Tyrosine phosphorylation of phospholipase C gamma in c-met/HGF receptor-stimulated hepatocytes: comparison with HepG2 hepatocarcinoma cells

Hepatocyte growth factor (HGF) stimulates inositol 1,4,5-trisphosphate (InsP3) formation in rat primary cultured hepatocytes, which is inhibited by the pretreatment with a tyrosine kinase inhibitor, genistein. This InsP3 production was coincident with tyrosine phosphorylation of phospholipase C gamma (PLC gamma), detected in immunoprecipitates with anti-PLC gamma, suggesting activation mechanism of PLC gamma by tyrosine phosphorylation. However, in human hepatocarcinoma HepG2 cells, HGF, which suppresses cell growth, causes neither phosphorylation of PLC gamma nor InsP3 formation. The results suggests that PLC gamma in normal hepatocytes was activated by HGF through tyrosine kinase of HGF receptor.     

Mapping a telomere using the translocation eT1(III;V) in Caenorhabditis elegans

TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase.     

P-glycoprotein associated expression of c-fos and c-jun products in human lung carcinomas

Surgical specimens of non-small cell lung carcinomas of 167 previously untreated patients were analyzed for expression of c-fos, c-jun, c-myc and c-neu products and for resistance to drugs. Because most of the patients were treated only by surgery, an in vitro test was used to determine the resistance. For the detection of the oncoproteins the streptavidin-biotin-peroxidase-complex method was used. An association between the resistance and c-fos and c-jun proteins was found (c-fos p = 0.01, c-jun p = 0.09), whereas a correlation between resistance and expression of c-neu and c-myc products was not observed. P-glycoprotein 170 was detected immunohistochemically in 91 tumors using the monoclonal antibody JSB-1. There was a significant correlation between the resistance measured by the in vitro test and P-glycoprotein 170 expression (p < 0.001). Also a significant correlation between the c-fos and c-jun proteins and the expression of P-glycoprotein was found (c-fos p = 0.017, c-jun p = 0.036). In contrast, no significant relationship was found between the expression of the c-neu or c-myc products and the expression of P-glycoprotein 170. Thus, there exists a significant relationship between resistance, P-glycoprotein 170, and c-fos and c-jun products in human non-small cell lung carcinomas. P-glycoprotein 170 may be regulated by the c-fos/c-jun protein complex, which binds specifically to AP-1.     

Somatic and social prognosis of patients with angina pectoris and normal coronary arteriography: a follow-up study

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1.     

Antioxidants and inflammatory cell response in preeclampsia

We have previously reported that gastrin induces a rapid and transient tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1) in association with inositol 1,4,5-trisphosphate (IP3) formation in rat colonic epithelial cells (34). In this study, we demonstrate that gastrin regulates IP3 formation mainly through PLC gamma 1 isozyme. Immunoblotting analysis revealed the expression of PLC beta 3 and -gamma 1, but not PLC beta 1, -beta 2, or -beta 4 in the rat colonic epitheliums. To explore what PLC isozyme(s) modulates gastrin effect on IP3, immunoneutralizing antibody to PLC beta 1, -beta 3, or -gamma 1 was introduced into the colonic cells using a lipid carrier. The gastrin-stimulated increase in IP3 concentration was specifically prevented by anti-PLC gamma 1 but not by anti-PLC beta 1 or -beta 3 antibody. Immunoprecipitation assays have also revealed that gastrin promoted an increase in tyrosine phosphorylation and co-precipitation of a 60 kDa src kinase with PLC gamma 1. Administration of antibody specific to pp60c-src into the colonic cells prevented the gastrin-stimulated increases in IP3. Tyrosine phosphorylation of PLC gamma 1 may be a major mechanism through which gastrin regulates IP3 level in the colonic cells. Pretreatment of cells with the tyrosine kinase inhibitor genistein abrogated gastrin's effect on IP3, while extended pretreatment with pertussis toxin, a G-protein inhibitor, did not affect the ability of gastrin to stimulate IP3 formation. Colonic cells expressed the G alpha i subunits1-3; however, immunoblotting analysis did not reveal any difference in G alpha i proteins' expression between control and gastrin treated cells. The results provide direct evidence that gastrin regulates IP3 level by a signaling mechanism that involves PLC gamma 1 and pp60c-src kinase.     

