJ macrophages, suggesting a defective calcium-related pathway in the C3H/HeJ macrophages as compared to the C3H/OuJ cells.     

Identification of three cAMP-dependent protein kinase (PKA) phosphorylation sites within the major intracellular domain of neuronal nicotinic receptor alpha4 subunits

This study determined whether all protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites on the alpha4 subunit of rat alpha4beta2 neuronal nicotinic receptors could be localized to the M3/M4 cytoplasmic domain of the protein, and investigated specific amino acid substrates for the kinases through two-dimensional phosphopeptide mapping and site-directed mutagenesis. Experiments were conducted using alpha4beta2 receptors expressed in Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(333-594) ). When oocytes expressing alpha4beta2 receptors were incubated with [(32) P]orthophosphate in order to label endogenous ATP stores, phosphorylation of alpha4 subunits was evident. Incubation of either immunoprecipitated receptors or the fusion protein with [(32) P]ATP and either PKA or PKC followed by trypsinization of the samples demonstrated that the kinases phosphorylated alpha4 subunits on multiple phosphopeptides, and that the phosphorylated full-length alpha4 protein and fusion protein produced identical phosphopeptide maps. Site-directed mutagenesis of Ser365, Ser472 and Ser491 to alanines in the fusion protein eliminated phosphopeptides phosphorylated by PKA, but not by PKC. Other mutations investigated, Ser470, Ser493, Ser517 and Ser590, did not alter the phosphopeptide maps. Results indicate that Ser365, Ser472 and Ser491 on neuronal nicotinic receptor alpha4 subunits are phosphorylated by PKA and are likely to represent post-translational regulatory sites on the receptor.     

Phosphorylation of the MARCKS Protein (P87), a Major Protein Kinase C Substrate, Is Not an Obligatory Step in the Mitogenic Signaling Pathway of Basic Fibroblast Growth Factor in Rat Oligodendrocytes

Basic fibroblast growth factor (bFGF) is a well-characterized peptide hormone that has mitogenic activity for various cell types and elicits a characteristic set of responses on the cell types investigated. In this report we confirmed that bFGF is a potent mitogen for rat brain-derived oligodendrocyte (OL) precursor cells as well as for differentiated OL in secondary culture. bFGF was shown to induce expression of the protooncogene c-fos in OL. The role of protein kinase C (PKC) in mediating bFGF-stimulated proliferation as well as c-fos expression in OL was investigated. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated c-fos expression but did not trigger cell proliferation. When PKC was down-regulated by pretreatment of OL with PMA for 20 h, the bFGF-mediated stimulations of OL proliferation and c-fos mRNA expression were still observed, whereas the induction of c-fos mRNA by PMA was totally inhibited. These data demonstrate that the bFGF mitogenic signaling pathway in OLs does not require PKC. On the other hand, bFGF was found to stimulate specifically the phosphorylation of a limited number of PKC substrates in oligodendroglial cells, including the MARCKS protein. The bFGF-dependent phosphorylation of MARCKS protein was totally inhibited when PKC was first down-regulated, indicating that the phosphorylation of this protein is PKC dependent. Tryptic digestion of the phosphorylated MARCKS protein revealed that bFGF stimulated specifically the phosphorylation of the MARCKS protein on a single phosphopeptide. We provide evidence that bFGF also stimulated fatty acylation of the MARCKS protein, which might explain the observed specific bFGF-dependent phosphorylation of this protein in OL. We propose that bFGF-dependent fatty acylation and phosphorylation of the MARCKS protein are not essential for the transduction of the bFGF mitogenic signal but are probably linked to differentiation processes elicited by bFGF on OL.     

Phosphorylation of proteins in human neutrophils activated with phorbol myristate acetate or with chemotactic factor

