Multiple kinases and signal transduction. Phosphorylation of the T cell antigen receptor complex

Multiple kinases interact at the multicomponent murine T cell antigen receptor. Antigen induces serine phosphorylation of the 21-kDa gamma glycoprotein and tyrosine phosphorylation of p21, a distinct 21-kDa chain. We demonstrate that tyrosine phosphorylation is due to kinase activation, and that all phosphorylated p21 is associated with the antigen receptor. We also show that antigen leads to polyphosphoinositide metabolism and subsequent protein kinase C activation. The two phosphorylation events can be dissociated by protein kinase C depletion, which eliminates phorbol 12-myristate 13-acetate-induced serine but not tyrosine phosphorylation. Activation of a third kinase, cyclic AMP-dependent protein kinase, inhibits both serine and tyrosine events, yet this inhibition can be modulated by addition of the protein kinase C activator, phorbol 12-myristate 13-acetate. Receptor-mediated signal transduction may be understood as the interaction of multiple stimulatory and inhibitory kinase activities.     

Vinculin phosphorylation in response to calcium and phorbol esters in intact cells

Vinculin phosphorylation in both chick embryo fibroblasts and Swiss 3T3 cells was increased by either calcium or biologically active phorbol esters. Increased phosphorylation of vinculin was noted as early as 10 min following phorbol 12-myristate 13-acetate treatment and was maximal at about 1 h. Maximal increases in phosphorylation were noted at approximately 100 nM phorbol 12-myristate 13-acetate. Phorbol 12,13-dibutyrate (80 nM), a less potent phorbol ester, resulted in smaller increases in vinculin phosphorylation than phorbol 12-myristate 13-acetate at equimolar concentrations. Phorbol, dibutyryl cAMP, and dibutyryl cGMP had no significant effect on phosphorylation. No correlation was found between vinculin phosphorylation and the morphological changes induced by phorbol esters. Tryptic peptide analysis of vinculin revealed multisite phosphorylation. Phosphorylation of only three of the peptides was significantly increased following phorbol 12-myristate 13-acetate treatment. Phosphoamino acid analysis revealed increases at both serine and threonine residues. The low level of phosphotyrosine present in control cells was not significantly increased by phorbol 12-myristate 13-acetate treatment. These findings combined with studies of vinculin phosphorylation by purified protein kinase C (Werth, D. K., Niedel, J. E., and Pastan I. (1983) J. Biol. Chem. 258, 11423-11426) suggest the hypothesis that protein kinase C may be involved in regulation of phosphorylation of vinculin, a cytoskeletal protein.     

Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.     

Phosphorylation of the CD20 phosphoprotein in resting B lymphocytes. Regulation by protein kinase C

CD20, a B cell integral membrane protein, regulates B cell activation and is differently phosphorylated in resting and activated cells. We have previously shown that CD20 phosphorylation is increased in activated cells and in phorbol ester-treated resting cells. Phosphorylation is also altered by agents which signal B cell proliferation, such as anti-IgM and a B cell growth factor. The present study was designed to address whether protein kinase C (PKC) or other kinases used CD20 as a substrate. When purified PKC was incubated with isolated CD20, both the 35- and 37-kDa CD20 proteins were phosphorylated in vitro. Intact resting B cells were next incubated with the protein kinase inhibitors H-7, H-8, and W-7. No change in basal CD20 phosphorylation was observed in the presence of W-7 and H-8, indicating that the protein cyclic nucleotide-dependent and calmodulin-dependent kinases were not actively phosphorylating CD20. Surprisingly, the PKC inhibitor H-7 increased CD20 phosphorylation at concentrations above 25-50 microM. To assess whether PKC either activated a phosphatase or inactivated a kinase affecting CD20 phosphorylation, tryptic phosphopeptide mapping of CD20 was performed. These studies revealed that addition of phorbol 12-myristate 13-acetate increased phosphorylation of some peptides differing from those which had increased phosphorylation following addition of H-7. Furthermore, signalling through surface immunoglobulin increased phosphorylation of CD20 peptides distinct from those hyperphosphorylated following addition of phorbol 12-myristate 13-acetate. These results demonstrate that 1) CD20 has multiple phosphorylation sites, as predicted from sequence data, and 2) whereas PKC can use CD20 as substrate, at least one other unidentified kinase phosphorylates CD20 in resting cells. Our data also predict that activation of B cells via the antigen receptor (surface IgM) may activate other protein kinases in addition to PKC.     