Impaired insulin signaling in skeletal muscles from transgenic mice expressing kinase-deficient insulin receptors

Transgenic mice which overexpress kinase-deficient human insulin receptors in muscle were used to study the relationship between insulin receptor tyrosine kinase and the in vivo activation of several downstream signaling pathways. Intravenous insulin stimulated insulin receptor tyrosine kinase activity by 7-fold in control muscle versus < or = 1.5-fold in muscle from transgenic mice. Similarly, insulin failed to stimulate tyrosyl phosphorylation of receptor beta-subunits or insulin receptor substrate 1 (IRS-1) in transgenic muscle. Insulin substantially stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase in control versus absent stimulation in transgenic muscles. In contrast, insulin-like growth factor 1 modestly stimulated PI 3-kinase in both control and transgenic muscle. The effects of insulin to stimulate p42 mitogen-activated protein kinase and c-fos mRNA expression were also markedly impaired in transgenic muscle. Specific immunoprecipitation of human receptors followed by measurement of residual insulin receptors suggested the presence of hybrid mouse-human heterodimers. In contrast, negligible hybrid formation involving insulin-like growth factor 1 receptors was evident. We conclude that (i) transgenic expression of kinase-defective insulin receptors exerts dominant-negative effects at the level of receptor auto-phosphorylation and kinase activation; (ii) insulin receptor tyrosine kinase activity is required for in vivo insulin-stimulated IRS-1 phosphorylation, IRS-1-associated PI 3-kinase activation, phosphorylation of mitogen-activated protein kinase, and c-fos gene induction in skeletal muscle; (iii) hybrid receptor formation is likely to contribute to the in vivo dominant-negative effects of kinase-defective receptor expression.     

Inhibition of G1 cyclin-dependent kinase activity in cell density-dependent growth arrest in human fibroblasts

The growth of normal fibroblasts in culture ceases as the cells reach saturation density. Although cells in dense cultures express functionally active growth factor receptors, they are essentially refractory to the mitogenic activity of growth factors. Northern blot analysis revealed that immediate early genes, c-myc, c-fos and c-jun are induced by mitogen in dense cultures. However, these cells fail to express the late G1 genes as E2F-1, cdc25A, and cyclin A in response to mitogen stimulation. Furthermore, because pRb-phosphorylation is a key event in G1 progression, here we show that in dense cultures, pRb remains in its active (hypophosphorylated) form after stimulation by mitogens. We also show that the kinase activity of cyclin-dependent kinases that are indispensable for the phosphorylation of pRb in late G1 phase was decreased on increasing cell density. The reduced kinase activity may be caused by the observed increase in cyclin-dependent kinase inhibitors and the reduction of cdc25A expression in dense cells.     

A specific increased expression of insulin receptor substrate 2 in pancreatic beta-cell lines is involved in mediating serum-stimulated beta-cell growth

Certain nutrients and growth factors can stimulate pancreatic beta-cell growth. However, the appropriate mitogenic signaling pathways in beta-cells have been relatively undefined. In this study, differential gene expression in NEDH rat insulinoma was compared with NEDH rat primary islet beta-cells. Differential mRNA display analysis revealed an elevated expression in insulinoma of VL30 transposons, S24 ribosomal protein, and cytochrome-C oxidaseVIIc that is typical for cells undergoing mitosis. A gene candidate approach revealed that mRNA levels of the oncogenes c-fos and c-jun were equivalently expressed in insulinoma and islet cells, as was the mRNA for the mitogenic signal transduction molecule insulin receptor substrate (IRS)-1. However, in contrast to that of IRS-1, IRS-2 gene expression was 60- to 70-fold higher in the insulinoma tissue compared with islets, which was reflected at the protein as well as the mRNA level. The specific elevated IRS-2 expression was a consistent observation across all rodent pancreatic beta-cell lines. To investigate whether IRS-2 was functional, serum-stimulated beta-cell proliferation was examined in isolated insulinoma cells. After a 48-h period of serum withdrawal, 24 h of serum refeeding rendered an 8- to 10-fold increase in [3H]thymidine incorporation into insulinoma cells. This serum-stimulated DNA synthesis was prevented by inhibitors of tyrosine protein kinase and phosphatidylinositol (PI) 3-kinase activities, as well as the activation of mitogen-activated protein (MAP) kinase and p70S6K. Examination of IRS-mediated signal transduction pathways indicated that after 10-15 min of serum refeeding, there was increased tyrosine phosphorylation of IRS-2 and pp60, and PI 3-kinase recruitment to IRS-2. Serum also increased the association of growth factor-bound protein 2/murine sons of sevenless 1 protein to a PI 3-kinase/IRS-2 protein complex. Moreover, serum also activated MAP-kinase (erk-1 and erk-2 isoforms) and 70 kD S6 kinase. Thus IRS-mediated signal transduction pathways are functional in pancreatic beta-cells. It is conceivable that IRS-2 expression in beta-cells contributes to maintaining the islet beta-cell population, complementary to observations in the IRS-2 knockout mouse in which beta-cell mass is markedly reduced.     

