Epidermal growth factor stimulates tyrosine phosphorylation of specific proteins in permeabilized human fibroblasts

We have investigated the epidermal growth factor (EGF)-stimulated tyrosine-specific protein kinase activity in quiescent cultures of diploid human fibroblasts that have a well characterized mitogenic response to EGF. We developed a method of permeabilizing cells with digitonin or other agents that permitted the rapid labeling of cellular proteins with exogenously added [gamma-32P]ATP while allowing only about 25% of marker cytosolic enzymes to escape from the cells. When phosphatases were inhibited with zinc and vanadate, EGF induced up to 8-fold stimulation of the incorporation of radioactivity from [gamma-32P]ATP into a 35-kDa band on sodium dodecyl sulfate gels. Alkali treatment of gels showed that EGF stimulated the phosphorylation of bands with apparent molecular masses of 170, 45, 35, 26, 22, and 21 kDa. Phosphoamino acid analysis was performed on the 170- and 35-kDa bands and revealed that the EGF-stimulated phosphorylation was on tyrosyl residues. The 35-kDa band was resolved into four spots by two-dimensional gel electrophoresis. The most acidic form was the most prominent and it was precipitated by an antiserum against a 35-kDa protein from A-431 cells; heretofore, this protein has only been reported to be phosphorylated in an EGF-dependent manner by A-431 membranes in vitro (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). This antiserum also precipitated a 35-kDa phospho-protein from extracts of intact [32P]orthophosphate-labeled fibroblasts which was phosphorylated on tyrosine in an EGF-dependent manner. None of the forms of the 35-kDa phosphoproteins labeled in permeabilized cells were immunologically related to the 34-kDa protein that is a substrate for the tyrosyl kinase encoded by Rous sarcoma virus. Other mitogens (serum, insulin, platelet-derived growth factor, and thrombin) did not detectably stimulate phosphorylation in permeabilized cells.     

Epidermal growth factor dependent phosphorylation of a 35-kilodalton protein in placental membranes

In human placental membranes isolated in the presence of ethylenediaminetetraacetic acid (EDTA), epidermal growth factor (EGF) stimulated the [gamma-32P]ATP-dependent phosphorylation of tyrosine residues on the 170-kilodalton (kDa) EGF receptor and on a 35-kDa protein. The initial rate of phosphorylation of these proteins in the presence of EGF was 5.2 and 3.5 nmol of phosphate min-1 (mg of receptor protein)-1, and this was approximately 10- and 6-fold higher than the basal rate, respectively. Half-maximal phosphorylation of both proteins occurred at about 2.5 nM EGF. In the presence of p-nitrophenyl phosphate, EGF stimulated the phosphorylation of the 35-kDa protein but not the EGF receptor, suggesting that hormone-stimulated autophosphorylation of the receptor/kinase was not required for kinase activation. The 35-kDa protein exists in two forms: (1) 35Keluate, which was associated with the membrane in the presence of Ca2+ but was eluted with EDTA, and (2) 35Kmemb, which was not eluted from membranes with EDTA. Both forms were immunologically related to a 35-kDa protein previously isolated from A431 cells. Antiserum against the 35-kDa protein also reacted with a protein with an apparent size of 66 kDa that was phosphorylated in an EGF-dependent manner. In phosphorylation reactions performed in the presence of Mg2+, Ca2+ was required for phosphorylation of the 35Keluate form, but Ca2+ was not required for phosphorylation of the 35Kmemb form. Phosphorylation appears to change the membrane-binding properties of the 35Kmemb form because 32P-labeled 35Kmemb could be eluted from the membrane by EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor stimulates serine and tyrosine phosphorylation in a 59-kD protein in purified plasma membranes from rat liver

Preincubation of purified plasma membranes from rat liver with EGF stimulates the level of phosphorylation on serine and tyrosine residues in a 59-kD protein. Such an increased phosphoserine and phosphotyrosine content of the 59-kD protein occurs at the expense of the phosphorylation on threonine residues. The effect is observed under conditions where the plasma membranes have been extracted at pH 10. It is not observed when the membranes are simply washed at pH 7.5 before further purification. A number of experiments, including TBR-IgG phosphorylation in immunoprecipitates and partial hydrolysis with varying concentrations of the V8 protease, suggest that the 59-kD protein modified upon EGF treatment could be a representative of the c-src gene product from hepatocytes.     