Protein kinase C phosphorylates glutamyl-tRNA synthetase in rabbit reticulocytes stimulated by tumor promoting phorbol esters

A high Mr synthetase core complex isolated from higher eukaryotes contains aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline. Previously, five of the synthetases were shown to be phosphorylated in reticulocytes, and the glutaminyl- and aspartyl-tRNA synthetases were shown to be selectively phosphorylated in response to 8-bromo cAMP (Pendergast, A. M., Venema, R. C., and Traugh, J. A. (1987) J. Biol. Chem. 262, 5939-5942). Exposure of reticulocytes to phorbol 12-myristate 13-acetate stimulates the selective phosphorylation of one synthetase in the complex, glutamyl-tRNA synthetase. Only the glutamyl-tRNA synthetase is modified to a significant extent when the purified complex is phosphorylated in vitro by protein kinase C; up to 0.7 mol of phosphate is incorporated per mol of synthetase. Two-dimensional phosphopeptide mapping shows a single tryptic phosphopeptide, which is identical for the enzyme modified in vitro by protein kinase C or in phorbol 12-myristate 13-acetate-stimulated cells. Phosphorylation in vivo is reproducibly accompanied by a 38 +/- 10% reduction in aminoacylation activity of partially purified glutamyl-tRNA synthetase assayed in vitro. Phosphorylation in vitro has no detectable effect on aminoacylation. This difference may be due to the absence of a required effector molecule which alters activity by interaction with the phosphorylated synthetase. Glutamyl-tRNA synthetase is one of a growing number of translational components, including initiation factors, which are coordinately modified by protein kinase C in response to phorbol 12-myristate 13-acetate.     

Antigen activation of murine T cells induces tyrosine phosphorylation of a polypeptide associated with the T cell antigen receptor

The antigen receptor complex on murine MHC class II-restricted T cells consists of disulfide-linked alpha and beta chains noncovalently associated with four additional polypeptides, two that are endoglycosaminidase F-sensitive, gp26 and gp21, and two that are endoglycosaminidase F-resistant, p25 and p16. We demonstrate here that treatment of murine T cell hybridomas with phorbol 12-myristate 13-acetate results in phosphorylation of p25 and gp21 on serine residues. However, activation of cells by antigen results in the phosphorylation of the gp21 chain and a heretofore unidentified 21 kd protein. This newly defined polypeptide, p21, is specifically immunoprecipitated with the antigen receptor complex, is endoglycosaminidase F-resistant, and is itself part of a disulfide-linked molecule. Unlike antigen-induced phosphorylation of gp21, which occurs on serine residues, phosphorylation of p21 occurs uniquely on tyrosine residues.     

Effect of phorbol 12-myristate 13-acetate and its analogue 4 alpha-phorbol 12,13-didecanoate on protein phosphorylation and lysosomal enzyme release in rabbit neutrophils

The co-carcinogenic compound phorbol 12-myristate 13-acetate but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate causes the phosphorylation of several rabbit neutrophil polypeptides whose molecular weights and isoelectric points (pI) are as follows: Mr = 40,000, pI = 6.4; Mr = 50,000, pI = 4.9; Mr = 55,000, pI = 6.3; Mr = 64,000, pI = 6.0; Mr = 70,000, pI = 5.6; Mr = 90,000, pI = 6.0. Most of these phosphorylated proteins are located exclusively in the cytosol; the 64,000 molecular weight protein is found both in the cytosol and the cytoskeleton, and the 40,000 molecular weight protein is found in the nuclear pellet. The 50,000 molecular weight protein is also phosphorylated in whole cells by the chemotactic peptide fMet-Leu-Phe and in cell-free systems by protein kinase C. Using limited proteolysis, one phosphopeptide fragment was phosphorylated by the three stimuli. In addition, phorbol 12-myristate 13-acetate but not 4 alpha-phorbol 12,13-didecanoate causes cell aggregation and the exocytotic release of the specific granules of rabbit neutrophils. In contrast, both compounds increase the amount of actin associated with the cytoskeleton. The divalent cation ionophore A23187 at low concentration and the compound phorbol 12-myristate 13-acetate act synergistically in causing neutrophil degranulation. Lysosomal enzyme release and the phosphorylation of the 50,000 molecular weight polypeptide produced by phorbl 12-myristate 13-acetate are inhibited by trifluoperazine, and these two responses seem to be causally related. These results are discussed in terms of the role of 1,2-diacylglycerol and activation of protein kinase C in specific granule release from rabbit neutrophils.     

