Tyrosine Phosphorylation Of PLCγ2 Research Articles

Protein kinase Cε may act as EGF-inducible scaffold protein for phospholipase Cγ1

Phospholipase Cγ1 (PLCγ1) represents a major downstream signalling component of the epidermal growth factor (EGF) receptor (EGFR) and is activated by tyrosine phosphorylation. Here we show for the first time that cellular knockdown of protein kinase Cε (PKCε) leads to decreased activation of PLCγ1 by EGF and that EGF induces tyrosine phosphorylation of PKCε as well as association of PKCε with both EGFR and PLCγ1. Using several mutants, co-immunoprecipitation and phosphopeptide-based pull-down experiments we found that in dependency on c-Src and EGF-stimulation PKCε may bind to the c-Src-specific phosphorylation site pY845-EGFR. Furthermore, we identified a single tyrosine residue, PKCε-Y573, within a consensus binding sequence of the C-terminal SH2 domain of PLCγ1 which is critical for both tyrosine phosphorylation of PKCε and its association with PLCγ1. Thus, in particular cells and independent of the kinase activity PKCε may form a signalling module with EGFR and PLCγ1. Thereby the tyrosine phosphorylation of PLCγ1 via the EGFR may be facilitated. This is a novel function of PKCε upstream of PLCγ1 and a novel paradigm for the EGF-induced formation of multi-protein complexes.

The role of phospholipase Cγ1 tyrosine phosphorylation during phasic myometrial contractions

Phospholipase Cgamma1 (PLCgamma1) is expressed in myometrium and is activated by tyrosine phosphorylation. These studies sought to determine the association between PLCgamma1 tyrosine phosphorylation and spontaneous uterine contractions. In vitro contraction studies were performed with spontaneously contracting rat uterine strips along with strips that were treated with potassium bisperoxo(1,10 phenanthroline)oxovanadate (bpV(phen), a protein tyrosine phosphatase inhibitor. Additional studies were performed with phenylarsine oxide (a PLCgamma inhibitor) and other inhibitors. Western blots were performed to determine the phosphotyrosine PLCgamma1 levels. Spontaneous contractile activity and tyrosine phosphorylation of PLCgamma1 (but not PLCgamma2) were increased significantly in response to bpV(phen); in contrast, oxytocin and thrombin produced comparable contractile activity but did not alter phosphotyrosine-PLCgamma1. Phenylarsine oxide and neomycin significantly decrease bpV(phen)-stimulated contractions and PLCgamma1 tyrosine phosphorylation; other inhibitors only suppressed contractions. These studies support the hypothesis that spontaneous myometrial contractions are associated with tyrosine phosphorylation of PLCgamma1; both of which are further enhanced by the inhibition of protein tyrosine phosphatase activity.

PKCδ regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation

AbstractProtein kinase Cδ (PKCδ) has been shown by pharmacologic approaches to negatively regulate collagen-induced platelet aggregation. Here we addressed the molecular and cellular mechanisms underlying this negative regulation. Using PKCδ–/– platelets, we show that the mechanism did not involve altered inside-out signaling to integrin αIIbβ3 and did not affect early signaling events downstream of GPVI, because there was no difference in tyrosine phosphorylation of PLCγ2 between wild-type and PKCδ–/– platelets. There was also no increase in secretion of dense granule content, in contrast to studies using rottlerin where secretion was enhanced. Importantly, however, there was marked enhancement of filopodia generation in PKCδ–/– platelets upon adhesion to collagen compared with wild-type platelets. Filopodia play an essential role regulating adhesive events leading to platelet aggregation by increasing platelet-platelet contact. We show that the critical effector for PKCδ is vasodilator-stimulated phosphoprotein (VASP), a major regulator of actin cytoskeleton dynamics. PKCδ physically interacts with VASP constitutively and regulates its phosphorylation on Ser157. In VASP–/– platelets, the enhancement of filopodia generation, actin polymerization, and platelet aggregation by rottlerin is ablated. PKCδ is therefore a critical negative regulator of filopodia, and hence platelet aggregation, through a functional interaction with the actin organizer VASP.