In human neutrophils stimulated with phorbol myristate acetate (PMA) or with the chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLF) a number of proteins are phosphorylated, including proteins recovered in the membrane fraction corresponding to molecular masses of 130, 78, 46, 40, and 34 kDa and proteins recovered in the cytosol fraction corresponding to molecular masses of 65, 55, 48, 38, 36, 30, and 22 kDa. Phosphorylation of the membrane proteins was fourfold greater in cells stimulated with PMA, as compared to cells stimulated with fMLF, whereas both activators induced similar phosphorylation of proteins recovered in the cytosol fraction. Phosphorylation of membrane proteins appeared to be mediated by native protein kinase C (PKC) translocated from the cytosol to the plasma membrane. Thus phosphate incorporation was inhibited by retinal and a similar pattern of incorporation was reproduced in a reconstituted system composed of isolated cell membranes and purified PKC. Phosphorylation of cytosol proteins, on the other hand, appeared to be mediated by the proteolytically modified form of PKC. In this case, phosphate incorporation was inhibited by leupeptin, which prevents the conversion of native PKC to the proteolytically modified form, The phosphorylation pattern was reproduced when isolated cytosol fractions were incubated with the proteolytically modified form of the enzyme but not with the native PKC. These results demonstrate that responses to stimuli such as PMA or fMLF are mediated by different forms of PKC and that the proteolytically modified form is responsible for the major responses elicited by fMLF.     

Effects of bacterial lipopolysaccharide on the hydrolysis of phosphatidylinositol-4,5-bisphosphate in murine peritoneal macrophages

LPS and lipid A initiated enhanced hydrolysis of PIP2 in macrophages. When murine peritoneal macrophages were labeled with [2-3H]myoinositol and stimulated with either LPS or lipid A, a rapid (within 10 sec) rise in Ins(1,4,5)P3 was observed. The breakdown pattern of Ins(1,4,5)P3 was complex; this included breakdown of Ins(1,4,5)P3 and formation of Ins(1,3,4,5)P4 (approximately 10 to 30 sec), and ultimately formation of Ins(1,3,4)P3 (approximately 60 sec). Within 10 sec after treatment, LPS caused an average increase of about fourfold to fivefold in Ins(1,4,5)P3, which declined over 5 min. When the total isomers of InsP3 were measured, levels rose about twofold in response to LPS or to lipid A and remained elevated for as long as 5 min. Lipid A, in the concentration range of 0.1 to 10 micrograms/ml, induced elevated intracellular levels of Ca2+ as quantified by fluorescence with Quin 2 or with Fura 2. When single, adherent Fura 2-loaded macrophages were treated with lipid A, basal levels of calcium rose over 10 sec from approximately 55 nM to almost 600 nM. LPS, paradoxically, did not cause such substantial increases in intracellular calcium (i.e., increases of approximately 26 nM) when judged by Fura 2 fluorescence. LPS treatment led to enhanced phosphorylation of a characteristic set of proteins, similar to those induced by stimulating protein kinase C (PKC) with phorbol myristate acetate as previously reported. The enhanced phosphorylation of pp28, pp33, and pp67 in macrophages was evident by 15 min and optimal by 30 min. Taken together, these observations indicate that LPS and lipid A cause increased breakdown of phosphatidylinositol 4,5-bisphosphate, which led to enhanced intracellular levels of calcium and also to enhanced protein phosphorylation, presumably mediated by PKC. The data thus suggest that one major intracellular signal transduction mechanism, initiated by LPS and lipid A in macrophages, is the rapid breakdown of PIP2.     

Phosphorylation-dependent regulation of phospholipase D2 by protein kinase C delta in rat Pheochromocytoma PC12 cells.

Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31-8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKC delta by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKC delta using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKC delta kinase activity in PC12 cells. PKC delta co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylated in vitro by PKC delta were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKC delta inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKC delta was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKC delta-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells.     

Protein kinase C plays a role in the induction of tyrosine phosphorylation of lymphoid microtubule-associated protein-2 kinase. Evidence for a CD3-associated cascade that includes pp56lck and that is defective in HPB-ALL.