Functional characterization of phosphorylation of 69-kDa human choline acetyltransferase at serine 440 by protein kinase C

Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and site-directed mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time- and dose-related increases in specific activity of the enzyme; in control and phorbol ester-treated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [(32)P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.     

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.     

Chromaffin cell F-actin disassembly and potentiation of catecholamine release in response to protein kinase C activation by phorbol esters is mediated through myristoylated alanine-rich C kinase substrate phosphorylation

The large majority of chromaffin vesicles are excluded from the plasma membrane by a cortical F-actin network. Treatment of chromaffin cells with phorbol 12-myristate 13-acetate produces disassembly of cortical F-actin, increasing the number of vesicles at release sites (Vitale, M. L., Seward, E. P., and Trifaró, J. M. (1995) Neuron 14, 353-363). Here, we provide evidence for involvement of myristoylated alanine-rich protein kinase C substrate (MARCKS), a protein kinase C substrate, in chromaffin cell secretion. MARCKS binds and cross-links F-actin, the latter is inhibited by protein kinase C-induced MARCKS phosphorylation. MARCKS was found in chromaffin cells by immunoblotting. MARCKS was also detected by immunoprecipitation. In intact or permeabilized cells MARCKS phosphorylation increased upon stimulation with 10(-7) m phorbol 12-myristate 13-acetate. This was accompanied by cortical F-actin disassembly and potentiation of secretion. MARCKS phosphorylation, cortical F-actin disassembly, and potentiation of Ca(2+)-evoked secretion were inhibited by a peptide (MARCKS phosphorylation site domain sequence (MPSD)) with amino acid sequence corresponding to MARCKS phosphorylation site. MPSD was phosphorylated in the process. A similar peptide (alanine-substituted phosphorylated site domain) with four serine residues of MPSD substituted by alanines was ineffective. These results provide the first evidence for MARCKS involvement in chromaffin cell secretion and suggest that regulation of cortical F-actin cross-linking might be involved in this process.     

Protein kinase C-stimulated phosphorylation in vitro of a Mr 80,000 protein phosphorylated in response to phorbol esters and growth factors in intact fibroblasts. Distinction from protein kinase C and prominence in brain

In previous studies in intact 3T3-L1 fibroblasts and adipocytes, we demonstrated that the phosphorylation state of an acidic, multicomponent Mr 80,000 protein appeared to be a specific and useful marker for the activation state of protein kinase C (Blackshear, P.J., Witters, L.A., Girard, P.R., Kuo, J.F., and Quamo, S.N. (1985) J. Biol. Chem. 260, 13304-13315). In the present studies, we demonstrate that the Mr 80,000 protein from rat adipose tissue was a substrate for protein kinase C in vitro, and co-migrated on two-dimensional gels with the analogous protein from murine 3T3-L1 adipocytes labeled by exposure of intact cells to 32Pi and phorbol 12-myristate 13-acetate. Partial proteolytic maps of the two 32P-proteins were nearly identical, supporting the postulate that the sites phosphorylated by protein kinase C in vitro, and in response to phorbol 12-myristate 13-acetate in vivo, were similar or identical. Despite their similar apparent molecular weights, we were able to distinguish between the Mr 80,000 protein and protein kinase C by several physical criteria. The Mr 80,000 protein kinase C substrate was found in fractions of all rat tissues examined, but was most prominent in rat brain. Phorbol 12-myristate 13-acetate also stimulated phosphorylation of the Mr 80,000 protein in several types of cultured neuronal cells, suggesting a possible role for this protein in cholinergic neurotransmission. The Mr 80,000 protein appears to be a useful marker for protein kinase C activation in a variety of cell types.     

Phosphorylated form of MacMARCKS is essential to LFA-1-dependent cell-cell adhesion of U937 monocytic cells.

MacMARCKS (MRP, F52), a protein kinase C (PKC) substrate, is involved in the activation of beta2-integrin. To determine the role of the PKC-mediated phosphorylation of MacMARCKS in this process, human U937 monocytic cells were transfected with cDNAs encoding wild type or mutant MacMARCKS. We observed that the expression of the exogenous wild type MacMARCKS greatly enhanced LFA-1-mediated cell-cell adhesion in U937 cells treated with phorbol 12-myristate 13-acetate (PMA). This MacMARCKS-stimulated adhesion depended on the phosphorylation status of MacMARCKS: whereas phosphorylated MacMARCKS enhanced adhesion, unphosphorylated MacMARCKS inhibited it. However, phosphorylated MacMARCKS alone could not induce LFA-1-mediated cell-cell adhesion unless phorbol esters were added, suggesting that the phosphorylation of other proteins might also be involved. Okadaic acid, a phosphatase inhibitor, induced LFA-1-mediated cell-cell adhesion only in the cells expressing wild type or phosphorylated MacMARCKS and not in the cells expressing unphosphorylated MacMARCKS. Therefore, we conclude that the phosphorylated form of MacMARCKS is essential to LFA-1-mediated cell-cell adhesion.     