Insulin-like growth factor I rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cells

The type I insulin-like growth factor (IGF) receptor, like the insulin receptor, contains a ligand-stimulated protein-tyrosine kinase activity in its beta-subunit. However, in vivo, no substrates have been identified. We used anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins which occur during IGF-I stimulation of normal rat kidney and Madin-Darby canine kidney cells labeled with ortho[32P]phosphate. Both cells provide a good system to study the function of the type I IGF receptors because they contain high concentrations of these receptors but no insulin receptors. In addition, physiological levels of IGF-I, but not insulin, stimulated DNA synthesis in growth-arrested cells. IGF-I stimulated within 1 min of tyrosine phosphorylation of two proteins. One of them, with a molecular mass between 97 and 102 kDa, was supposed to be the beta-subunit of the type I IGF receptor previously identified. The other protein had an approximate molecular mass of 185 kDa, which resembled, by several criteria, pp 185, originally identified during the initial response of Fao cells to insulin binding (White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186). These data suggest that tyrosine phosphorylation of pp 185 may occur during activation of both the type I IGF receptor and the insulin receptor, and it could be a common substrate that transmits important metabolic signals during ligand binding.     

Epidermal growth factor, but not nerve growth factor, stimulates tyrosine-specific protein-kinase activity in pheochromocytoma (PC12) plasma membranes

Rat pheochromocytoma (PC12) cells contain specific plasma membrane receptors for both epidermal growth factor (EGF) and nerve growth factor (NGF). Whereas EGF addition to PC12 cells causes a persistent enhancement of proliferation. NGF addition induces a transient stimulation of growth, followed by growth arrest and neuronal differentiation. Despite these differences in biological response, EGF and NGF share a number of early receptor-mediated responses, which are likely te be related to their effect on cell proliferation. In this paper we show that EGF, but not NGF, is able to stimulate the phosphorylation of membrane proteins. In addition, EGF was able to stimulate phosphorylation of a synthetic peptide (RR-SRC) by PC12 membranes in a concentration-dependent manner. Kinetic analysis of the phosphorylation reaction indicated that EGF increased the Vmax from 13 to 70 pmoles/min/mg protein, while no change was observed in Km. Furthermore, EGF was able to stimulate tyrosine phosphorylation of angiotensin I and II, to the same extent as RR-SRC. In contrast no effects of NGF on peptide phosphorylation by PC12 membranes were observed. Cross-linking experiments demonstrated the presence of receptors for both NGF and EGF in PC12 membranes. These different effects of NGF and EGF on activation of membrane-associated protein-kinase activity demonstrate that NGF might be able to stimulate growth transiently without stimulating protein kinase activity.     

Epidermal growth factor-specific protein tyrosine phosphorylation in preimplantation embryo development.

We examined whether epidermal growth factor (EGF)-induced preimplantation mouse embryo development and function are mediated by EGF-specific protein tyrosine phosphorylation (PTP). In situ cross-linking and autophosphorylation studies showed that EGF receptor (EGF-R) in Day 4 mouse blastocysts is a protein of approximately 170 kDa that is phosphorylated when exposed to EGF and ATP. Furthermore, EGF induced about a twofold increase in protein tyrosine kinase (PTK) activity in Day 4 blastocysts when incubated in the presence of a peptide substrate with a tyrosine moiety and ATP. RG 50864, a specific inhibitor of EGF-dependent PTK, diminished autophosphorylation of the 170-kDa protein and completely blocked PTK activity in the blastocyst induced by EGF. However, this inhibitor did not affect EGF binding to the embryonic cell surface. In contrast, an inactive tyrphostin compound, RG 50862, did not alter EGF-induced PTK activity in the blastocyst. These findings led us to examine the effects of these tyrphostin compounds on preimplantation mouse embryo development and blastocyst hatching in vitro. RG 50864, in a dose-dependent manner, inhibited EGF-dependent development of 2-cell embryos to blastocysts and the number of cells per blastocyst. This inhibitor also antagonized EGF-induced zona-hatching of blastocysts formed from 8-cell embryos in culture. However, the inhibitor was not effective in deterring transforming growth factor-beta 1-induced blastocyst formation. The inactive compound, RG 50862, had no effects on EGF-dependent blastocyst formation or zona-hatching. The data show that the effects of RG 50864 are specific and mediated by inhibition of EGF-specific PTK activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells

Addition of EGF to A431 cells at physiological concentrations causes a rapid three- to four-fold increase in the abundance of phosphotyrosine in cellular protein. The increase is essentially complete within 1 min and is maintained for several hours. No change in phosphotyrosine levels is found with fibroblast growth factor or insulin. Two phosphoproteins (molecular weights of 39 and 81 kd) containing phosphotyrosine appear de novo upon administration of EGF to A431 cells. The EGF receptor itself is a phosphoprotein containing phosphotyrosine as well as phosphoserine and phosphothreonine. Changes in the phosphorylation pattern of the EGF receptor are seen upon treatment of A431 cells with EGF. Increased phosphorylation of tyrosine is the most rapid response of cells to EGF known, and may play an important role in the biological effects of EGF.     

Epidermal growth factor and sphingosine-1-phosphate stimulate Na+/H+ exchanger activity in the human placental syncytiotrophoblast

The Na+/H+ exchanger (NHE) has a key role in intracellular pH ([pH]i) regulation of the syncytiotrophoblast in the human placenta and may have a role in the life cycle of this cell. In other cells the NHE (actually a family of up to 9 isoforms) is regulated by a variety of factors, but its regulation in the syncytiotrophoblast has not been studied. Here, we tested the hypotheses that EGF and sphingosine-1-phosphate (S1P), both of which affect trophoblast apoptosis and, in other cell types, NHE activity, stimulate syncytiotrophoblast NHE activity. Villous fragments from term human placentas were loaded with the pH-sensitive dye, BCECF. NHE activity was measured by following the recovery of syncytiotrophoblast [pH]i following an imposed acid load, in the presence and absence of EGF, S1P, and specific inhibitors of NHE activity. Both EGF and S1P caused a dose-dependent upregulation of NHE activity in the syncytiotrophoblast. These effects were blocked by amiloride 500 microM (a nonspecific NHE blocker) and HOE694 100 microM (NHE blocker with NHE1 and 2 isoform selectivity). Effects of EGF were also reduced by the NHE3 selective blocker S3226 (used at 1 microM). These data provide the first evidence that both EGF and S1P stimulate NHE activity in the syncytiotrophoblast; they appear to do so predominantly by activating the NHE1 isoform.     

Epidermal growth factor-stimulated tyrosine phosphorylation of caveolin-1. Enhanced caveolin-1 tyrosine phosphorylation following aberrant epidermal growth factor receptor status

Caveolin-1 is the major coat protein of caveolae and has been reported to interact with various intracellular signaling molecules including the epidermal growth factor (EGF) receptor. To investigate the involvement of caveolin-1 in EGF receptor action, we used mouse B82L fibroblasts transfected with (a) wild type EGF receptor, (b) a C-terminally truncated EGF receptor at residue 1022, (c) a C-terminally truncated EGF receptor at residue 973, or (d) a kinase-inactive EGF receptor (K721M). Following EGF treatment, there was a distinct electrophoretic mobility shift of the caveolin-1 present in cells expressing the truncated forms of the EGF receptor, but this shift was not detectable in cells bearing either normal levels of the wild type EGF receptor or a kinase-inactive receptor. This mobility shift was also not observed following the addition of other cell stimuli, such as platelet-derived growth factor, insulin, basic fibroblast growth factor, or phorbol 12-myristate 13-acetate. Analysis of caveolin-1 immunoprecipitates from EGF-stimulated or nonstimulated cells demonstrated that the EGF-induced mobility shift of caveolin-1 was associated with its tyrosine phosphorylation in cells expressing truncated EGF receptors. Maximal caveolin-1 phosphorylation was achieved within 5 min after exposure to 10 nM EGF and remained elevated for at least 2 h. Additionally, several distinct phosphotyrosine-containing proteins (60, 45, 29, 24, and 20 kDa) were co-immunoprecipitated with caveolin-1 in an EGF-dependent manner. Furthermore, the Src family kinase inhibitor, PP1, does not affect autophosphorylation of the receptor, but it does inhibit the EGF-induced mobility shift and phosphorylation of caveolin-1. Conversely, the MEK inhibitors PD98059 and UO126 could attenuate EGF-induced mitogen-activated protein kinase activation, they do not affect the EGF-induced mobility shift of caveolin-1. Because truncation and overexpression of the EGF receptor have been linked to cell transformation, these results provide the first evidence that the tyrosine phosphorylation of caveolin-1 occurs via an EGF-sensitive signaling pathway that can be potentiated by an aberrant activity or expression of various forms of the EGF receptor.     

