Inhibition of CD3-linked phospholipase C by phorbol ester and by cAMP is associated with decreased phosphotyrosine and increased phosphoserine contents of PLC-gamma 1.

The mechanisms by which phorbol 12-myristate 13-acetate (PMA) and cAMP attenuate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) induced by ligation of the T-cell antigen receptor complex (TCR) was studied in the human Jurkat T-cell line. It has previously been shown that stimulation of Jurkat cells with antibodies to CD3, components of the TCR, elicits a rapid and transient phosphorylation of phospholipase C (PLC)-gamma 1, the predominant PLC isozyme in Jurkat cells, at multiple tyrosine residues and that such tyrosine phosphorylation leads to activation of PLC-gamma 1. Prior incubation of Jurkat cells with PMA or forskolin, which increases intracellular cAMP concentrations, prevented tyrosine phosphorylation of PLC-gamma 1 as well as the hydrolysis of PtdIns 4,5-P2 induced by ligation of CD3. Dose-response curves of PMA and of forskolin for the inhibition of PLC-gamma 1 tyrosine phosphorylation and of PtdIns 4,5-P2 hydrolysis were similar. These results suggest that the inhibition of PtdIns 4,5-P2 hydrolysis by PMA and cAMP is attributable to reduced tyrosine phosphorylation of PLC-gamma 1. Treatment of Jurkat cells with PMA or forskolin stimulated the phosphorylation of PLC-gamma 1 at serine 1248. PMA treatment also elicited the phosphorylation of PLC-gamma 1 at an unidentified serine site. Phosphopeptide map analysis indicated that the sites of PLC-gamma 1 phosphorylated in Jurkat cells treated with PMA and forskolin are the same as those phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), respectively. Stimulation of Jurkat cells with antibodies to CD3 also elicited phosphorylation of PLC-gamma 1 at serine 1248 and at the unidentified serine site phosphorylated in PLC-gamma 1 from PMA-treated cells. Thus, phosphorylation of PLC-gamma 1 by PKC or PKA at serine 1248 may modulate the interaction of PLC-gamma 1 with the protein tyrosine kinase or the protein tyrosine phosphatase; this altered interaction may, at least in part, be responsible for the decreased tyrosine phosphorylation of PLC-gamma 1 seen in PMA- and forskolin-treated Jurkat cells. Furthermore, in the absence of PMA, activation of PKC by diacylglycerol provides a negative feedback signal responsible for reducing the phosphotyrosine contents of PLC-gamma 1.     

cAMP-mediated modulation of signal transduction of epidermal growth factor (EGF) receptor systems in human epidermoid carcinoma A431 cells. Depression of EGF-dependent diacylglycerol production and EGF receptor phosphorylation.

While a cAMP-dependent protein kinase (protein kinase A) has been suggested to phosphorylate epidermal growth factor (EGF) receptor in vitro, both intrinsic and EGF- or potent phorbol tumor promoter-induced phosphorylation of EGF receptor were found to be depressed in human epidermoid carcinoma A431 cells by prior incubation of the cells with various protein kinase A activators (e.g. cholera toxin, forskolin, cAMP analogues, or a combination of prostaglandin E1 and 3-isobutyl-1-methylxanthine). Protein kinase A activators did not change significantly either the number of EGF receptors or their affinity for EGF. The tryptic phosphopeptide map of EGF receptors from cells treated with cholera toxin alone or cholera toxin followed by EGF revealed unique peptides whose serine phosphorylation was preferentially depressed. However, the catalytic subunit of protein kinase A phosphorylated no threonine and little serine in the EGF receptors in the plasma membranes of isolated A431 cells in vitro, while serine residues in an unidentified 170-kDa membrane protein(s) other than EGF receptor were heavily phosphorylated. Pretreatment of the cells with forskolin blocked 1,2-diacylglycerol induction by EGF; growth inhibition by nanomolar levels of EGF could be partially restored by the presence of forskolin. These results indicate that an increase in intracellular cAMP modulates the EGF receptor signal transduction system by reducing EGF-induced production of diacylglycerol without direct phosphorylation of EGF receptors by protein kinase A in A431 cells.     