B cell receptor-induced cAMP-response element-binding protein activation in B lymphocytes requires novel protein kinase Cdelta

The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKCdelta (nPKCdelta) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor G?6976, which targets conventional PKCs and PKCmu, has no effect on CREB phosphorylation, whereas the nPKCdelta inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKCdelta depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKCdelta blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKCdelta-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase Cgamma2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase Cgamma2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKCdelta. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKCdelta and p90 RSK in mature B cells.     

Protein kinase D-mediated phosphorylation and nuclear export of sphingosine kinase 2

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling.     

Insulin activation of protein kinase C: a reassessment

Although insulin is known to activate several protein serine/threonine protein kinases, its ability to activate protein kinase C remains controversial. We reinvestigated this question, taking advantage of several technical advances such as the development of fibroblast cell lines that overexpress normal human insulin receptors, and the development of antibodies to and expression vectors for the myristoylated, alanine-rich C kinase substrate (MARCKS) protein, a major cellular substrate for protein kinase C. In HIR 3.5 cells, a mouse 3T3 cell derivative that expresses about 6 x 10(6) human insulin receptors/cell, insulin (70 nM for 10 min) stimulated phosphorylation of the MARCKS protein by approximately 2-fold (p less than 0.005). This phosphorylation was not further increased by different times of insulin exposure, different insulin concentrations, or longer periods of serum deprivation. The insulin stimulation represented about 14% of the response to phorbol 12-myristate 13-acetate and about 17% of the response to 10% fetal calf serum. No significant stimulation of MARCKS protein phosphorylation was seen in four other insulin-sensitive cell lines, in which insulin is known to activate other protein serine/threonine kinases: HIRC-B, BC3H-1, 3T3-L1 adipocytes, and H35 rat hepatoma cells made to stably express the MARCKS protein. In these four cell lines, serum and/or phorbol 12-myristate 13-acetate exerted a large stimulatory effect on MARCKS protein phosphorylation. We conclude that insulin may activate protein kinase C to a minor extent in certain cell types that vastly overexpress insulin receptors; however, we believe that this effect of insulin is unlikely to be of physiological importance.     

Phosphorylation of translation initiation factor eIF-4E is induced in a ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation.

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Consistent with this, overexpression of eIF-4E in fibroblasts results in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylation on a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor (NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympathetic neurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment, resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermal growth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhance eIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarily a protein kinase C-independent response. The NGF-induced enhancement of eIF-4E phosphorylation is abrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating that eIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effects translation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation may play a role in switching the pattern of gene expression during the differentiation of PC12 cells.     

Activation of a human c-K-ras oncogene

The human lung carcinomas PR310 and PR371 contain activated c-K-ras oncogenes. The oncogene of PR371 was found to present a mutation at codon 12 of the first coding exon which substitutes cysteine for glycine in the encoded p21 protein. We report here that the transforming gene of PR310 tumor contains a mutation in the second coding exon. An A----T transversion at codon 61 results in the incorporation of histidine instead of glutamine in the c-K-ras gene product. By constructing c-K-ras/c-H-ras chimeric genes we show that this point mutation is sufficient to confer transforming potential to ras genes, and that a hybrid ras gene coding for a protein mutant at both codons 12 and 61 is also capable of transforming NIH3T3 cells. The relative transforming potency of p21 proteins encoded by ras genes mutant at codons 12, 61 or both has been analyzed. Our studies also show that the coding exons of ras genes, including the fourth, can be interchanged and the chimeric p21 ras proteins retain their oncogenic ability in normal rodent established cell lines.     