Epidermal growth factor and epidermal growth factor receptor-dependent phosphorylation of a Mr = 34,000 protein substrate for pp60src

A Mr = 34,000 protein present in the 100,000 X g supernatant fraction from A431 human epidermoid carcinoma cells is the major radiolabeled phosphate acceptor from [gamma-32P]ATP in a cell-free system requiring epidermal growth factor (EGF) and EGF receptor kinase. This protein is immunoprecipitated by IgG directed against avian Mr = 34,000 cellular substrate for pp60src. Phosphoamino acid analysis of the Mr = 34,000 protein labeled with 32Pi from [gamma-32P]ATP in a cell-free system requiring EGF and EGF receptor kinase yielded radiolabeled phosphotyrosine with no detectable radioactivity in phosphoserine or phosphothreonine.     

Insulin stimulates the tyrosine phosphorylation of a Mr = 160,000 glycoprotein in rat adipocyte plasma membranes

The action of insulin on tyrosine phosphorylation of plasma membrane-associated proteins in rat adipocytes was investigated. Incubation of plasma membranes from insulin-treated adipocytes with [gamma-32P] ATP results in a marked increase in tyrosine phosphorylation of Mr = 160,000 (P160) and Mr = 92,000 proteins when compared to controls. Based on the immunoreactivities of these two proteins with anti-insulin receptor antibodies, the Mr = 92,000 species is identified as the insulin receptor beta subunit while P160 is unrelated to the receptor structure. P160 appears to be a glycoprotein as evidenced by its adsorption to wheat germ agglutinin-agarose. The tyrosine phosphorylation of P160 exhibits a rapid response to insulin (maximal within 2 min at 37 degrees C) and is readily reversed following removal of the free hormone by anti-insulin serum. The time courses of insulin-stimulated phosphorylation as well as the dephosphorylation of P160 coincide with those of the activation and deactivation of the insulin receptor kinase in the same plasma membrane preparation. Concanavalin A and hydrogen peroxide mimic insulin stimulation of the insulin receptor kinase and enhance the tyrosine phosphorylation of P160. Isoproterenol, epidermal growth factor, and phorbol diester are without effects. Analysis of the insulin dose-response relationship between P160 tyrosine phosphorylation and insulin receptor kinase activity reveals that maximal phosphorylation of P160 occurs when only a fraction (25%) of the receptor kinase is activated by the hormone. A similar relationship between these two parameters is observed for the insulinomimetic agent hydrogen peroxide. The close correlation between the level of P160 phosphorylation and insulin receptor kinase activity suggests that P160 may be tyrosine phosphorylated by the receptor kinase following receptor kinase activation by the hormone or insulin-like agents. This hypothesis is further supported by the finding that the insulin receptor kinase is the only insulin-sensitive tyrosine kinase detectable in adipocyte plasma membranes under the conditions of our experiments.     

Epidermal growth factor stimulates the phosphorylation of pyruvate kinase in freshly isolated rat hepatocytes.

Epidermal growth factor (EGF) has previously been shown to stimulate gluconeogenesis in rat liver by decreasing the activity of pyruvate kinase [(1988) Biochem. J. 255, 361-364]. Here we investigate the mechanism underlying the inactivation of the enzyme. EGF was found to increase the incorporation of phosphate into pyruvate kinase, with maximal phosphorylation achieved only after 10 min in the presence of the growth factor. The increase in phosphorylation was not additive with that caused by cyclic AMP. Phosphoamino acid analysis of pyruvate kinase isolated from cells treated with EGF indicated that EGF increases phosphorylation solely on serine residues. The exact site of EGF-mediated phosphorylation has yet to be identified.     

c-Kit-kinase induces a cascade of protein tyrosine phosphorylation in normal human melanocytes in response to mast cell growth factor and stimulates mitogen-activated protein kinase but is down-regulated in melanomas.