Phosphorylation of phospholipase C-gamma by cAMP-dependent protein kinase

The mechanism by which cAMP modulates the activity of phosphoinositide-specific phospholipase C (PLC) was studied. Elevation of cAMP inhibited both basal and norepinephrine-stimulated phosphoinositide breakdown in C6Bu1 cells which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of C6Bu1 cells with cAMP-elevating agents (cholera toxin, isobutylmethylxanthine, forskolin, and 8-bromo-cAMP) increased serine phosphate in PLC-gamma, but the phosphate contents in PLC-beta and PLC-delta were not changed. In addition, cAMP-dependent protein kinase selectively phosphorylated purified PLC-gamma among the three isozymes and added a single phosphate at serine. The serine phosphorylation, nevertheless, did not affect the activity of PLC-gamma in vitro. We propose, therefore, that the phosphorylation of PLC-gamma by cAMP-dependent protein kinase alters its interaction with putative modulatory proteins and leads to its inhibition.     

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

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

The SH2/SH3 domain-containing protein Nck is recognized by certain anti-phospholipase C-gamma 1 monoclonal antibodies, and its phosphorylation on tyrosine is stimulated by platelet-derived growth factor and epidermal growth factor treatment.

In the course of our investigation of phospholipase C (PLC)-gamma 1 phosphorylation by using a set of anti-PLC-gamma 1 monoclonal antibodies (P.-G. Suh, S. H. Ryu, W. C. Choi, K.-Y. Lee, and S. G. Rhee, J. Biol. Chem. 263:14497-14504, 1988), we found that some of these antibodies directly recognize a 47-kDa protein. We show here that this 47-kDa protein is identical to the SH2/SH3-containing protein Nck (J. M. Lehmann, G. Riethmuller, and J. P. Johnson, Nucleic Acids Res. 18:1048, 1990). Nck was found to be constitutively phosphorylated on serine in resting NIH 3T3 cells. Platelet-derived growth factor (PDGF) treatment led to increased Nck phosphorylation on both tyrosine and serine. Nck was also found to be phosphorylated on tyrosine in epidermal growth factor (EGF)-treated A431 cells and in v-Src-transformed NIH 3T3 cells. Multiple sites of serine phosphorylation were detected in Nck from resting cells, and no novel sites were found upon PDGF or EGF treatment. A single major tyrosine phosphorylation site was found in Nck in both PDGF- and EGF-treated cells and in v-Src-transformed cells. This same tyrosine was phosphorylated in vitro by purified PDGF and EGF receptors and also by pp60c-src. We compared the phosphorylation of Nck and PLC-gamma 1 in several cell lines transformed by oncogenes with different modes of transformation. Although PLC-gamma 1 and Nck have significant amino acid identity, particularly in their SH3 regions, and both associate with growth factor receptors in a ligand-dependent manner, they were not always phosphorylated on tyrosine in a coincident manner.     

Tyrosine kinase activity is essential for the association of phospholipase C-gamma with the epidermal growth factor receptor.

Epidermal growth factor (EGF) treatment of NIH 3T3 cells transfected with wild-type EGF receptor induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). The EGF receptor and PLC-gamma were found to be physically associated such that antibodies directed against PLC-gamma or the EGF receptor coimmunoprecipitated both proteins. The association between PLC-gamma and wild-type EGF receptor was dependent on the concentration of EGF, but EGF did not enhance the association between PLC-gamma and a kinase-negative mutant of the EGF receptor. Oligomerization of the EGF receptor was not sufficient to induce association of the EGF receptor with PLC-gamma, since the kinase-negative mutant receptor underwent normal dimerization in response to EGF yet did not associate with PLC-gamma. The form of PLC-gamma associated with the EGF receptor appeared to be primarily the non-tyrosine-phosphorylated form. It is concluded that the kinase activity of the EGF receptor is essential for association of PLC-gamma with the EGF receptor, possibly by stimulating receptor autophosphorylation.     