Inhibition of Platelet-derived Growth Factor-induced Cell Growth Signaling by a Short Interfering RNA for EWS-Fli1 via Down-regulation of Phospholipase D2 in Ewing Sarcoma Cells

EWS-Fli1, a fusion gene resulting from a chromosomal translocation t(11;22, q24;q12) and found in Ewing sarcoma and primitive neuroectodermal tumors, encodes a transcriptional activator and promotes cellular transformation. However, the precise biological functions of its products remain unknown. To investigate the role of EWS-Fli1 in cell growth signaling, we transfected Ewing sarcoma TC-135 cells with short interfering RNAs for EWS-Fli1. EWS-Fli1 knockdown reduced cell growth and platelet-derived growth factor (PDGF)-BB-induced activation of the growth signaling enzymes. Interestingly, phospholipase D2 (but not the PDGF-BB receptor) showed marked down-regulation in the EWS-Fli1-knocked down TC-135 cells compared with the control cells. In Ewing sarcoma TC-135 cells, the PDGF-BB-induced phosphorylation of growth signaling involving extracellular signal-regulated kinase, Akt, p70S6K, and the expression of cyclin D3 were markedly inhibited by transfection with short interfering RNA phospholipase (PL)-D2. The PDGF-BB-induced activation of growth signaling was also suppressed by 1-butanol, which prevents the production of phosphatidic acid by phospholipase D (but not by t-butyl alcohol), thereby implicating PLD2 in PDGF-BB-mediated signaling in TC-135 cells. These results suggest that EWS-Fli1 may play a role in the regulation of tumor proliferation-signaling enzymes via PLD2 expression in Ewing sarcoma cells.

Inhibitory effect of GBH on platelet aggregation through inhibition of intracellular Ca 2+ mobilization in activated human platelets

Geiji-Bokryung-Hwan (GBH) was studied on antiplatelet activity in human platelet suspensions. GBH consists of the 5 herbs Cinnamomi Ramulus, Poria Cocos, Mountan Cortex Radicis, Paeoniae Radix, and Persicae Semen, which have been used in herbal medicine for thousands of years for atherosclerosis. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH inhibited platelet aggregation and Ca 2+ mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. GBH had no inhibitory effect on thromboxane B2 (TXB2) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca 2+ mobilization is regulated by phospholipase C-2 (PLC-γ2) activation. We evaluated the effect of GBH on tyrosine phosphorylation of PLC-γ2 and the production of inositol-1,4,5-trisphosphate (IP3). GBH at concentrations that inhibited platelet aggregation and Ca 2+ mobilization had no effects on tyrosine phosphorylation of PLC-γ2 or on the formation of IP3 induced by CRP. Similar results were obtained with thrombin-induced platelet activation. GBH inhibited platelet aggregation and Ca 2+ mobilization induced by thrombin without affecting the production of IP3. We then evaluated the effect of GBH on the binding of IP3 to its receptor. GBH at high concentrations partially blocked the binding of IP3 to its receptor. Therefore, the results suggested that GBH suppresses Ca 2+ mobilization at a step distal to IP3 formation. GBH may provide a good tool for investigating Ca 2+ mobilization.