Differential inhibition of epidermal growth factor signaling pathways in rat hepatocytes by long-term ethanol treatment

BACKGROUND & AIMS: Long-term ethanol intake suppresses liver regeneration in vivo and ethanol interferes with epidermal growth factor (EGF)-induced DNA synthesis in vitro. Therefore, the effects of long-term ethanol treatment on EGF-activated signaling reactions in rat hepatocytes were investigated. METHODS: Hepatocytes from long-term ethanol-fed rats and pair-fed controls were stimulated with EGF (0.5-20 nmol/L) for 15-120 seconds. Tyrosine phosphorylation of EGF receptor (EGFR), Shc, and phospholipase-C gamma1 (PLC gamma), and growth factor receptor binding protein 2 (Grb2) coprecipitation with EGFR and Shc were analyzed by Western blotting. RESULTS: EGFR autophosphorylation was suppressed at all EGF concentrations in ethanol-fed cells compared with pair-fed cells, without significant differences in total EGFR protein or EGFR tyrosine kinase activity detected in cell lysates, suggesting that intracellular factors suppressed EGFR function. EGF-induced PLC gamma tyrosine phosphorylation and inositol 1,4,5-trisphosphate (InsP3) formation were suppressed, but cytosolic [Ca2+]c elevation was little affected, indicating enhanced InsP3-mediated intracellular Ca2+ release in ethanol-fed cells. Grb2 binding to EGFR was suppressed, but EGF-induced Shc tyrosine phosphorylation and Grb2 association with Shc were not significantly decreased. CONCLUSIONS: Long-term ethanol feeding suppressed EGF-induced receptor autophosphorylation in rat hepatocytes with differential inhibition of downstream signaling processes mediated by PLC gamma, Shc, and Grb2. Altered patterns of downstream signals emanating from EGFR may contribute to deficient liver regeneration in chronic alcoholism.     

Tyrosine phosphorylation and association with phospholipase C gamma-1 of the GAP-associated 62-kD protein after CD2 stimulation of Jurkat T cell

Numerous substrates are tyrosine phosphorylated upon CD2 stimulation of human Jurkat T cells using a mitogenic pair of CD2 monoclonal antibodies, including the phospholipase C (PLC)gamma-1-p35/36 complex. Most of these substrates are identically tyrosine phosphorylated after CD3 ligation, suggesting that both stimuli share the same biochemical pathway. We show, however, in this report that a 63-kD protein is specifically phosphorylated on tyrosine residues after ligation of the CD2 molecule. The tyrosine phosphorylation of p63 can be induced independently of other substrates when using a single CD2 mAb recognizing the D66 epitope of the molecule. Importantly, this CD2-induced tyrosine phosphorylation of p63 can also occur in the absence of the CD3 zeta chain membrane expression, and is also distinct from the protein tyrosine kinases p56lck and p59fyn. We demonstrate, moreover, that p63 is physically linked with PLC gamma-1 and p35/36 upon CD2 stimulation. Finally, we also show that a 62-kD protein coimmunoprecipitating with the p21ras GTPase activating protein (GAP) is heavily tyrosine phosphorylated only after CD2 stimulation. This ultimately suggests that p63 may represent in fact the 62-kD protein that associates with GAP after tyrosine phosphorylation. Taken together, these results demonstrate the occurrence in Jurkat cells of a tyrosine kinase pathway specifically coupled to the CD2 molecule. They also suggest a function of the p62-GAP-associated protein as a link between PLC gamma-1 and p21ras activation pathways after CD2 activation.     

APS, an adaptor protein containing PH and SH2 domains, is associated with the PDGF receptor and c-Cbl and inhibits PDGF-induced mitogenesis

Previously we cloned a novel adaptor protein, APS (adaptor molecules containing PH and SH2 domains) which was tyrosine phosphorylated in response to c-kit or B cell receptor stimulation. Here we report that APS was expressed in some human osteosarcoma cell lines, markedly so in SaOS-2 cells, and was tyrosine-phosphorylated in response to several growth factors, including platelet derived growth factor (PDGF), insulin-like growth factor (IGF), and granulocyte-macrophage colony stimulating factor (GM-CSF). Ectopic expression of the wild type APS, but not C-terminal truncated APS, in NIH3T3 fibroblasts suppressed PDGF-induced MAP kinase (Erk2) activation, c-fos and c-myc induction as well as cell proliferation. In vitro binding experiments suggest that APS bound to the beta type PDGF receptor, mainly via phosphotyrosine 1021 (pY1021). Indeed, tyrosine phosphorylation of PLC-gamma, which has been demonstrated to bind to pY1021, but not that of PI3 kinase and associated proteins, was reduced in APS transformants. PDGF induced phosphorylation of the tyrosine residue of APS close to the C-terminal end. In vitro and in vivo binding experiments indicate that the tyrosine phosphorylated C-terminal region of APS bound to c-Cbl, which has been shown to be a negative regulator of tyrosine kinases. Since coexpression of c-Cbl with wild type APS, but not C-terminal truncated APS, synergistically inhibited PDGF-induced c-fos promoter activation, c-Cbl could be a mechanism of inhibitory action of APS on PDGF receptor signaling.     