The tyrosine phosphorylated carboxyterminus of the EGF receptor is a binding site for GAP and PLC-gamma.

Phospholipase C-gamma (PLC-gamma) and GTPase activating protein (GAP) are substrates of EGF, PDGF and other growth factor receptors. Since either PLC-gamma or GAP also bind to the activated receptors it was suggested that their SH2 domains are mediating this association. We attempted to delineate the specific region of the EGF receptor that is responsible for the binding, utilizing EGF receptor mutants, PLC-gamma, and a bacterially expressed TRP E fusion protein containing the SH2 domains of GAP. As previously shown, tyrosine autophosphorylation of the wild-type receptor wsa crucial in mediating the association and in agreement, a kinase negative EGF receptor could bind PLC-gamma or TRP E GAP SH2, but only when cross tyrosine phosphorylated by an active EGF receptor kinase. The importance of autophosphorylation for association was confirmed by demonstrating that a carboxy-terminal deletion of the EGFR missing four autophosphorylation sites bound these proteins poorly. To study the role of EGF receptor autophosphorylation further, a 203 amino acid EGF receptor fragment was generated with cyanogen bromide that contained all known tyrosine autophosphorylation sites. This fragment bound both TRP E GAP SH2 and PLC-gamma but only when tyrosine phosphorylated. This data localizes a major binding site for SH2 domain containing proteins to the carboxy-terminus of the EGF receptor and points to the importance of tyrosine phosphorylation in mediating this association.     

Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP.

The 47-kDa protein coimmunoprecipitated with phospholipase C (PLC)-gamma 1 by anti-PLC-gamma 1 monoclonal antibodies is proved to be Nck, a protein composed almost exclusively of one SH2 and three SH3 domains. Nck and PLC-gamma 1 are recognized by certain anti-PLC-gamma 1 monoclonal antibodies because Nck and PLC-gamma 1 share an epitope that likely is located in their SH3 domains. Nck is widely distributed in rat tissues, with an especially high level of expression in testes. The expression levels of Nck remains unchanged during the development of rat brain, whereas PLC-gamma 1 decreases during the same developmental period. Stimulation of A431 cells with epidermal growth factor elicits the tight association of Nck with the epidermal growth factor receptor and phosphorylation of Nck on both serine and tyrosine residues. The phosphorylation of Nck is also enhanced in response to stimulation of the nerve growth factor receptor in PC12 cells, the T-cell receptor complex in Jurkat cells, the membrane immunoglobulin M in Daudi cells, and the low-affinity immunoglobulin G receptor (Fc gamma RII) in U937 cells. The phosphorylation of Nck was also enhanced following treatment of A431 cells with phorbol 12-myristate 13-acetate or forskolin. These results suggest that Nck is a target for a variety of protein kinases that might modulate the postulated role of Nck as an adaptor for the physical and functional coordination of signalling proteins.     

Pertussis toxin-sensitive Gi protein involvement in epidermal growth factor-induced activation of phospholipase C-gamma in rat hepatocytes.

Treatment of rat hepatocytes with epidermal growth factor (EGF) produced an enhanced tyrosine phosphorylation of the EGF receptor and phospholipase C-gamma (PLC-gamma) in conjunction with the mobilization of Ca2+. Approximately 30% of the total PLC-gamma was tyrosine-phosphorylated with a maximum being reached after 30 s of incubation with EGF. Pretreatment of the rats with pertussis toxin prior to isolation of the hepatocytes blocked EGF-induced tyrosine phosphorylation of PLC-gamma and Ca2+ mobilization but had no effect on autophosphorylation of the EGF receptor or Ca2+ responses elicited by angiotensin II or phenylephrine. Under these conditions Gi protein alpha subunits were fully ADP-ribosylated. A 41-kDa Gi protein alpha subunit was found to be present in the anti-PLC-gamma immune complex after EGF stimulation as shown by in vitro ADP-ribosylation using [32P]NAD+ and activated pertussis toxin. The kinetics of association between PLC-gamma with Gi alpha protein reached a maximum after 1 min of incubation with EGF. Antibodies specific for the EGF receptor also coimmunoprecipitated a Gi protein alpha subunit. Treatment of hepatocytes with EGF caused first an increase and then a decrease in the amount of Gi protein alpha subunit associated with the EGF receptor. In contrast, studies with cultured rat liver (WB) cells, a cell line in which EGF stimulation of phosphoinositide hydrolysis is not inhibited by pertussis toxin, showed that a stable complex of Gi alpha was not formed with either PLC-gamma or EGF receptor immunoprecipitates. These results indicate that a pertussis toxin-sensitive Gi protein is uniquely involved in the signal transduction pathway mediating EGF-induced activation of PLC-gamma and Ca2+ mobilization in hepatocytes.     