Tec regulates platelet activation by GPVI in the absence of Btk

AbstractThe Tec family kinase Btk plays an important role in the regulation of phospholipase Cγ2 (PLCγ2) downstream of the collagen receptor glycoprotein VI (GPVI) in human platelets. Platelets also express a second member of this family, Tec; however, its function has not been analyzed. To address the role of Tec, we analyzed Btk-/-, Tec-/-, and Btk/Tec double-deficient (Btk-/-/Tec-/-) platelets. Tec-/- platelets exhibit a minor reduction in aggregation to threshold concentrations of collagen or the GPVI-specific agonist collagen-related peptide (CRP), whereas responses to higher concentrations are normal. Tyrosine phosphorylation of PLCγ2 by collagen and CRP is not altered in Tec-/- platelets. However, Btk-/-/Tec-/- platelets exhibit a greater reduction in PLCγ2 phosphorylation than is seen in the absence of Btk, thus revealing an important role for Tec in this situation. Furthermore, Btk-/-/Tec-/- platelets fail to undergo an increase in Ca2+, aggregation, secretion, and spreading in response to collagen or CRP, whereas they aggregate normally to adenosine diphosphate (ADP) and spread on fibrinogen. A residual GPVI signal exists in the Btk-/-/Tec-/- platelets as CRP synergizes with ADP to mediate aggregation. These results demonstrate an essential requirement for Tec and Btk in platelet activation by GPVI and reveal a functional role for Tec in the regulation of PLCγ2 in the absence of Btk. (Blood. 2003;102: 3592-3599)

Pleiotropic contributions of phospholipase C-gamma1 (PLC-gamma1) to T-cell antigen receptor-mediated signaling: reconstitution studies of a PLC-gamma1-deficient Jurkat T-cell line.

Phospholipase C-gamma1 (PLC-gamma1) plays a crucial role in the coupling of T-cell antigen receptor (TCR) ligation to interleukin-2 (IL-2) gene expression in activated T lymphocytes. In this study, we have isolated and characterized two novel, PLC-gamma1-deficient sublines derived from the Jurkat T-leukemic cell line. The P98 subline displays a >90% reduction in PLC-gamma1 expression, while the J.gamma1 subline contains no detectable PLC-gamma1 protein. The lack of PLC-gamma1 expression in J.gamma1 cells caused profound defects in TCR-dependent Ca(2+) mobilization and NFAT activation. In contrast, both of these responses occurred at normal levels in PLC-gamma1-deficient P98 cells. Unexpectedly, the P98 cells displayed significant and selective defects in the activation of both the composite CD28 response element (RE/AP) and the full-length IL-2 promoter following costimulation with anti-TCR antibodies and phorbol ester. These transcriptional defects were reversed by transfection of P98 cells with a wild-type PLC-gamma1 expression vector but not by expression of mutated PLC-gamma1 constructs that lacked a functional, carboxyl-terminal SH2 [SH2(C)] domain or the major Tyr(783) phosphorylation site. On the other hand, the amino-terminal SH2 [SH2(N)] domain was not essential for reconstitution of RE/AP- or IL-2 promoter-dependent transcription but was required for the association of PLC-gamma1 with LAT, as well as the tyrosine phosphorylation of PLC-gamma1 itself, in activated P98 cells. These studies demonstrate that the PLC-gamma1 SH2(N) and SH2(C) domains play functionally distinct roles during TCR-mediated signaling and identify a non-Ca(2+)-related signaling function linked to the SH2(C) domain, which couples TCR plus phorbol ester-CD28 costimulation to the activation of the IL-2 promoter in T lymphocytes.

Inhibition by diallyl trisulfide, a garlic component, of intracellular Ca 2+ mobilization without affecting inositol-1,4,5-trisphosphate (IP 3) formation in activated platelets

Garlic has been used in herbal medicine for thousands of years. Some reports have shown that garlic has protective effects against atherosclerosis and inhibits platelet function. In this study, we investigated the mechanism by which diallyl trisulfide (DT), a component of garlic, inhibits platelet function. DT inhibited platelet aggregation and Ca 2+ mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. DT also had no inhibitory effects on thromboxane A 2 (TXA 2) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca 2+ mobilization is regulated by phospholipase C-γ2 (PLC-γ2) activation. We evaluated the effect of DT on tyrosine phosphorylation of PLC-γ2 and the production of inositol-1,4,5-trisphosphate (IP 3). DT at concentrations that inhibited platelet aggregation and Ca 2+ mobilization had no effects on tyrosine phosphorylation of PLC-γ2 or on the formation of IP 3 induced by CRP. Similar results were obtained with thrombin-induced platelet activation. DT inhibited platelet aggregation and Ca 2+ mobilization induced by thrombin without affecting the production of IP 3. We then evaluated the effect of DT on the binding of IP 3 to its receptor. DT at high concentrations partially blocked the binding of IP 3 to its receptor. Taken together, our findings suggest that the agent suppresses Ca 2+ mobilization at a step distal to IP 3 formation. DT may provide a good tool for investigating Ca 2+ mobilization.