Full activation of the platelet-derived growth factor beta-receptor kinase involves multiple events

Activation of receptor tyrosine kinases is thought to involve ligand-induced dimerization, which promotes receptor transphosphorylation and thereby increases the receptor's phosphotransferase activity. We used two platelet-derived growth factor beta-receptor (beta-PDGFR) mutants to identify events that are required for full engagement (autophosphorylation and activation of the kinase activity) of the beta-PDGFR kinase. The F79/81 receptor (Tyr to Phe substitution at 579 and 581 in the juxtamembrane domain of the receptor) was capable of only very modest ligand-dependent autophosphorylation and also failed to associate with numerous SH2 domain-containing proteins. Furthermore, stimulation with platelet-derived growth factor (PDGF) did not increase the kinase activity of the F79/81 mutant toward exogenous substrates. However, the F79/81 receptor had basal kinase activity and could be artificially stimulated by incubation with ATP. Because the low kinase activity of the F857 mutant (Tyr to Phe substitution at 857 in the putative activation loop) could not be increased by incubation with ATP, failure to phosphorylate Tyr-857 may be the reason why the F79/81 mutant has low kinase activity. Surprisingly, the F857 mutant underwent efficient PDGF-dependent autophosphorylation. Thus the PDGF-dependent increase in the kinase activity of the receptor is not required for autophosphorylation. We conclude that full activation of the beta-PDGFR kinase requires at least two, apparently distinct events.     

In vitro toxicity of methyl mercury to fathead minnow cells

We have reported previously that serotonin (5-HT) stimulates the mitogenesis of bovine pulmonary artery smooth muscle cells (SMCs) through active transport of 5-HT and cellular signaling that includes elevation of superoxide (O2.-) and enhancement of protein tyrosine phosphorylation. Ginkgo biloba extract 501 (EGb 501), which has been demonstrated to act as an antioxidant, was found to block both the elevated O2.- and the proliferative and hypertrophic influences of 5-HT on SMCs, but not to directly inhibit the associated activation of NAD(P)H oxidase or the stimulation of phosphorylation of GTPase-activating protein (GAP). A similar effect of Ginkgo biloba extract 501 occurred on Chinese hamster lung fibroblasts (CCL-39), where 5-HT receptor, as opposed to transporter, action has been associated with mitogenesis. We conclude from these studies that Ginkgo biloba extract 501 quenches O2.- formation by 5-HT, thereby blocking its mitogenic effect. Stimulation of protein tyrosine phosphorylation of GAP by 5-HT appears to precede the elevation of O2.-.     

Beta 1 integrin ligation stimulates tyrosine phosphorylation of phospholipase C gamma 1 and elevates intracellular Ca2+ in pancreatic acinar cells

We have recently reported increased tyrosine (TYR) phosphorylation of a number of pancreatic acinar cell proteins following antibody ligation of beta 1 integrins (Wrenn and Herman, Biochem, Biophys. Res. Commun. 208, 1995, 978-984). Concurrent with this TYR phosphorylation was a marked activation of protein kinase C (PKC). This led us to investigate phospholipase C gamma 1 (PLC gamma 1), a key enzyme responsible for diacylglycerol generation, as a target for integrin-mediated TYR phosphorylation. Staining with antiphosphotyrosine antibodies revealed increased TYR phosphorylation of immunoprecipitated PLC gamma 1 prepared from beta 1 integrin-ligated acinar cells. Subsequent stripping and reprobing of Western blots with polyclonal anti-PLC gamma 1 was confirmatory. Over this same time period, intracellular [Ca2+] increased from < 100 nM to 600 nM, further suggesting a functional relevance of integrin-linked phosphorylation as a regulatory mechanism in exocrine pancreas.     