Phosphorylation of coronin 1B by protein kinase C regulates interaction with Arp2/3 and cell motility

Coronins are a conserved family of WD repeat-containing, actin-binding proteins that regulate cell motility in a variety of model organisms. Our results show that Coronin 1B is a ubiquitously expressed member of the mammalian Coronin gene family that co-localizes with the Arp2/3 complex at the leading edge of fibroblasts, and co-immunoprecipitates with this complex. Pharmacological experiments show that the interaction between Coronin 1B and the Arp2/3 complex is regulated by protein kinase C (PKC) phosphorylation. Coronin 1B is phosphorylated by PKC both in vitro and in vivo. Using tryptic peptide mapping and mutagenesis, we have identified serine 2 (Ser-2) on Coronin 1B as the major residue phosphorylated by PKC in vivo. Rat2 fibroblasts expressing the Coronin 1B S2A mutant show enhanced ruffling in response to phorbol 12-myristate 13-acetate (PMA) and increased speed in single cell tracking assays. Cells expressing the Coronin 1B S2D mutant have attenuated PMA-induced ruffling and slower cell speed. Expression of the S2A mutant partially protects cells from the inhibitory effects of PMA on cell speed, whereas expression of the S2D mutant renders cells hypersensitive to its effects. These data demonstrate that Coronin 1B regulates leading edge dynamics and cell motility in fibroblasts, and that its ability to control motility and interactions with the Arp2/3 complex are regulated by PKC phosphorylation at Ser-2. Furthermore, Coronin 1B phosphorylation is responsible for a significant fraction of the effects of PMA on fibroblast motility.     

Biosynthesis and post-translational modification of CD6, a T cell signal-transducing molecule

CD6 (T12) is a 130-kDa glycoprotein present on the surface of human T cells. Previously, we demonstrated that the anti-T12 and anti-2H1 monoclonal antibodies recognized different epitopes on CD6, and both were capable of transducing activation signals to T cells. Anti-T12 augmented suboptimal signaling via the TCR/CD3 complex and directly activated separated CD4+ but not CD8+ cells. Structural characterization of CD6 revealed that it contained intrachain disulfide bonds, was N-glycosylated, and in activated cells was phosphorylated on serine. Given the functional significance of CD6 and its involvement in signaling via CD3 and CD2 pathways, we examined in detail the biosynthesis, structural characteristics, and phosphorylation properties of this receptor-like molecule. These studies demonstrate that the nascent CD6 polypeptide on both T cells and thymocytes in 88 kDa, and the immature N-glycosylated form is 110 kDa. After maturation of N-linked glycan and addition of sulfated O-linked oligosaccharide, CD6 appears on the cell surface as a molecule of 130 kDa. CD6 is phosphorylated in resting cells and can be hyperphosphorylated when stimulated by phorbol 12-myristate 13-acetate, indicating that it may participate in the major common signaling pathway mediated through protein kinase C. Concanavalin A-activated cells are phosphorylated at an additional site(s) on the molecule and cannot be hyperphosphorylated with phorbol 12-myristate 13-acetate. These physical features reveal additional clues about the physiological role of CD6 and its mechanism of signal transduction and strongly suggest that CD6 represents a physiologically important membrane receptor involved in T cell activation.     

Increased rate of phosphorylation-dephosphorylation of the translational initiation factor eIF-4E correlates with the induction of protein and glycoprotein biosynthesis in activated B lymphocytes

A 10-50-fold, biphasic increase in the rate of 32Pi labeling of eIF-4E was closely correlated with the induction of protein and glycoprotein biosynthesis when resting murine splenic B lymphocytes (B cells) were activated by bacterial lipopolysaccharide or the combination of phorbol 12-myristate 13-acetate and ionomycin. The fraction of eIF-4E which was phosphorylated only increased from 46% in resting cells to 83% in lipopolysaccharide-activated cells. This discrepancy between the increase in the fraction of phosphorylated eIF-4E and the increase in 32Pi labeling suggested that the phosphoryl group of eIF-4E turns over slowly in resting B cells compared with activated cells. The turnover rate for the eIF-4E phosphate moiety in lipopolysaccharide-activated cells was rapid (t1/2 = 2 h) in comparison to the eIF-4E polypeptide chain, which did not turn over detectably in 6 h. Neither protein kinase C nor a cyclic nucleotide-dependent protein kinase appeared to be involved in eIF-4E phosphorylation in B cells, based on the observations that the metabolic labeling of eIF-4E by 32Pi was insensitive to the protein kinase inhibitors H-7 and HA1004, and that maximal labeling occurred after protein kinase C activity was "down-regulated" to very low levels in phorbol 12-myristate 13-acetate/ionomycin-activated cells. Dephosphorylation in vivo was blocked by okadaic acid (IC50 = 200 nM). These results indicate that a rapid phosphorylation-dephosphorylation of eIF-4E is associated with high translation rates during the activation of B cells, and implicate protein phosphatase-1 (or possibly-2A) in the dephosphorylation of the initiation factor.