Chronic ethanol consumption disrupts complexation between EGF receptor and phospholipase C-gamma1: relevance to impaired hepatocyte proliferation

We have previously shown that chronic ethanol consumption inhibits liver regeneration by impairing EGF receptor (EGFR)-operated phospholipase C-gamma1 (PLC-gamma1) activation and resultant intracellular Ca2+ signalling. Activation of PLC-gamma1 by EGFR requires the EGFR to bind to PLC-gamma1 after its translocation from cytosol to cytoskeleton. In order to understand the mechanism by which ethanol impairs PLC-gamma1 activation, we examined the effect of alcohol on interactions between EGFR and PLC-gamma1. In cultured hepatocytes from control rats, EGF rapidly induced tyrosine phosphorylation of both the EGFR and of PLC-gamma1. EGF also stimulated PLC-gamma1 translocation from cytosol to a cytoskeletal compartment where PLC-gamma1 interacted with EGFR. In hepatocytes from rats fed ethanol for 16 weeks, the above reactions were substantially inhibited. Tyrphostin AG1478, an EGFR-specific tyrosine kinase inhibitor, mimicked the effects of chronic ethanol on EGFR phosphorylation, PLC-gamma1 translocation and interactions between EGFR and PLC-gamma1 in the cytoskeleton. Further, tyrphostin AG1478 also inhibited EGF-induced DNA synthesis. These results indicate that ethanol impairs EGFR-operated [Ca2+]i signaling by disrupting the interactions between EGFR and PLC-gamma1.     

cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.     

Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor.

Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction.     

Cell-specific and developmental expression of phospholipase C-gamma and diacylglycerol in fetal lung

Epidermal growth factor (EGF) receptor (EGFR) regulates development of cell-cell communication in fetal lung, but the signal transduction mechanisms involved are unknown. We hypothesized that, in late-gestation fetal rat lung, phospholipase C-gamma (PLC-gamma) expression and activation by EGF is cell specific and developmentally regulated. PLC-gamma immunolocalized to cuboidal epithelium and mesenchymal clusters underlying developing saccules. PLC-gamma protein increased from day 17 to day 19 and then decreased. In cultured fetal lung fibroblasts, EGF stimulated PLC-gamma phosphorylation 2.6-fold (day 17), 10.8-fold (day 19), and 4.2-fold (day 21). EGF stimulated (3)H-labeled diacylglycerol production in fibroblasts (beginning on day 18 in female and on day 19 in male rats), but not in type II cells at any time during gestation. EGFR blockade abrogated the observed stimulation of PLC-gamma phosphorylation by EGF. In conclusion, PLC-gamma expression and activation by EGF in fetal lung are cell specific, corresponding to the development of EGFR expression. EGF induces diacylglycerol production in a cell- and gestation-specific manner. PLC-gamma activation by EGFR in fetal lung fibroblasts may be involved in EGF control of lung development.     

Epidermal growth factor, but not insulin, stimulates tyrosine phosphorylation of an endogenous protein of Mr 95,000 in triton extracts of human placental syncytiotrophoblast membranes.

1. Triton extracts of syncytiotrophoblast membranes were incubated with [gamma-32P]ATP, MgCl2 and MnCl2. Addition of epidermal growth factor (EGF) resulted in increased phosphorylation not only of the EGF receptor and a Mr-35,000 protein as previously described, but also a protein of Mr 95,000 on both tyrosine and serine residues. In addition, a small increase in the phosphorylation of a protein of Mr 105,000 was observed. Spermine had a similar effect on the phosphorylation of the Mr-95,000 protein, without affecting the phosphorylation of the other proteins. In the absence of MnCl2, the effect of spermine on the phosphorylation of Mr-95,000 protein was still evident, whereas that of EGF was greatly diminished. 2. The Mr-95,000 protein bound poorly to wheat-germ-lectin-Sepharose and was not precipitated by antisera specific for insulin and EGF receptors. The protein continued to exhibit serine and tyrosine phosphorylation on addition of [gamma-32P]ATP, MgCl2 and MnCl2 to a glycoprotein-depleted fraction prepared by chromatography on wheat-germ-lectin-Sepharose. The extent of phosphorylation was no longer increased by spermine or EGF, but was inhibited by heparin. 3. It is suggested that the Mr-95,000 protein not only is a possible direct substrate for the EGF-receptor (but not the insulin receptor) tyrosine kinase but is a substrate for other endogenous kinases, including a protein tyrosine kinase which is probably not a glycoprotein, and a protein serine kinase with properties similar to those of casein kinase II.     

Epidermal growth factor receptor threonine and serine residues phosphorylated in vivo

The epidermal growth factor (EGF) receptor is regulated by EGF-stimulated autophosphorylation and by phorbol ester-stimulated, protein kinase C (Ca2+/phospholipid-dependent enzyme) mediated phosphorylation at identified sites. The EGF receptor contains additional phosphorylation sites including a prominent phosphothreonine and several phosphoserines which account for the majority of phosphate covalently bound to the receptor in vivo. We have identified three of these sites in EGF receptor purified from 32P-labeled A431 cells. The major phosphothreonine was identified as threonine 669 in the EGF receptor sequence. Phosphoserine residues were identified as serines 671 and 1046/1047 of the EGF receptor. Two other phosphoserine residues were localized to tryptic peptides containing multiple serine residues located carboxyl-terminal to the conserved protein kinase domain. The amino acid sequences surrounding the three identified phosphorylation sites are highly conserved in the EGF receptor and the protein products of the v-erb B and neu oncogenes. Analysis of predicted secondary structure of the EGF receptor reveals that all of the phosphorylation sites are located near beta turns. In A431 cells phosphorylation of the serine residues was dependent upon serum. In mouse B82 L cells transfected with a wild type human EGF receptor. EGF increased the 32P content in all tryptic phosphopeptides. A mutant EGF receptor lacking protein tyrosine kinase activity was phosphorylated only at threonine 669. Regulated phosphorylation of the EGF receptor at these threonine and serine residues may influence aspects of receptor function.     

Mechanism of tyrosine phosphorylation and activation of phospholipase C-gamma 1. Tyrosine 783 phosphorylation is not sufficient for lipase activation

Phospholipase C-gamma 1 (PLC-gamma 1) is phosphorylated on three tyrosine residues: Tyr-771, Tyr-783, and Tyr-1253. With the use of antibodies specific for each of these phosphorylation sites, we have now determined the kinetics and magnitude of phosphorylation at each site. Phosphorylation of Tyr-783, which is essential for lipase activation, was observed in all stimulated cell types examined. The extent of phosphorylation of Tyr-1253 was approximately 50 to 70% of that of Tyr-783 in cells stimulated with platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), but Tyr-1253 phosphorylation was not detected in B or T cell lines stimulated through B- and T-cell antigen receptors, respectively. Tyr-771 was phosphorylated only at a low level in all cells studied. In cells stimulated with PDGF, phosphorylation and dephosphorylation of Tyr-783 and of Tyr-1253 occurred with similar kinetics; the receptor kinase appeared to phosphorylate both sites, albeit with Tyr-783 favored over Tyr-1253, before the bound PLC-gamma 1 was released, and phosphorylation at the two sites occurred independently. PDGF and EGF induced similar levels of phosphorylation of Tyr-783 and of Tyr-1253 in a cell line that expressed receptors for both growth factors. However, only PDGF, not EGF, elicited substantial PLC activity, suggesting that Tyr-783 phosphorylation was not sufficient for enzyme activation. Finally, concurrent production of phosphatidylinositol 3,4,5-trisphosphate was found to contribute to the activation of phosphorylated PLC-gamma 1.     

Increasing the cAMP content of IM-9 cells alters the phosphorylation state and protein kinase activity of the insulin receptor

The effect of 8-bromo-cAMP and forskolin on the phosphorylation state and protein kinase activity of the insulin receptor was evaluated in cultured IM-9 lymphoblasts. 8-Bromo-cAMP (1 mM) or forskolin (10 microM) enhanced the phosphorylation of the insulin receptor purified from 32P-labeled cells by affinity chromatography on wheat germ agglutinin-agarose and immunoprecipitation with monoclonal antibody. In the absence of insulin, phosphorylation of the beta subunit of the receptor was increased approximately 2-fold by raising intracellular cAMP. Phosphoamino acid analysis of the beta subunit following treatment of cells with forskolin revealed an increase in phosphoserine and phosphothreonine residues. In contrast, the insulin-stimulated phosphorylation of the receptor occurred on serine, threonine, and tyrosine residues and was diminished by prior exposure of cells to forskolin. Pulse-chase experiments indicated that forskolin did not enhance the turnover of phosphate on the receptor of cells previously exposed to insulin. Furthermore, extracts from forskolin-treated cells did not differ from control extracts in their capacity to dephosphorylate 32P-labeled receptor isolated from cells treated with insulin. The insulin-dependent tyrosine protein kinase activity of the receptor isolated from forskolin-treated cells was approximately 50% as active as the receptor isolated from either control or insulin-treated cells. This was assessed using both histone and a peptide synthesized in accordance with the deduced amino acid sequence of a potential autophosphorylation site of the human receptor (Thr-Arg-Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg-Lys) as substrates for the protein kinase reaction. These results suggest that agents that raise intracellular cAMP increase phosphorylation of the insulin receptor on serine and threonine residues, reduce insulin-mediated receptor phosphorylation on tyrosine, serine, and threonine residues, and inhibit the insulin-dependent tyrosine protein kinase activity of the receptor. Thus cAMP may attenuate insulin action by altering the state of phosphorylation of the insulin receptor.     

Phospholipase C-gamma 1 and phospholipase C-gamma 2 are substrates of the B cell antigen receptor associated protein tyrosine kinase.

Cross-linking the antigen receptor on B cells results in a rapid increase in protein tyrosine kinase activity as detected by increased phosphorylation on tyrosine residues of multiple proteins. Although the identity of most of this substrates remains unknown, some have been proposed. One possible substrate of the antigen receptor-associated kinase is phospholipase C (PLC). Since multiple isoforms of PLC have been identified, we have studied which isoforms are targets of the antigen receptor. PLC-gamma 1 and PLC-gamma 2 but not PLC-beta 1 or PLC-delta 1 were detected in human B cells. Immunoprecipitation with antibodies against PLC-gamma 1 or PLC-gamma 2 and subsequent Western blotting with anti-phosphotyrosine antibodies revealed that both PLC-gamma 1 and PLC-gamma 2 are tyrosine phosphorylated in stimulated but not in resting B cells. This was confirmed by experiments whereby B cell lysates were immunoprecipitated with anti-phosphotyrosine antibody and subsequently blotted with antibodies against PLC-gamma 1 or PLC-gamma 2. Further, the specific protein tyrosine kinase inhibitors, tyrphostins, which block phospholipase-C activation and proliferation of B cells also inhibited tyrosine phosphorylation on both PLC-gamma 1 and PLC-gamma 2. We conclude that both isoforms PLC-gamma 1 and PLC-gamma 2 are targets of the antigen receptor-associated protein tyrosine kinase.     

Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex.

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.