Actin filaments facilitate insulin activation of the src and collagen homologous/mitogen-activated protein kinase pathway leading to DNA synthesis and c-fos expression

The exact mechanism of the spatial organization of the insulin signaling pathway leading to nuclear events remains unknown. Here, we investigated the involvement of the actin cytoskeleton in propagation of insulin signaling events leading to DNA synthesis and expression of the immediate early genes c-fos and c-jun in L6 muscle cells. Insulin reorganized the cellular actin network and increased the rate of DNA synthesis and the levels of c-fos mRNA, but not those of c-jun mRNA, in undifferentiated L6 myoblasts. Similarly, insulin markedly elevated the levels of c-fos mRNA but not of c-jun mRNA in differentiated L6 myotubes. Disassembly of the actin filaments by cytochalasin D, latrunculin B, or botulinum C2 toxin significantly inhibited insulin-mediated DNA synthesis in myoblasts and abolished stimulation of c-fos expression by the hormone in myoblasts and myotubes. Actin disassembly abolished insulin-induced phosphorylation and activation of extracellulor signal-regulated kinases, activation of a 65-kda member of the p21-activated kinases, and phosphorylation of p38 mitogen-activated protein kinases but did not prevent activation of phosphatidylinositol 3-kinase and p70(S6k). Under these conditions, insulin-induced Ras activation was also abolished, and Grb2 association with the Src and collogen homologous (Shc) molecule was inhibited without inhibition of the tyrosine phosphorylation of Shc. We conclude that the actin filament network plays an essential role in insulin regulation of Shc-dependent signaling events governing gene expression by facilitating the interaction of Shc with Grb2.     

Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein-coupled receptors transactivate growth factor receptors to transmit mitogenic effects. In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II-induced extracellular signal-regulated kinase (ERK) activation, c-fos gene expression, and DNA synthesis in cardiac fibroblasts. Ang II induced a rapid tyrosine phosphorylation of EGF-R in association with phosphorylation of Shc protein and ERK activation. Specific inhibition of EGF-R function by either a dominant-negative EGF-R mutant or selective tyrphostin AG1478 completely abolished Ang II-induced ERK activation. Induction of c-fos gene expression and DNA synthesis were also abolished by the inhibition of EGF-R function. Calmodulin or tyrosine kinase inhibitors, but not protein kinase C (PKC) inhibitors or downregulation of PKC, completely abolished transactivation of EGF-R by Ang II or the Ca2+ ionophore A23187. Epidermal growth factor (EGF) activity in concentrated supernatant from Ang II-treated cells was not detected, and saturation of culture media with anti-EGF antibody did not affect the Ang II-induced transactivation of EGF-R. Conditioned media in which cells were incubated with Ang II could not induce phosphorylation of EGF-R on recipient cells. Platelet-derived growth factor-beta receptor was not phosphorylated on Ang II stimulation, and Ang II-induced c-jun gene expression was not affected by tyrphostin AG1478. Our results demonstrated that in cardiac fibroblasts Ang II-induced ERK activation and its mitogenic signals are dominantly mediated by EGF-R transactivated in a Ca2+/calmodulin-dependent manner and suggested that the effects of Ang II on cardiac fibroblasts should be interpreted in association with the signaling pathways regulating cellular proliferation and/or differentiation by growth factors.     

Focal adhesion kinase-related fakB is regulated by the integrin LFA-1 and interacts with the SH3 domain of phospholipase C gamma 1

Signal transduction through integrin molecules expressed on platelets and nonlymphoid cells involves activation of the intracellular focal adhesion kinase ppI25FAK (FAK) to phosphorylate substrate proteins on tyrosine residues. Similar mechanisms are also functional in T-lymphocytes through the beta 1-integrin VLA-4. A putative FAK-related phosphoprotein (fakB) was identified that is responsive to intracellular signals induced through ligation of antigen receptors on both T- and B-lymphocytes, and whose induced tyrosine phosphorylation is augmented by TCR costimulation through the adhesion/costimulatory receptors CD2 and CD4. In this report, fakB is shown to respond to extracellular signals through the beta 2-integrin LFA-1 in the absence of primary signals through the TCR. Protein-protein complex formation was observed involving an association between fakB, phospholipase C gamma 1 (PLC gamma 1), and the tyrosine phosphoprotein pp35-36. Evidence is provided here that fakB interacts with PLC gamma 1 through its SH3 domain. The association between fakB and PLC gamma 1 does not appear to require T-cell activation, whereas the induced tyrosine phosphorylation of the protein complex components occurs following engagement of LFA-1. These data indicate that the beta2-integrin LFA-1 expressed on T-lymphocytes stimulates a novel, FAK-related molecule that may function in the interplay between adhesion receptors and intracellular signaling enzymes responsible for downstream second messenger generation.