In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nck adapters are involved in the formation of dorsal ruffles, cell migration, and Rho signaling downstream of the platelet-derived growth factor beta receptor

The SH3 and SH2 domain-containing adapter proteins Nck1 and Nck2 are known to function downstream of activated tyrosine kinase receptors, such as the platelet-derived growth factor (PDGF) receptors. The SH2 domain of Nck1 binds to phosphorylated tyrosine residue 751 in PDGFbeta receptor and has been suggested to have a role in the PDGF-induced mobilization of the actin filament system. Because Tyr-751 is a site for additional receptor interactors, it has been difficult to discriminate the signaling from Nck from signaling via other molecules. For this reason we have used mouse embryonic fibroblasts derived from mice in which the genes for Nck1 and Nck2 have been inactivated by gene targeting (knock-out (KO) cells). The mutant cells had a reduced ability to form edge ruffles in response to PDGF, and the presence of Nck was obligatory for the formation of dorsal ruffles. In addition, the KO cells had a reduced chemotactic and migratory potential. Importantly, KO cells had reduced cell attachment properties and a reduced ability to form focal adhesions in response to serum stimulation. Moreover, signaling involving the Rho GTPases was defective in KO cells. In summary, our observations suggest that the Nck adapters are needed for signaling to Rho GTPases and actin dynamics downstream of the PDGFbeta receptor.     

Nckbeta adapter regulates actin polymerization in NIH 3T3 fibroblasts in response to platelet-derived growth factor bb

The SH3-SH3-SH3-SH2 adapter Nck represents a two-gene family that includes Nckalpha (Nck) and Nckbeta (Grb4/Nck2), and it links receptor tyrosine kinases to intracellular signaling networks. The function of these mammalian Nck genes has not been established. We report here a specific role for Nckbeta in platelet-derived growth factor (PDGF)-induced actin polymerization in NIH 3T3 cells. Overexpression of Nckbeta but not Nckalpha blocks PDGF-stimulated membrane ruffling and formation of lamellipoda. Mutation in either the SH2 or the middle SH3 domain of Nckbeta abolishes its interfering effect. Nckbeta binds at Tyr-1009 in human PDGF receptor beta (PDGFR-beta) which is different from Nckalpha's binding site, Tyr-751, and does not compete with phosphatidylinositol-3 kinase for binding to PDGFR. Microinjection of an anti-Nckbeta but not an anti-Nckalpha antibody inhibits PDGF-stimulated actin polymerization. Constitutively membrane-bound Nckbeta but not Nckalpha blocks Rac1-L62-induced membrane ruffling and formation of lamellipodia, suggesting that Nckbeta acts in parallel to or downstream of Rac1. This is the first report of Nckbeta's role in receptor tyrosine kinase signaling to the actin cytoskeleton.     

Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells.

The activated platelet-derived growth factor (PDGF) receptor physically associates with p85, a subunit of phosphatidylinositol-3 kinase. Although this interaction may activate phosphatidylinositol-kinase and is crucial for PDGF-induced mitogenesis, it has not been shown whether p85 is modified in the process. p85 contains two SH2 (Src homology) domains, designated SH2-N and SH2-C. Recent experiments have shown that the SH2-C domain alone determines high-affinity binding of p85 to the PDGF receptor. The function of SH2-N, which binds receptors with lower affinity, is unknown. In this study, using a receptor-blotting technique, we find that p85 is modified by PDGF stimulation of intact cells. This modification involves inhibition of binding of the SH2-N region of p85 to the PDGF receptor. Studies with vanadate suggest that tyrosine phosphorylation of p85 is responsible for the modification of p85 detected by receptor blotting. Furthermore, recombinant p85 is modified in a similar manner when it is tyrosine phosphorylated in vitro by PDGF receptors. Tyrosine phosphorylation of p85 does not block binding of the SH2-C domain and therefore does not release p85 from high-affinity binding sites on the receptor in vitro. Instead, phosphorylation may regulate the ability of the SH2-N of p85 to bind to a different portion of the PDGF receptor or to another molecule in the signaling complex. This study provides the first evidence that p85 is tyrosine phosphorylated upon PDGF stimulation of cells and suggests that tyrosine phosphorylation of p85 regulates its activity or its interaction with other proteins.     

Molecular Dynamics Simulations Reveal that Tyr-317 Phosphorylation Reduces Shc Binding Affinity for Phosphotyrosyl Residues of Epidermal Growth Factor Receptor

The Src homology 2 (SH2) and collagen domain protein Shc plays a pivotal role in signaling via tyrosine kinase receptors, including epidermal growth factor receptor (EGFR). Shc binding to phospho-tyrosine residues on activated receptors is mediated by the SH2 and phospho-tyrosine binding (PTB) domains. Subsequent phosphorylation on Tyr-317 within the Shc linker region induces Shc interactions with Grb2-Son of Sevenless that initiate Ras-mitogen-activated protein kinase signaling. We use molecular dynamics simulations of full-length Shc to examine how Tyr-317 phosphorylation controls Shc conformation and interactions with EGFR. Our simulations reveal that Shc tyrosine phosphorylation results in a significant rearrangement of the relative position of its domains, suggesting a key conformational change. Importantly, computational estimations of binding affinities show that EGFR-derived phosphotyrosyl peptides bind with significantly more strength to unphosphorylated than to phosphorylated Shc. Our results unveil what we believe is a novel structural phenomenon, i.e., tyrosine phosphorylation of Shc within its linker region regulates the binding affinity of SH2 and PTB domains for phosphorylated Shc partners, with important implications for signaling dynamics.     

Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.     

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.     

The SH2 and SH3 domain-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors.

Signalling proteins such as phospholipase C-gamma (PLC-gamma) or GTPase-activating protein (GAP) of ras contain conserved regions of approximately 100 amino acids termed src homology 2 (SH2) domains. SH2 domains were shown to be responsible for mediating association between signalling proteins and tyrosine-phosphorylated proteins, including growth factor receptors. Nck is an ubiquitously expressed protein consisting exclusively of one SH2 and three SH3 domains. Here we show that epidermal growth factor or platelet-derived growth factor stimulation of intact human or murine cells leads to phosphorylation of Nck protein on tyrosine, serine, and threonine residues. Similar stimulation of Nck phosphorylation was detected upon activation of rat basophilic leukemia RBL-2H3 cells by cross-linking of the high-affinity immunoglobulin E receptors (Fc epsilon RI). Ligand-activated, tyrosine-autophosphorylated platelet-derived growth factor or epidermal growth factor receptors were coimmunoprecipitated with anti-Nck antibodies, and the association with either receptor molecule was mediated by the SH2 domain of Nck. Addition of phorbol ester was also able to stimulate Nck phosphorylation on serine residues. However, growth factor-induced serine/threonine phosphorylation of Nck was not mediated by protein kinase C. Interestingly, approximately fivefold overexpression of Nck in NIH 3T3 cells resulted in formation of oncogenic foci. These results show that Nck is an oncogenic protein and a common target for the action of different surface receptors. Nck probably functions as an adaptor protein which links surface receptors with tyrosine kinase activity to downstream signalling pathways involved in the control of cell proliferation.     

YXXL motifs in SH2-Bbeta are phosphorylated by JAK2, JAK1, and platelet-derived growth factor receptor and are required for membrane ruffling

SH2-Bbeta binds to the activated form of JAK2 and various receptor tyrosine kinases. It is a potent stimulator of JAK2, is required for growth hormone (GH)-induced membrane ruffling, and increases mitogenesis stimulated by platelet-derived growth factor (PDGF) and insulin-like growth factor I. Its domain structure suggests that SH2-Bbeta may act as an adapter protein to recruit downstream signaling proteins to kinase.SH2-Bbeta complexes. SH2-Bbeta is tyrosyl-phosphorylated in response to GH and interferon-gamma, stimulators of JAK2, as well as in response to PDGF and nerve growth factor. To begin to elucidate the role of tyrosyl phosphorylation in the function of SH2-Bbeta, we used phosphopeptide mapping, mutagenesis, and a phosphotyrosine-specific antibody to identify Tyr-439 and Tyr-494 in SH2-Bbeta as targets of JAK2 both in vitro and in intact cells. SH2-Bbeta lacking Tyr-439 and Tyr-494 inhibits GH-induced membrane ruffling but still activates JAK2. We provide evidence that JAK1, like JAK2, phosphorylates Tyr-439 and Tyr-494 in SH2-Bbeta and that PDGF receptor phosphorylates SH2-Bbeta on Tyr-439. Therefore, phosphorylated Tyr-439 and/or Tyr-494 in SH2-Bbeta may provide a binding site for one or more proteins linking cytokine receptor.JAK2 complexes and/or receptor tyrosine kinases to the actin cytoskeleton.     

Phosphospecific Antibodies Reveal Temporal Regulation of Platelet-Derived Growth Factor β Receptor Signaling

The platelet-derived growth factor β receptor (βPDGFR) is a receptor tyrosine kinase involved in multiple aspects of cell growth and differentiation. Upon activation, βPDGFR is phosphorylated at up to nine different tyrosine residues. Phosphorylation of the receptor results in at least two different outcomes: recruitment of signaling molecules and activation of intrinsic receptor kinase activity. In order to evaluate the phosphorylation state of the receptor, phosphospecific antibodies were generated against peptides encompassing βPDGFR phospho-Y751, phospho-Y771, or phospho-Y857. When phosphorylated, these sites enable the receptor to recruit signaling molecules PI3K or RasGAP, or enhance the receptor's kinase activity, respectively. We found that receptors phosphorylated at Y751, Y771, and Y857 display distinct temporal and spatial distribution by immunofluorescence. Subsequent biochemical studies revealed that receptor function corresponding to each of the phosphorylated sites was regulated as a function of time. Within the first 10 min, PDGF enhanced the receptor's kinase activity and initiated recruitment of PI3K and RasGAP. After prolonged exposure to PDGF, PI3K binding persisted to approximately 85% of the amount bound at 10 min, whereas binding of RasGAP and the exogenous kinase activity of the receptor diminished to less than 15% of the levels displayed at 10 min. We conclude that the phosphorylation state of the receptor, as well as its signaling capacity, is dynamic and changes as cells are continuously exposed to PDGF.     

Phosphospecific antibodies reveal temporal regulation of platelet-derived growth factor beta receptor signaling

The platelet-derived growth factor beta receptor (betaPDGFR) is a receptor tyrosine kinase involved in multiple aspects of cell growth and differentiation. Upon activation, betaPDGFR is phosphorylated at up to nine different tyrosine residues. Phosphorylation of the receptor results in at least two different outcomes: recruitment of signaling molecules and activation of intrinsic receptor kinase activity. In order to evaluate the phosphorylation state of the receptor, phosphospecific antibodies were generated against peptides encompassing betaPDGFR phospho-Y751, phospho-Y771, or phospho-Y857. When phosphorylated, these sites enable the receptor to recruit signaling molecules PI3K or RasGAP, or enhance the receptor's kinase activity, respectively. We found that receptors phosphorylated at Y751, Y771, and Y857 display distinct temporal and spatial distribution by immunofluorescence. Subsequent biochemical studies revealed that receptor function corresponding to each of the phosphorylated sites was regulated as a function of time. Within the first 10 min, PDGF enhanced the receptor's kinase activity and initiated recruitment of PI3K and RasGAP. After prolonged exposure to PDGF, PI3K binding persisted to approximately 85% of the amount bound at 10 min, whereas binding of RasGAP and the exogenous kinase activity of the receptor diminished to less than 15% of the levels displayed at 10 min. We conclude that the phosphorylation state of the receptor, as well as its signaling capacity, is dynamic and changes as cells are continuously exposed to PDGF.     

A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.

Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown.     

Tyrosine-phosphorylated SOCS3 interacts with the Nck and Crk-L adapter proteins and regulates Nck activation

Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr(221) within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr(221) forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins.     

A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase.

Autophosphorylated growth factor receptors provide binding sites for the src homology 2 domains of intracellular signaling molecules. In response to epidermal growth factor (EGF), the activated EGF receptor binds to a complex containing the signaling protein GRB2 and the Ras guanine nucleotide-releasing factor Sos, leading to activation of the Ras signaling pathway. We have investigated whether the platelet-derived growth factor (PDGF) receptor binds GRB2-Sos. In contrast with the EGF receptor, the GRB2 does not bind to the PDGF receptor directly. Instead, PDGF stimulation induces the formation of a complex containing GRB2; 70-, 80-, and 110-kDa tyrosine-phosphorylated proteins; and the PDGF receptor. Moreover, GRB2 binds directly to the 70-kDa protein but not to the PDGF receptor. Using a panel of PDGF beta-receptor mutants with altered tyrosine phosphorylation sites, we identified Tyr-1009 in the PDGF receptor as required for GRB2 binding. Binding is inhibited by a phosphopeptide containing a YXNX motif. The protein tyrosine phosphatase Syp/PTP1D/SHPTP2/PTP2C is approximately 70 kDa, binds to the PDGF receptor via Tyr-1009, and contains several YXNX sequences. We found that the 70-kDa protein that binds to the PDGF receptor and to GRB2 comigrates with Syp and is recognized by anti-Syp antibodies. Furthermore, both GRB2 and Sos coimmunoprecipitate with Syp from lysates of PDGF-stimulated cells, and GRB2 binds directly to tyrosine-phosphorylated Syp in vitro. These results indicate that GRB2 interacts with different growth factor receptors by different mechanisms and the cytoplasmic phosphotyrosine phosphatase Syp acts as an adapter between the PDGF receptor and the GRB2-Sos complex.     