Ligation of the CD3 receptor induces multiple signal transduction events that modify the activation state of the T cell. We have compared two lines that express biologically active CD3 receptors but differ in their biochemical activation pathways during ligation of this receptor. Jurkat cells respond to anti-CD3 with Ca2+ mobilization, PKC activation, induction of protein tyrosine phosphorylation, and activation of newly characterized lymphoid microtubule associated protein-2 kinase (MAP-2K). MAP-2K itself is a 43-kDa phosphoprotein that requires tyrosine phosphorylation for activation. Although ligation of the CD3 receptor in HPB-ALL could stimulate tyrosine phosphorylation of a 59- kDa substrate, there was no associated induction of [Ca2+]i flux, PKC, or MAP-2K activation. A specific PKC agonist, PMA, which bypasses the CD3 receptor, could, however, activate MAP-2K in HPB-ALL cells. This implies that defective stimulation of PKC by the CD3 receptor is responsible for its failure to activate MAP-2K in HPB-ALL. The defect in PKC activation is likely distal to the CD3 receptor as A1F14- failed to activate MAP-2K in HPB-ALL but was effective in Jurkat cells. The stimulatory effect of PMA on MAP-2K activity in HPB-ALL was accompanied by tyrosine phosphorylation of this kinase which implies that PKC may, in some way, regulate tyrosine phosphorylation of MAP-2K. A candidate for this role is pp56lck which underwent posttranslational modification (seen as mobility change on SDS-PAGE) during anti-CD3 and PMA stimulation in Jurkat or PMA treatment in HPB-ALL. There was, in fact, exact coincidence between induction of PKC activity, posttranslational modification of lck and tyrosine phosphorylation/activation of MAP-2K. Lck kinase activity in an immune complex kinase assay was unchanged during PMA treatment. An alternative explanation is that modification of lck may alter its substrate profile. We therefore looked at the previously documented ability of PKC to dissociate lck from the CD4 receptor and found that PMA could reduce the stoichiometry of the lck interaction with CD4 in HPB-ALL and to a lesser extent in Jurkat cells. These results imply the existence of a kinase cascade that is initiated by PKC and, in the course of which, lck and MAP-2K may interact.     

Characteristics of the F52 protein, a MARCKS homologue.

A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.     

Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C

The kinetics of phosphorylation of an integral membrane enzyme, Na+/K(+)-ATPase, by calcium- and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic alpha-subunit of Na+/K(+)-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (Ka approximately 1.0 microM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 microM). PKC phosphorylation of Na+/K(+)-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+/K(+)-ATPase as a substrate was 0.5 microM for dog kidney enzyme and 0.3 microM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+/K(+)-ATPase greater than 1.0 microM. Phosphorylation of purified kidney and salt-gland Na+/K+ ATPases occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated alpha subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+/K(+)-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+/K(+)-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins.     

Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor.

Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pasteurella multocida toxin, a potent mitogen, stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells

Pasteurella multocida toxin, either native or recombinant (rPMT), is an extremely effective mitogen for Swiss 3T3 cells and acts at picomolar concentrations (Rozengurt, E., Higgins, T. E., Chanter, N., Lax, A. J., and Staddon, J. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 123-127). Here, we show that similar concentrations of rPMT markedly stimulated the phosphorylation of an acidic 80-kDa protein in [32P]Pi-labeled Swiss 3T3 cells. Co-migration on one- and two-dimensional gels and phosphopeptide analysis indicated that this phosphoprotein was indistinguishable from 80K, a known protein kinase C substrate. In parallel cultures, the stimulation of 80K phosphorylation by rPMT (5-10-fold) was comparable to that induced by bombesin or phorbol dibutyrate (PBt2). However, the increase in phosphorylation by rPMT occurred after a pronounced lag period (1-3 h, depending upon the concentration of rPMT) in contrast to the relatively immediate stimulation by PBt2 or bombesin. Early, but not late, addition of either PMT antiserum or the lysosomotrophic agent methylamine selectively inhibited 80K phosphorylation in response to rPMT. 80K phosphorylation persisted after removal of free toxin and was not inhibited by cycloheximide. It appears that rPMT enters the cells via an endocytotic pathway to initiate and perpetuate events leading to 80K phosphorylation. rPMT, like PBt2, also stimulated the phosphorylation of 87-kDa and 33-kDa proteins in Swiss 3T3 cells. Phosphorylation of the 80K and 87-kDa proteins by rPMT or PBt2 were greatly attenuated in cells depleted of protein kinase C. In contrast, phosphorylation of the 33-kDa protein by rPMT, but not by PBt2, persisted in the absence of protein kinase C. rPMT, like bombesin, caused a translocation of protein kinase C to the cellular particulate fraction. The toxin enhanced the cellular content of diacylglycerol. rPMT also caused a time- and dose-dependent decrease in the binding of 125I-epidermal growth factor to its receptor which was blocked by methylamine and dependent only in part upon the presence of protein kinase C. We conclude that rPMT stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells.