Regulation of phospholipase C-gamma 2 via phosphatidylinositol 3-kinase in macrophages.

Phospholipase C-gamma (PLC-gamma) isoforms are thought to be activated by both tyrosine phosphorylation and phosphatidylinositol 3,4,5 trisphosphate (PtdIns 3,4,5 P(3)), the product of phosphatidylinositol 3-kinase (PtdIns 3-kinase). In this study, we show that stimulation of mouse macrophages with either zymosan beads or bacteria (Prevotella intermedia) induced tyrosine phosphorylation of PLC-gamma 2. Zymosan stimulation also induced translocation to membrane and cytoskeleton fractions, which was inhibited by the PtdIns 3-kinase inhibitors wortmannin and LY 294002. However, the tyrosine phosphorylation of PLC-gamma 2 induced by zymosan was not affected by the inhibitors wortmannin and LY 294002. In contrast to zymosan and bacteria, PLC-gamma 2 was not phosphorylated by stimulation with lipopolysaccharide (LPS), phorbol ester or calcium ionophore. Moreover, the PLC-gamma 1 isoform was not detected in mouse macrophages. These data indicate that PtdIns 3-kinase is critical for the translocation but not for the tyrosine phosphorylation of PLC-gamma 2 in mouse macrophages and that the latter may be insufficient for enzyme activation.     

EGF-dependent association of phospholipase C-gamma1 with c-Cbl

The structure of phospholipase Cgamma1 (PLC-gamma1) contains two SH2 domains and one SH3 domain. While the function of the SH2 domains in PLC-gamma1 are well described, to date no growth factor-dependent function for the SH3 domain has been presented. To assess SH3 domain function in the context of the full-length PLC-gamma1, this domain was deleted and the mutant was stably expressed in Plcg1 null mouse embryonic fibroblasts. Following EGF treatment of cells, the PLC-gamma1DeltaSH3 mutant displayed the same increased level of tyrosine phosphorylation and association with EGF receptor as wild-type PLC-gamma1. Also, the SH3 mutant demonstrated membrane translocation and mediated the mobilization of intracellular Ca(2+) in response to EGF. c-Cbl is shown to associate with tyrosine phosphorylated PLC-gamma1 in an EGF-dependent manner, but no association was detected with the PLC-gamma1DeltaSH3 mutant. Interestingly, PDGF, which also tyrosine phosphorylates PLC-gamma1, failed to induce c-Cbl association with PLC-gamma1 and also provoked no c-Cbl tyrosine phosphorylation. This suggests that c-Cbl tyrosine phosphorylation is necessary for its interaction with PLC-gamma1. Evidence of a direct association of c-Cbl with PLC-gamma1 was provided by pull-down and overlay experiments, using glutathione S-transferase fusion proteins that contain the SH3 domain of PLC-gamma1. The data, therefore, show an EGF-inducible direct association of PLC-gamma1 with c-Cbl in vivo that is mediated by the SH3 domain of PLC-gamma1.     

Phosphatidylinositol 3-kinase regulates glycosylphosphatidylinositol hydrolysis through PLC-gamma(2) activation in erythropoietin-stimulated cells

Erythropoietin (Epo)-induced glycosylphosphatidylinositol (GPI) hydrolysis was previously described to be correlated with phospholipase C-gamma 2 (PLC-gamma2) activation. Here, we analyzed the involvement of phosphatidylinositol (PtdIns) 3-kinase in GPI hydrolysis through PLC-gamma2 tyrosine phosphorylation in response to Epo in FDC-P1 cells transfected with a wild type (WT) erythropoietin-receptor (Epo-R). We showed that phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor LY294002 inhibits Epo-induced hydrolysis of endogenous GPI and Epo-induced PLC-gamma2 tyrosine phosphorylation in a dose-dependent manner. Wortmannin, another PtdIns 3-kinase inhibitor, also suppressed Epo-induced PLC-gamma2 tyrosine phosphorylation. We also present evidence that PLC-gamma2 translocation to the membrane fraction on Epo stimulation is completely inhibited by LY294002. Upon Epo stimulation, the tyrosine-phosphorylated PLC-gamma2 was found to be associated with the tyrosine-phosphorylated Grb2-associated binder (GAB)2, SHC and SHP2 proteins. LY294002 cell preincubation did not affect GAB2, SHC and SHP2 tyrosine phosphorylation but inhibited the binding of PLC-gamma2 to GAB2 and SHP2. Taken together, these results show that PtdIns 3-kinase controls Epo-induced GPI hydrolysis through PLC-gamma2.     

Leukotriene D(4) triggers an association between gbetagamma subunits and phospholipase C-gamma1 in intestinal epithelial cells

The proinflammatory mediator leukotriene D(4) (LTD(4)) binds to the seven-transmembrane receptor CYSLT(1). Although this leukotriene plays an important biological role, its intracellular signaling pathways are only partly known. In previous experiments, we found that LTD(4) induced tyrosine phosphorylation and translocation of phospholipase (PLC)-gamma1 to a plasma membrane fraction in a human epithelial cell line (Int 407). In the present study, we further examined these signaling events and found that LTD(4) induced a rapid interaction between Gbetagamma subunits and PLC-gamma1; results obtained with GST fusion proteins of PLC-gamma1 suggest that this interaction is mediated via the pleckstrin homology domain of PLC-gamma1. Moreover, LTD(4) induced an increased association of c-Src with PLC-gamma1, and the selective Src family tyrosine kinase inhibitor PP1 blocked both LTD(4)-induced tyrosine phosphorylation of PLC-gamma1 and the association of PLC-gamma1 with Gbetagamma subunits. The relevance of these observations in intracellular calcium signaling was investigated by microinjecting cells with anti-Gbeta, anti-PLC-gamma1, or anti-c-Src antibodies and by pretreatment with PP1. LTD(4)-induced calcium mobilization was blocked by each of the indicated antibodies (but not isotype-matched control antibodies) and by PP1. Our data suggest that Gbetagamma subunits can, directly or indirectly, serve as membrane-bound partners for PLC-gamma1 and c-Src and that each of these proteins is essential for LTD(4)-induced downstream PLC-gamma1 signaling.     

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.     

IgE-induced tyrosine phosphorylation of phospholipase C-gamma 1 in rat basophilic leukemia cells.

Stimulation of rat basophilic leukemia (RBL-2H3) cells with oligomeric IgE elicited a rapid and transient phosphorylation of phospholipase C (PLC)-gamma 1 on tyrosine residues. Prior incubation of RBL-2H3 cells with a protein tyrosine kinase inhibitor, herbimycin A, prevented the tyrosine phosphorylation of PLC-gamma 1 as well as the hydrolysis of phosphatidylinositol 4,5-bisphosphate induced by oligomeric IgE. However, 5'-(N-ethyl)carboxamidoadenosine, which is known to activate PLC through a G protein, did not elicit tyrosine phosphorylation of PLC-gamma 1. These results, together with previous findings showing that tyrosine phosphorylation of PLC-gamma 1 enhances its catalytic activity, indicate that phosphorylation of PLC-gamma 1 by a nonreceptor tyrosine kinase is the mechanism by which IgE receptor aggregation triggers PLC activation.     

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.     

Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.     

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.     

Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro

Phospholipase C-gamma (PLC-gamma) was rapidly phosphorylated on tyrosines and serines following PDGF and EGF treatment of quiescent 3T3 mouse fibroblasts and A431 human epidermoid cells, respectively, PDGF treatment increased PLC-gamma phosphorylation within 30 sec. This lasted for up to 1 hr, and occurred at high stoichiometry. Continuous receptor occupancy was required to maintain this phosphorylation. Three major sites of tyrosine phosphorylation were detected in PLC-gamma, two of which were phosphorylated in EGF-treated A431 cells. Under certain conditions PDGF receptor coimmunoprecipitated with PLC-gamma, suggesting that PDGF receptor can phosphorylate PLC-gamma directly. Indeed, purified PDGF or EGF receptor phosphorylated purified PLC-gamma on tyrosines identical to those phosphorylated in vivo. Tyrosine phosphorylation of PLC-gamma was not induced by bombesin, TPA, or insulin. Stimulation of PLC-gamma tyrosine phosphorylation and the reported ability of PDGF and EGF to induce phosphatidylinositol turnover in different cells were strongly correlated. We propose that tyrosine phosphorylation of PLC-gamma by PDGF and EGF receptors leads to its activation, and a consequent increase in phosphatidylinositol turnover.     

Phospholipase C-gamma 1 is translocated to the membrane of rat basophilic leukemia cells in response to aggregation of IgE receptors.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the surface of mast cells results in the rapid hydrolysis of membrane inositol phospholipids by phospholipase C (PLC). Although at least seven isoenzymes of PLC have been characterized in different mammalian cells, the isoenzyme involved in Fc epsilon RI-mediated signal transduction and the mechanism of its activation have not been demonstrated. We now report that PLC-gamma 1 is translocated to the membrane of mast cells after aggregation of Fc epsilon RI. Activation of rat basophilic leukemia cells, a rat mast cell line, with oligomeric IgE resulted in an increase in PLC activity in washed membrane preparations in a cell free assay containing exogenous [3H]phosphatidylinositol (PI). The increase in PLC activity has the same dose-response to oligomeric IgE as receptor mediated hydrolysis of inositol lipids (PI hydrolysis) in intact cells. Analysis by Western blot probed with anti-PLC-gamma 1 antibody revealed that there is a three- to fourfold increase in PLC-gamma 1 in membranes from activated cells. The increase in PLC activity is augmented a further 20% by the addition of orthovanadate to the incubation medium suggesting that a tyrosine phosphatase is involved in the down-regulation of this phenomenon. These findings demonstrate translocation of PLC-gamma 1 to the membrane following activation of a receptor which does not contain intrinsic tyrosine kinase activity. Activation of PLC-gamma 1 by this pathway may account for Fc epsilon RI-mediated PI hydrolysis.     

Nerve growth factor stimulates phosphorylation of phospholipase C-gamma in PC12 cells.

PC12 cells contain at least three immunologically distinct phospholipase C (PLC) isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of PC12 cells with nerve growth factor (NGF) leads to an increase in the phosphorylation of PLC-gamma, but not of PLC-beta or PLC-delta. This increase can be seen in as little as 1 minute. The increased phosphorylation occurs on both serine and tyrosine residues, with the major increase being in the former. This result suggests the possibility that the NGF-dependent increase in phosphoinositide hydrolysis in PC12 cells is due to selective phosphorylation of PLC-gamma by serine and tyrosine protein kinases associated with the NGF receptor.     

Role of tyrosine phosphorylation of phospholipase C gamma1 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 Ca2+ mobilization from the Ca2+ 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) gamma1. This tyrosine phosphorylation of PLC-gamma1 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-gamma1. 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-gamma1 plays, at least in part, an important role for the development of simvastatin-induced cell death.     

Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2.

Upon binding to its cell surface receptor, platelet-derived growth factor (PDGF) causes the tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and stimulates the production of diacylglycerol and inositol 1,4,5-triphosphate. We showed that following stimulation by PDGF, rat-2 cells overexpressing PLC-gamma 1 display an increase in the levels of both tyrosine-phosphorylated PLC-gamma 1 and inositol phosphates compared with the parental rat-2 cells. This increased responsiveness to PDGF is a direct effect of PLC-gamma 1 overexpression, as a cell line expressing similar levels of an enzymatically inactive point mutant of PLC-gamma 1, PLC-gamma 1 335Q, did not show elevated inositol phosphate production in response to PDGF. Hematopoietic cells express PLC-gamma 2, a PLC isoform that is closely related to PLC-gamma 1. When rat-2 cells overexpressing PLC-gamma 2 were treated with PDGF, an increase in both the tyrosine phosphorylation and the in vivo activity of PLC-gamma 2 was observed. Aluminum fluoride (AIF4-), a universal activator of PLC linked to G-proteins, did not produce an increase in the levels of inositol phosphates in either of the overexpressing cell lines compared with parental rat-2 cells, demonstrating that PLC-gamma isoforms respond specifically to a receptor with tyrosine kinase activity.     

Erythropoietin induces glycosylphosphatidylinositol hydrolysis. Possible involvement of phospholipase c-gamma(2)

We showed that erythropoietin induced rapid glycosylphosphatidylinositol (GPI) hydrolysis and tyrosine phosphorylation of phospholipase C (PLC)-gamma(2) in FDC-P1 cells transfected with the wild-type erythropoietin-receptor. Erythropoietin-induced tyrosine phosphorylation of PLC-gamma(2) was time- and dose-dependent. By using FDC-P1 cells transfected with an erythropoietin receptor devoid of tyrosine residues, we showed that both effects required the tyrosine residues of intracellular domain on the erythropoietin receptor. Erythropoietin-activated PLC-gamma(2) hydrolyzed purified [(3)H]GPI indicating that GPI hydrolysis and PLC-gamma(2) activation under erythropoietin stimulation were correlated. Results obtained on FDC-P1 cells transfected with erythropoietin receptor mutated on tyrosine residues suggest that tyrosines 343, 401, 464, and/or 479 are involved in erythropoietin-induced GPI hydrolysis and tyrosine phosphorylation of PLC-gamma(2), whereas tyrosines 429 and/or 431 seem to be involved in an inhibition of both effects. Thus, our results suggest that erythropoietin regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-gamma(2) activation.     

Potential role of Gab1 and phospholipase C-gamma in osmotic shock-induced glucose uptake in 3T3-L1 adipocytes.

Osmotic shock induces GLUT4 translocation and glucose uptake through a mechanism independent of PI 3-kinase, but dependent on tyrosine phosphorylation of cellular proteins. To identify the tyrosine phosphorylated proteins required for osmotic shock-stimulated glucose uptake, we examined tyrosine phosphorylation of candidate proteins, and found that the 60-80kDa species including paxillin and the 120-130kDa species including p130Cas, PYK2, FAK and Gab1 were tyrosine-phosphorylated in response to osmotic shock. Inhibition of actin polymerization by cytochalasin D significantly decreased the tyrosine phosphorylation of paxillin, p130Cas, PYK2 and FAK but not Gab1, but had no effect on 2-deoxyglucose (DOG) uptake, suggesting a role for Gab1 in osmotic shock-induced glucose transport. Also, we found that osmotic shock increases the association of phospholipase C-gamma (PLC-gamma) with Gab1 and stimulates tyrosine phosphorylation of PLC-gamma itself. The PLC inhibitor, U73122, inhibited osmotic shock-induced 2-DOG uptake. These results suggest that tyrosine phosphorylation of Gab1 and subsequent recruitment and activation of PLC-gamma may play a role in osmotic shock-induced glucose transport.     

Dual role of SLP-76 in mediating T cell receptor-induced activation of phospholipase C-gamma1

Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.     

Phospholipase C-gamma, a substrate for PDGF receptor kinase, is not phosphorylated on tyrosine during the mitogenic response to CSF-1.

Quiescent mouse NIH3T3 cells expressing a transduced human c-fms gene encoding the receptor for colony stimulating factor-1 (CSF-1) were stimulated with mitogenic concentrations of platelet-derived growth factor (PDGF) or CSF-1. Immunoprecipitated phospholipase C-gamma (PLC-gamma) was phosphorylated on tyrosine and calcium was mobilized following treatment of intact cells with PDGF. In contrast, only trace amounts of phosphotyrosine were incorporated into PLC-gamma and no intracellular calcium signal was detected after CSF-1 stimulation. Similarly, CSF-1 treatment did not stimulate phosphorylation of PLC-gamma on tyrosine in a CSF-1-dependent. SV40-immortalized mouse macrophage cell line that expresses high levels of the CSF-1 receptor. In fibroblasts, antiserum to PLC-gamma co-precipitated a fraction of the tyrosine phosphorylated form of the PDGF receptor (PDGF-R) after ligand stimulation, implying that phosphorylated PDGF-R and PLC-gamma were associated in a stable complex. Pre-treatment of cells with orthovanadate also led to tyrosine phosphorylation of PLC-gamma which was significantly enhanced by PDGF, but not by CSF-1. Thus, although the PDGF and CSF-1 receptors are structurally related and appear to be derived from a single ancestor gene, only PDGF-induced mitogenesis in fibroblasts correlated with tyrosine phosphorylation of PLC-gamma.     

IGF-I induces caveolin 1 tyrosine phosphorylation and translocation in the lipid rafts

Caveolin 1, a component of caveolae, regulates signalling pathways compartmentalization interacting with tyrosine kinase receptors and their substrates. The role of caveolin 1 in the Insulin Receptor (IR) signalling has been well investigated. On the contrary, the functional link between caveolin 1 and IGF-I Receptor (IGF-IR) remains largely unknown. Here we show that (1) IGF-IR colocalizes with caveolin 1 in the lipid rafts enriched fractions on plasmamembrane in R-IGF-IR(WT) cells, (2) IGF-I induces caveolin 1 phosphorylation at the level of tyrosine 14, (3) this effect is rapid and results in the translocation of caveolin 1 and in the formation of membrane patches on cell surface. These actions are IGF-I specific since we did not detect caveolin 1 redistribution in insulin stimulated R(-) cells overexpressing IRs.     

Phospholipase C-gamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk.

Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-gamma (PLC-gamma) isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)] domain of PLC-gamma1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells phosphorylated PLC-gamma1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same B cells phosphorylated PLC-gamma1 only on Tyr-771. The ability of Syk to phosphorylate PLC-gamma1 required antigen receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-gamma1 SH2(C) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamma1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding of this variant chimera to PLC-gamma1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-gamma1 SH(C) and completely eliminated its ability to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamma1 are required for the tyrosyl phosphorylation of PLC-gamma1.