The proto-oncogene c-Kit, a transmembrane receptor tyrosine kinase, is an important regulator of cell growth whose constitutively active oncogenic counterpart, v-kit, induces sarcomas in cats. Mutations in murine c-kit that reduce the receptor tyrosine kinase activity cause deficiencies in the migration and proliferation of melanoblasts, hematopoietic stem cells, and primordial germ cells. We therefore investigated whether c-Kit regulates normal human melanocyte proliferation and plays a role in melanomas. We show that normal human melanocytes respond to mast cell growth factor (MGF), the Kit-ligand that stimulates phosphorylation of tyrosyl residues in c-Kit and induces sequential phosphorylation of tyrosyl residues in several other proteins. One of the phosphorylated intermediates in the signal transduction pathway was identified as an early response kinase (mitogen-activated protein [MAP] kinase). Dephosphorylation of a prominent 180-kDa protein suggests that MGF also activates a phosphotyrosine phosphatase. In contrast, MGF did not induce proliferation, the cascade of protein phosphorylations, or MAP kinase activation in the majority of cells cultured from primary nodular and metastatic melanomas that grow independently of exogenous factors. In the five out of eight human melanoma lines expressing c-kit mRNAs, c-Kit was not constitutively activated. Therefore, although c-Kit-kinase is a potent growth regulator of normal human melanocytes, its activity is not positively associated with malignant transformation.     

Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases

Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites.     

Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.     

Elevation of cAMP, but not cGMP, inhibits thrombin-stimulated tyrosine phosphorylation in human platelets.

Platelets have abundant tyrosine kinase activities, and activation of platelets results in the increased tyrosine phosphorylation of numerous protein substrates. The stimulation of tyrosine phosphorylation elicited by thrombin can be completely inhibited by preincubation with 10nm prostacyclin (PGI2), 1 microM PGD2, or 1mM N2,2'-O-dibutyryl-cAMP. In contrast, incubation of platelets with agents that increase cGMP (sodium nitroprusside or with 1mM 8-Bromo-cGMP) was without effect. The inhibition by prostacyclin was dose dependent, with an IC50 of approximately 3nM, corresponding to the dose range necessary to inhibit other platelet activation processes. These results demonstrate a novel pathway by which agents which raise cAMP may inhibit platelet signal transduction and differential mechanism of action between compounds which raise cAMP and those which elevate cGMP.     

Vanadate stimulates tyrosine phosphorylation of two proteins in Raji human lymphoblastoid cell membranes

A membrane fraction from Raji human lymphoblastoid cells exhibited tyrosine-specific kinase activity. Vanadate increased tyrosine phosphorylation up to 5-fold; serine and threonine phosphorylation were unchanged. The stimulation was detectable within 15 s at 0 degrees C and at concentrations of vanadate (0.3 and 1.0 microM) present in normal tissues and blood. The tyrosine phosphorylation of two substrates, M1 61 000 and 55 000, was dependent upon vanadate and incorporation into these substrates represented the majority of the vanadate-sensitive tyrosine phosphorylation.     

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).     

Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3

Here, we report the identification of a new tensin family member, tensin3, and its role in epidermal growth factor (EGF) signaling pathway. Human tensin3 cDNA encodes a 1445 amino acid sequence that shares extensive homology with tensin1, tensin2, and COOH-terminal tensin-like protein. Tensin3 is expressed in various tissues and in different cell types such as endothelia, epithelia, and fibroblasts. The potential role of tensin3 in EGF-induced signaling pathway is explored. EGF induces tyrosine phosphorylation of tensin3 in MDA-MB-468 cells in a time- and dose-dependent manner, but it is independent of an intact actin cytoskeleton or phosphatidylinositol 3-kinase. Activation of EGF receptor is necessary but not sufficient for tyrosine phosphorylation of tensin3. It also requires Src family kinase activities. Furthermore, tensin3 forms a complex with focal adhesion kinase and p130Cas in MDA-MB-468 cells. Addition of EGF to the cells induces dephosphorylation of these two molecules, leads to disassociation of the tensin3-focal adhesion kinase-p130Cas complex, and enhances the interaction between tensin3 and EGF receptor. Our results demonstrate that tensin3 may function as a platform for the disassembly of EGF-related signaling complexes at focal adhesions.