The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes.

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.     

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

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

Reconstitution of the high affinity epidermal growth factor receptor on cell-free membranes after transmodulation by platelet-derived growth factor

We have prepared plasma membranes from Balb/c 3T3 fibroblasts to study the transmodulation of the high affinity epidermal growth factor (EGF) receptor. Although phorbol esters do not transmodulate the high affinity EGF receptors on these membranes, the addition of platelet-derived growth factor (PDGF) or EGF to the membranes leads to the loss of high affinity EGF binding and to the phosphorylation of several membrane proteins, including the EGF receptor. The EGF receptor is phosphorylated at tyrosine residues although we have not yet established if this represents direct phosphorylation by the PDGF receptor kinase or is mediated by activation of other cell membrane-associated tyrosine kinases. Upon treatment of the membranes with PDGF, four major phosphoproteins (of apparent molecular masses of 69, 56, 38, and 28 kDa) are released from the membrane and can be retrieved from the supernatant fluid using a reversed-phase cartridge. As assessed by immunoprecipitation with an anti-phosphotyrosine antibody, all four proteins appear to be phosphorylated on tyrosine. The time course of dissociation of these proteins from the membranes closely parallels the loss of high affinity EGF receptors. The high affinity EGF receptor can be reconstituted on PDGF-transmodulated membranes by treating the supernatant fluid with alkaline phosphatase and adding the mixture to the membranes. It appears that dephosphorylation of the released proteins is sufficient to allow reassociation with the membranes and formation of the high affinity EGF receptor complex.     

Characterization of the interaction of 5'-p-fluorosulfonylbenzoyl adenosine with the epidermal growth factor receptor/protein kinase in A431 cell membranes

Treatment of membrane vesicles from A431 cells, a human epidermoid carcinoma line, with the affinity label 5'-p-fluorosulfonylbenzoyl [8-14C]adenosine (5'-p-FSO2Bz[14C]Ado) results in an inhibition of the epidermal growth factor (EGF)-stimulable protein kinase and in the modification of proteins having the same molecular weight (Mr = 170,000 and 150,000) as the receptor for EGF (Buhrow, S. A., Cohen, S., and Staros, J. V. (1982) J. Biol. Chem. 257, 4019-4022). Modification of the vesicles with 5'-p-FSO2BzAdo inhibits not only the EGF-stimulated phosphorylation of endogenous membrane proteins but also the EGF-stimulated phosphorylation of an exogenous synthetic tyrosine-containing peptide substrate. This indicates that the EGF-stimulable protein kinase is modified by 5'-p-FSO2BzAdo at a site affecting catalytic activity. Membrane vesicles were treated with 5'-p-FSO2Bz-[14C]Ado to affinity label the kinase, then the EGF receptor was purified by affinity chromatography on immobilized EGF. The EGF receptor thus purified contains the 5'-p-SO2Bz[14C]Ado moiety. These data strongly support our hypothesis that the EGF receptor and EGF-stimulable kinase are two parts of the same polypeptide chain.     

Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni

The epidermal growth factor receptor (EGF-R) plays an important role in development and cell differentiation, and homologues of EGF-R have been identified in a broad range of vertebrate and invertebrate organisms. This work concerns the functional characterization of SER, the EGF-R-like molecule previously identified in the helminth parasite Schistosoma mansoni. Transactivation assays performed in epithelial Madin-Darby canine kidney cells co-transfected with SER and a Ras-responsive reporter vector indicated that SER was able to trigger a Ras/ERK pathway in response to human epidermal growth factor (EGF). These results were confirmed in Xenopus oocytes showing that human EGF induced meiosis reinitiation characterized by germinal vesicle breakdown in SER-expressing oocytes. Germinal vesicle breakdown induced by EGF was dependent on receptor kinase activity and shown to be associated with phosphorylation of SER and of downstream ERK proteins. (125)I-EGF binding experiments performed on SER-expressing oocytes revealed high affinity (2.9 x 10(-9) M) of the schistosome receptor for human EGF. Phosphorylation of the native SER protein present in S. mansoni membranes was also shown to occur upon binding of human EGF. These data demonstrate the ability of the SER schistosome receptor to be activated by vertebrate EGF ligands as well as to activate the classical ERK pathway downstream, indicating the conservation of EGF-R function in S. mansoni. Moreover, human EGF was shown to increase protein and DNA synthesis as well as protein phosphorylation in parasites, supporting the hypothesis that host EGF could regulate schistosome development. The possible role of SER as a receptor for host EGF peptides and its implication in host-parasite signaling and parasite development are discussed.     

Specific association of annexin 1 with plasma membrane-resident and internalized EGF receptors mediated through the protein core domain

Phosphorylation of the Ca2+ and membrane-binding protein annexin 1 by epidermal growth factor (EGF) receptor tyrosine kinase has been thought to be involved in regulation of the EGF receptor trafficking. To elucidate the interaction of annexin 1 during EGF receptor internalization, we followed the distribution of annexin 1-GFP fusion proteins at sites of internalizing EGF receptors. The observed association of annexin 1 with EGF receptors was confirmed by immunoprecipitation. We found that this interaction was independent of a functional phosphorylation site in the annexin 1 N-terminal domain but mediated through the Ca2+ binding core domain.     

Phosphorylation-induced conformational changes regulate GGAs 1 and 3 function at the trans-Golgi network

The GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) are a family of multidomain proteins implicated in protein trafficking between the Golgi and the endosomes. All three GGAs (1, 2, and 3) bind to the mannose 6-phosphate receptor tail via their VHS domains, as well as to the adaptor protein complex-1 via their hinge domains. The latter interaction has been proposed to be important for cooperative packaging of cargo into forming clathrin-coated carriers at the trans-Golgi network. Here we present evidence that GGA1 function is highly regulated by cycles of phosphorylation and dephosphorylation. Cell fractionation showed that the phosphorylated pool of GGA1 resided predominantly in the cytosol and that recruitment onto membranes was associated with dephosphorylation. Okadaic acid inhibition studies and in vitro dephosphorylation assays indicated that dephosphorylation is mediated by a protein phosphatase 2A-like phosphatase. Dephosphorylation of GGA1 induced a change in the conformation to an "open" form as measured by gel filtration and sucrose gradient analyses. This was associated with enhanced binding to ligands because of release of autoinhibition and increased binding to the adaptor protein complex-1 gamma-appendage. A model is proposed for the regulation of GGA1 function at the trans-Golgi network.     

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)     

Clonal variants of differentiated P19 embryonal carcinoma cells exhibit epidermal growth factor receptor kinase activity

Differentiated clonal cell lines were isolated from pluripotent P19 embryonal carcinoma (EC) cells treated as aggregates with retinoic acid. Two were characterized in detail. The lines differ in morphology, proliferation rate, the production of plasminogen activator, and in their mitogenic response to insulin but both produce extracellular matrix proteins and can be serially passaged over extended periods, in contrast to differentiated derivatives of many other EC lines. Further, both lines have receptors for and respond mitogenically to epidermal growth factor (EGF). Endogenous phosphorylation of several proteins, including the EGF receptor (150 kDa) and a 38-kDa protein, is induced by EGF in membranes isolated from these cells. Preincubation of membranes with EGF renders them able to catalyze phosphorylation of tyrosine residues in exogenously added peptide substrates. High voltage electrophoresis confirmed the tyrosine specificity of the phosphorylation on the 150- and 38-kDa bands. By contrast, similar experiments in undifferentiated cells showed that intact P19 EC neither bind nor respond to EGF mitogenically and EGF induces no changes in phosphorylation in isolated membranes.     

The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.     

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

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

A calcium-dependent 35-kilodalton substrate for epidermal growth factor receptor/kinase isolated from normal tissue

We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, can serve as a substrate for the epidermal growth factor (EGF) receptor/tyrosine kinase (Fava, R.A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). We now report the detection of an antigenically related 35-kDa protein in a number, but not all, of rat, pig, and human tissues. These antigenically related proteins also can serve as substrates for the EGF receptor/kinase in the presence of Ca2+. All of these proteins share the property of reversible, Ca2+-dependent binding to the particulate fraction (presumably membranes) of cell homogenates. We have isolated the 35-kDa substrate from porcine lung and have demonstrated that it is a Ca2+-binding protein. The amino-terminal sequence and the site of tyrosine phosphorylation therein have been determined. The positions of the acidic amino acid residues amino-terminal to the tyrosine phosphorylation site bear a distinct resemblance to the sequence in the homologous region of a number of other substrates for tyrosine kinases. Based on available data, the 35-kDa protein clearly differs from the protein I complex derived from intestinal mucosa and thought to be related to the proteins isolated herein (Gerke, V., and Weber, K. (1985) J. Biol. Chem. 260, 1688-1695). Finally, we report a striking sequence homology between the porcine 35-kDa described herein and human lipocortin, a phospholipase A2 inhibitor.     

Mechanism of phosphorylation of the epidermal growth factor receptor at threonine 669

The major site of phosphorylation of the epidermal growth factor (EGF) receptor after treatment of cells with EGF is threonine 669. Phosphorylation of this site is also associated with the transmodulation of the EGF receptor caused by platelet-derived growth factor and phorbol ester. A distinctive feature of the primary sequence surrounding threonine 669 is the proximity of 2 proline residues (-Pro-Leu-Thr669-Pro-). This site is not a substrate for phosphorylation by protein kinase C. To investigate the mechanism of the increased phosphorylation of the EGF receptor at threonine 669, in vitro assays were used to measure protein kinase and protein phosphatase activities present in homogenates prepared from cells treated with and without EGF. No evidence for the regulation of protein phosphatase activity was obtained in experiments using the [32P]phosphate-labeled EGF receptor as a substrate. A synthetic peptide corresponding to residues 663-681 of the EGF receptor was used as a substrate for protein kinase assays. Incubation of murine 3T3 L1 pre-adipocytes and human WI-38 fibroblasts with EGF caused a rapid increase (3-10-fold) in the level of threonine protein kinase activity detected in cell homogenates. Similar results were obtained after EGF treatment of Chinese hamster ovary cells expressing wild-type (Thr669) and mutated (Ala669) human EGF receptors. Activation of the threonine protein kinase activity was also observed in cells treated with platelet-derived growth factor, serum, and phorbol ester. Insulin-like growth factor-1 caused no significant change in protein kinase activity. Together these data indicate a role for the regulation of the activity of a threonine protein kinase in the control of the phosphorylation state of the EGF receptor at threonine 669. The significance of the identification of a growth factor-stimulated threonine protein kinase to the mechanism of signal transduction is discussed.     

Interplay between PKC and the MAP kinase pathway in Connexin43 phosphorylation and inhibition of gap junction intercellular communication

Gap junction channels are made of a family proteins called connexins. The best-studied type of connexin, Connexin43 (Cx43), is phosphorylated at several sites in its C-terminus. The tumor-promoting phorbol ester TPA strongly inhibits Cx43 gap junction channels. In this study we have investigated mechanisms involved in TPA-induced phosphorylation of Cx43 and inhibition of gap junction channels. The data show that TPA-induced inhibition of gap junction intercellular communication (GJIC) is dependent on both PKC and the MAP kinase pathway. The data suggest that PKC-induced activation of MAP kinase partly involves Src-independent trans-activation of the EGF receptor, and that TPA-induced shift in SDS-PAGE gel mobility of Cx43 is caused by MAP kinase phosphorylation, whereas phosphorylation of S368 by PKC does not alter gel migration of Cx43. We also show that TPA, in addition to phosphorylation of S368, also induces phosphorylation of S255 and S262, in a MAP kinase-dependent manner. The data add to our understanding of the molecular mechanisms involved in the interplay between signaling pathways in regulation of GJIC.     

SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.

Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Inhibition of Src family kinases blocks epidermal growth factor (EGF)-induced activation of Akt, phosphorylation of c-Cbl, and ubiquitination of the EGF receptor

Stimulation of T47D cells with epidermal growth factor (EGF) results in the activation of the intrinsic tyrosine kinases of the receptor and the phosphorylation of multiple cellular proteins including the receptor, scaffold molecules such as c-Cbl, adapter molecules such as Shc, and the serine/threonine protein kinase Akt. We demonstrate that EGF stimulation of T47D cells results in the activation of the Src protein-tyrosine kinase and that the Src kinase inhibitor PP1 blocks the EGF-induced phosphorylation of c-Cbl but not the activation/phosphorylation of the EGF receptor itself. PP1 also blocks EGF-induced ubiquitination of the EGF receptor, which is presumably mediated by phosphorylated c-Cbl. Src is associated with c-Cbl, and we have previously demonstrated that the Src-like kinase Fyn can phosphorylate c-Cbl at a preferred binding site for the p85 subunit of phosphatidylinositol 3'-kinase. PP1 treatment blocks EGF-induced activation of the anti-apoptotic protein kinase Akt suggesting that Src may regulate activation of Akt, perhaps by a Src --> c-Cbl --> phosphatidylinositol 3'-kinase --> Akt pathway.     

Effects of substitution of threonine 654 of the epidermal growth factor receptor on epidermal growth factor-mediated activation of phospholipase C

Addition of epidermal growth factor (EGF) to many cell types activates phospholipase C resulting in increased levels of diacylglycerol and intracellular Ca2+ which may lead to activation of protein kinase C. EGF treatment of cells can also lead to phosphorylation of the EGF receptor at threonine 654 (a protein kinase C phosphorylation site) which appears to attenuate some aspects of receptor signaling. Thus, a feedback loop involving the EGF receptor, phospholipase C, and protein kinase C may regulate EGF receptor function. In this report, the role of phosphorylation of threonine 654 of the EGF receptor in regulation of EGF-stimulated activation of phospholipase C was investigated. NIH-3T3 cells expressing the normal human EGF receptor or expressing EGF receptor in which an alanine residue had been substituted at residue 654 of the receptor were used. Addition of EGF to cells expressing wild-type receptor induced a rapid, but transient, increase in phosphorylation of threonine 654. EGF addition also caused the rapid accumulation of inositol phosphates in these cells. EGF-stimulated accumulation of inositol phosphates was significantly higher in cells expressing Ala-654 receptors compared to control cells. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulated phosphorylation of threonine 654 to a greater degree than EGF, completely inhibited EGF-dependent inositol phosphate accumulation in cells expressing wild-type receptor, but caused only a 20-30% inhibition in Ala-654 expressing cells. EGF stimulated phosphorylation of phospholipase C-gamma on serine and tyrosine residues in cells expressing wild-type of Ala-654 receptors. However, TPA treatment of cells inhibited EGF-induced tyrosine phosphorylation of phospholipase C-gamma only in cells expressing wild-type receptors. Similarly, TPA inhibited tyrosine-specific autophosphorylation of the EGF receptor and tyrosine phosphorylation of several other proteins in wild-type receptor cells, but not in Ala-654 cells. TPA treatment abolished high affinity binding of EGF to cells expressing wild-type receptors, while decreasing the number of high affinity binding sites 20-30% in Ala-654 cells. These data suggest that phosphorylation of threonine 654 can regulate early events in EGF receptor signal transduction such as phosphoinositide turnover, probably through a feedback mechanism involving protein kinase C. Subsequent dephosphorylation of threonine 654 could reactivate the EGF receptor for participation in later signaling events.     

PKB-mediated negative feedback tightly regulates mitogenic signalling via Gab2.

Heregulin (HRG)-induced tyrosine phosphorylation of the Gab2 docking protein was enhanced by pretreatment with wortmannin, indicating negative regulation via a PI3-kinase-dependent pathway. This represents phosphorylation by the serine/threonine kinase protein kinase B (PKB), since PKB constitutively associates with Gab2, phosphorylates Gab2 on a consensus phosphorylation site, Ser159, in vitro and inhibits Gab2 tyrosine phosphorylation. However, expression of Gab2 mutated at this site (S159A Gab2) not only enhanced HRG-induced Gab2 tyrosine phosphorylation and association with Shc and ErbB2, but also markedly increased tyrosine phosphorylation of ErbB2 and other cellular proteins and amplified activation of the ERK and PKB pathways. The impact of this negative regulation was further emphasized by a potent transforming activity for S159A Gab2, but not wild-type Gab2, in fibroblasts. These studies establish Gab2 as a proto-oncogene, and a model in which receptor recruitment of Gab2 is tightly regulated via an intimate association with PKB. Release of this negative constraint enhances growth factor receptor signalling, possibly since Gab2 binding limits dephosphorylation and disassembly of receptor-associated signalling complexes.