Independent SH2-binding sites mediate interaction of Dok-related protein with RasGTPase-activating protein and Nck.

A murine embryonic cDNA library was screened for potential substrates of the Src family kinase, Lyn, using a phosphorylation-screening strategy. One cDNA that we identified encodes Dok-related protein (DokR), a protein with homology to p62(dok) (Dok), and members of the insulin receptor substrate-1 family of proteins. Analysis of murine tissue extracts with DokR-specific antisera revealed that DokR protein is expressed at highest levels in lymphoid tissues. Co-expression of a FLAG epitope-tagged form of DokR (FLAG-DokR) with Lyn in embryonic kidney 293T cells resulted in constitutive phosphorylation of FLAG-DokR on tyrosine residues and consequential physical association with RasGTPase-activating protein (GAP) and the Nck adaptor protein. Stimulation of BaF/3 hematopoietic cells co-expressing the epidermal growth factor (EGF) receptor tyrosine kinase and FLAG-DokR with EGF also induced phosphorylation of FLAG-DokR and promoted its association with GAP. Immunoprecipitation experiments using DokR-specific antibodies revealed an interaction between endogenous DokR and a 150-kDa protein that is tyrosine-phosphorylated in EGF-stimulated BaF/3 cells. The molecular basis of the interactions involving DokR with GAP and Nck was investigated using a novel glutathione S-transferase fusion protein binding assay and/or site-directed mutagenesis. Tandem SH2-binding sites containing Tyr-276 and Tyr-304 were shown to mediate binding of DokR to GAP, whereas Tyr-351 mediated the binding of DokR to Nck. These results suggest that DokR participates in numerous signaling pathways.     

Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins

We have identified two platelet-derived growth factor (PDGF)-dependent autophosphorylation sites in the beta subunit of the human PDGF receptor (PDGF-R). The major site of phosphorylation (Tyr-857) corresponds to the major autophosphorylation site in many other tyrosine kinases. Tyr-751, which lies within the kinase insert region, is a second in vivo site and the major in vitro site. Immunoprecipitates of wild-type PDGF-Rs prepared from PDGF-treated cells contained a phosphatidylinositol (PI) 3 kinase activity and three specific polypeptides as well as the PDGF-R. Mutation of Tyr-751 to Phe or Gly, or mutation of the catalytic domain to abolish kinase activity, blocked association of the PDGF-R with the PI kinase and the three proteins. These results suggest that autophosphorylation in the kinase insert region triggers the binding of the activated PDGF-R to specific cellular proteins, including a PI kinase whose activity is known to be stimulated by PDGF. Thus autophosphorylation may play a novel role in signal transduction via the PDGF-R.     

The SH2/SH3 domain-containing protein Nck is recognized by certain anti-phospholipase C-gamma 1 monoclonal antibodies, and its phosphorylation on tyrosine is stimulated by platelet-derived growth factor and epidermal growth factor treatment.

In the course of our investigation of phospholipase C (PLC)-gamma 1 phosphorylation by using a set of anti-PLC-gamma 1 monoclonal antibodies (P.-G. Suh, S. H. Ryu, W. C. Choi, K.-Y. Lee, and S. G. Rhee, J. Biol. Chem. 263:14497-14504, 1988), we found that some of these antibodies directly recognize a 47-kDa protein. We show here that this 47-kDa protein is identical to the SH2/SH3-containing protein Nck (J. M. Lehmann, G. Riethmuller, and J. P. Johnson, Nucleic Acids Res. 18:1048, 1990). Nck was found to be constitutively phosphorylated on serine in resting NIH 3T3 cells. Platelet-derived growth factor (PDGF) treatment led to increased Nck phosphorylation on both tyrosine and serine. Nck was also found to be phosphorylated on tyrosine in epidermal growth factor (EGF)-treated A431 cells and in v-Src-transformed NIH 3T3 cells. Multiple sites of serine phosphorylation were detected in Nck from resting cells, and no novel sites were found upon PDGF or EGF treatment. A single major tyrosine phosphorylation site was found in Nck in both PDGF- and EGF-treated cells and in v-Src-transformed cells. This same tyrosine was phosphorylated in vitro by purified PDGF and EGF receptors and also by pp60c-src. We compared the phosphorylation of Nck and PLC-gamma 1 in several cell lines transformed by oncogenes with different modes of transformation. Although PLC-gamma 1 and Nck have significant amino acid identity, particularly in their SH3 regions, and both associate with growth factor receptors in a ligand-dependent manner, they were not always phosphorylated on tyrosine in a coincident manner.     

Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.

Sphingosine-1-phosphate, a sphingolipid metabolite, is involved in the mitogenic response of platelet-derived growth factor (PDGF) and is formed by activation of sphingosine kinase. We examined the effect of PDGF on sphingosine kinase activation in TRMP cells expressing wild-type or various mutant betaPDGF receptors. Sphingosine kinase was stimulated by PDGF in cells expressing wild-type receptors but not in cells expressing kinase-inactive receptors (R634). Cells expressing mutated PDGF receptors with phenylalanine substitutions at five major tyrosine phosphorylation sites 740/751/771/1009/1021 (F5 mutants), which are unable to associate with PLCgamma, phosphatidylinositol 3-kinase, Ras GTPase-activating protein, or protein tyrosine phosphatase SHP-2, not only failed to increase DNA synthesis in response to PDGF but also did not activate sphingosine kinase. Moreover, mutation of tyrosine-1021 of the PDGF receptor to phenylalanine, which impairs its association with PLCgamma, abrogated PDGF-induced activation of sphingosine kinase. In contrast, PDGF was still able to stimulate sphingosine kinase in cells expressing the PDGF receptor mutated at tyrosines 740/751 and 1009, responsible for binding of phosphatidylinositol 3-kinase and SHP-2, respectively. In agreement, PDGF did not stimulate sphingosine kinase activity in F5 receptor 'add-back' mutants in which association with the Ras GTPase-activating protein, phosphatidylinositol 3-kinase, or SHP-2 was individually restored. However, a mutant PDGF receptor that was able to bind PLCgamma (tyrosine-1021), but not other signaling proteins, restored sphingosine kinase sensitivity to PDGF. These data indicate that the tyrosine residue responsible for binding of PLCgamma is required for PDGF-induced activation of sphingosine kinase. Moreover, calcium mobilization downstream of PLCgamma, but not protein kinase C activation, appears to be required for stimulation of sphingosine kinase by PDGF.-Olivera, A., Edsall, J., Poulton, S., Kazlauskas, A., Spiegel, S. Platelet-derived growth factor-induced activation of sphingosine kinase requires phosphorylation of the PDGF receptor tyrosine residue responsible for binding of PLCgamma.     

Recruitment of Dok-R to the EGF receptor through its PTB domain is required for attenuation of Erk MAP kinase activation.

Dok (for downstream of tyrosine kinases) proteins are a newly identified family of docking molecules that are characterized by the presence of an amino-terminal pleckstrin homology (PH) domain, a central putative phosphotyrosine-binding (PTB) domain and numerous potential sites of tyrosine phosphorylation [1] [2] [3] [4] [5] [6]. Here, we explore the potential role of the Dok family member Dok-R (also known as p56(Dok2) or FRIP) in signaling pathways mediated by the epidermal growth factor (EGF) receptor. An intact PTB domain in Dok-R was critical for its association with two PTB-binding consensus sites on the EGF receptor and the PH domain further contributed to stable in vivo binding and tyrosine phosphorylation of Dok-R. Multiple sites on Dok-R were tyrosine-phosphorylated following EGF stimulation; phosphorylated Tyr276 and Tyr304 are proposed to dock the tandem Src homology 2 (SH2) domains of the p21(Ras) GTPase-activating protein rasGAP and Tyr351 mediates an association with the SH2 domain of the adapter protein Nck. Interestingly, we have found that Dok-R could attenuate EGF-stimulated mitogen-activated protein (MAP) kinase activation independently of its association with rasGAP. Together, these results suggest that Dok-R has an important role downstream of growth factor receptors as a potential negative regulator of signal transduction.     

Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.