Antigen receptor signalling in apoptosis-resistant mutants of WEHI 231 cells.

Ligation of membrane immunoglobulin M (mIgM) induces cell cycle arrest and apoptosis in the WEHI 231 B-lymphoma cell line. The molecular mechanisms which link receptor ligation and the nuclear events that underlie this response, have yet to be fully elucidated. Here we have examined the signals induced following mIgM cross-linking in variants of WEHI 231 that no longer undergo apoptosis in response to this stimulus. Tyrosine phosphorylation of cellular substrates in two of the variants is identical to that seen in wild-type cells but in one of the mutants, VS2.12, a restricted set of substrates becomes tyrosine phosphorylated. In a second variant (E8), mIgM cross-linking does not induce elevation of intracellular Ca2+, although tyrosine phosphorylation of PLCgamma2 is induced to an equivalent extent to that seen in WEHI 231 cells. A third variant, 2E10.F9, is resistant to apoptosis despite the fact that all signals analysed appear to be similar to those induced in wild-type cells. Our findings show that resistance to apoptosis can arise as a result of mutations affecting discrete stages of the mIgM signalling pathway. The mutant lines reported here show defects that have not yet been identified in previous studies and are likely to be useful tools in dissecting the signalling of cell death in B lymphocytes.

Altered activation of phospholipase D by lysophosphatidic acid and endothelin-1 in mouse embryo fibroblasts lacking phospholipase C-γ1

Lysophosphatidic acid (LPA) and endothelin-1 (ET-1) activate phospholipase D (PLD) in many cell types. To see if phospholipase C-γ1 plays a role, we used embryonic fibroblasts from mice in which the PLCγ1 gene was disrupted. Surprisingly, the effect of LPA on inositol phosphate accumulation was increased in these PLCγ1 −/− cells, whereas that of ET-1 was completely abrogated. When PLD activity was measured, the response to LPA was also enhanced and the response to ET-1 lost in the PLCγ1 −/− cells. Treatment of these cells with ionomycin and oleoyl acetyl glycerol to mimic PLC stimulation restored PLD activity. Treatment of either PLCγ1 +/+ and PLCγ1 −/− cells with tyrosine kinase inhibitors did not inhibit LPA- or ET-1-induced PLD activity. Moreover, LPA and ET-1 treatment of PLCγ1 +/+ and PLCγ1 −/− cells did not cause tyrosine phosphorylation of PLC-γ1 or PLC-γ2. In summary, these results show that the altered PLD responses to LPA and ET-1 in PLCγ1 −/− are due to changes in PLC activity and do not involve tyrosine kinase activity.

A collagen-related peptide regulates phospholipase Cγ2 via phosphatidylinositol 3-kinase in human platelets

The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)10GCP*G]n; P* = hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca2+ ([Ca2+]i) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca2+ by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4,5)P3, but not the R28C (Arg28 → Cys) mutant which binds to PI(3,4,5)P3 with low affinity, also inhibits elevation of [Ca2+]i in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCγ2. These results demonstrate that PI 3-kinase is required for full activation of PLCγ2 by GPVI in platelets and megakaryocytes.

A collagen-related peptide regulates phospholipase Cγ2 via phosphatidylinositol 3-kinase in human platelets

The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)(10)GCP*G](n); P*=hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 3,4-bisphosphate [PI(3, 4)P(2)] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca(2+) ([Ca(2+)](i)) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca(2+) by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4, 5)P(3), but not the R28C (Arg(28)-->Cys) mutant which binds to PI(3, 4,5)P(3) with low affinity, also inhibits elevation of [Ca(2+)](i) in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCgamma2. These results demonstrate that PI 3-kinase is required for full activation of PLCgamma2 by GPVI in platelets and megakaryocytes.

Phosphatidylinositol 3′-kinase and tyrosine-phosphatase activation positively modulate Convulxin-induced platelet activation. Comparison with collagen

In this report we have studied the role of phosphatidylinositol 3′-kinase (PI3-K) and tyrosine phosphatase activation on platelet activation by Convulxin (Cvx). Wortmannin, a specific PI3-K inhibitor, and phenylarsine oxide (PAO), a sulfhydryl reagent that inhibits tyrosine phosphatase (PTPase), block Cvx-induced platelet aggregation, granule secretion, inositol phosphate production, and increase in [Ca 2+] i. However, PAO does not inhibit Cvx-induced tyrosine phosphorylation of platelet proteins, including Syk and PLCγ2, but blocked collagen-induced platelet aggregation as well as tyrosine phosphorylation of PLCγ2. In contrast, Cvx-induced PLCγ2 tyrosyl phosphorylation was partially inhibited by wortmannin. We conclude that (i) although Cvx and collagen activate platelets by a similar mechanism, different regulatory processes are specific to each agonist; (ii) mechanisms other than tyrosine phosphorylation regulate PLCγ2 activity; and (iii) besides protein tyrosine kinases, PI3-K (and PTPase) positively modulate platelet activation by both Cvx and collagen, and this enzyme is required for effective transmission of GPVI-Fc receptor γ chain signal to result in full activation and tyrosine phosphorylation of PLCγ2 in Cvx-stimulated platelets.

Role of tyrosine phosphorylation of phospholipase C γ1 in the signaling pathway of HMG-CoA reductase inhibitor-induced cell death of L6 myoblasts

Our previous studies have shown that the HMG-CoA reductase (HCR) inhibitor (HCRI), simvastatin, kills L6 myoblasts by involving Ca 2+ mobilization from the Ca 2+ pool in the cells but not by influx from extracellular space. More recently, we found that HCRI induced tyrosine phosphorylation of several cellular proteins, followed by apoptotic cell death of L6 myoblasts. The present study was aimed to elucidate the molecular target(s) of these tyrosine phosphorylations induced by HCRI and demonstrated that simvastatin induces tyrosine phosphorylation of phospholipase C (PLC) γ1. This tyrosine phosphorylation of PLC-γ1 caused the increment of the intracellular inositol triphosphate (IP3) levels in L6 myoblasts. Pretreatment of the cells with herbimycin A, a specific inhibitor of protein tyrosine kinase, inhibited a simvastatin-induced increase in IP3 level in the cells as well as tyrosine phosphorylation of PLC-γ1. Interestingly, pretreatment of the cells with U-73122, a specific inhibitor of PLC, prevented simvastatin-induced cell death. Thus, these results strongly suggest that simvastatin-induced tyrosine phosphorylation of PLC-γ1 plays, at least in part, an important role for the development of simvastatin-induced cell death.

Interleukin-4 overcomes the negative influence of cyclic amp accumulation on antigen receptor stimulated B lymphocytes

Activation of protein kinase A (PKA) in B lymphocytes prior to the ligation of the B cell antigen receptor (BCR) results in a profound inhibition of BCR induced proliferation. The major effect of increased PKA activity in B lymphocytes was the induction of apoptosis leading to a reduced BCR induced growth response. The growth promoting cytokine IL-4 rescued B lymphocytes from PKA mediated negative effects. IL-4 protected BCR stimulated cells from PKA mediated inhibition primarily by preventing apoptosis and growth arrest. PKA-activation caused a downregulation of anti-IgM induced expression of Bcl-xL protein, that was restored by IL-4. Previous studies have shown that PKA-activation blocks BCR induced phospholipase Cγ-activation and calcium mobilization. IL-4 was unable to overcome the block in anti-IgM mediated calcium mobilization due to PKA-activation. B cell apoptosis induced by PKA-activation was also seen in CD72 stimulated cells, although CD72 mediated B-lymphocyte proliferation was not affected. PKA mediated block in phospholipase γ-activation and calcium mobilization were not due to alterations in the activation of tyrosine kinases lyn, blk and syk. Moreover, BCR mediated tyrosine phosphorylation of PLC γ2 and CD19 were also unaffected by cAMP accumulation. These observations are in contrast to the ability of PKA to drastically reduce the activity of ZAP-70 and syk in T lymphocytes and neutrophils, respectively. The IL-4 mediated protection appears to be due to a change in late events in BCR signaling, which are important for Bcl-xL expression.

Regulatory role of the engagement of MHC class II on the activation and proliferation of human T cells. ♦ 56

We have employed specific monoclonal antibodies to engage the major histocompatibility complex (MHC) class II on the T cell surface, and assayed for several intracellular events to study the role of MHC class II in human activated T cells. We have characterized in the engaged T cells the phosphorylation of cellular proteins, of the enzymes phosphotyrosine kinases(PTK) and phospholipase C (PLC), the Ca++ mobilization, and the antigen-specific proliferation. The engagement of surface MHC class II was associated with an overall increase in cellular protein tyrosine phosphorylation. There was an increase in the tyrosine phosphorylated p56lck and p59fyn, but not ZAP-70. The tyrosine phosphorylation of PLC-γ1, which co-immunoprecipitated with surface MHC class II in an apparent complex with the adaptor protein Grb-2, was significantly augmented after the engagement of MHC class II. The engagement of MHC class II, either with or without cross-linking ligation by anti-species antibody, increased the intracellular Ca++ mobilization at a comparable level to anti-CD3 treatment. In contrast, such engagement of MHC class II on T cells partially inhibited antigen-specific proliferation. These data suggest that surface MHC class II involves both the PTK and PLC pathways, probably via a complex comprising MHC class II, the enzymes, and the adaptor protein Grb-2. We conclude that the engagement of surface MHC class II, which leads to T cell activation but partially inhibits its proliferation, could play a role in the regulation of immunological responses.

Human FcγRIIIA-mediated tyrosine phosphorylation of PLCγ-1 by its cross-linking in mouse monocytic cell line P388D1

Human FcγRIIIA-mediated tyrosine phosphorylation of PLCγ-1 by its cross-linking in mouse monocytic cell line P388D1

Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases

Treatment of PC 12 cells with nerve growth factor (NGF) induces a rapid increase in tyrosine phosphorylation of multiple cellular proteins. Expression of a dominant inhibitory Ras mutant specifically blocked NGF- and TPA-induced tyrosine phosphorylation of two proteins of approximately 42 and 44 kd. Conversely, expression of an oncogenic variant of Ras induced tyrosine phosphorylation of the same 42 and 44 kd proteins. The 44 kd protein was immunoprecipitated with an antibody directed against extracellular signal-regulated kinase 1/mitogen-activated protein kinase (MAPK) and the 42 kd protein comigrated with a 42 kd MAPK, indicating that at least one and probably both Ras-regulated phosphoproteins are MAPKs. In addition, MAPK activation, as measured by in vitro phosphorylation of myelin basic protein, was also regulated by Ras. Ras was not required for NGF-induced activation of Trk or tyrosine phosphorylation of PLC-γ1. Thus, NGF-induced tyrosine phosphorylation occurs both prior to and following Ras action, and Ras plays a critical role in the NGF- and TPA-induced tyrosine phosphorylation of MAPKs.