Src Homology-2 Domain-containing Protein Research Articles

Mouse and Human SHPS-1: Molecular Cloning of cDNAs and Chromosomal Localization of Genes

SHPS-1 (SHP substrate-1) is a glycosylated receptor-like protein with three immunoglobulin-like domains in its extracellular region and four YXX(L/V/I) motifs, potential tyrosine phosphorylation and SRC homology 2 (SH2) domain binding sites, in its cytoplasmic region. Various mitogens and cell adhesion induce tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2, an SH2 domain-containing protein tyrosine phosphatase, suggesting that SHPS-1 plays a role in cell signaling in response to both growth factors and cell adhesion. The mouse and human cDNAs encoding SHPS-1 have now been isolated. The deduced amino acid sequences of rat , human, and mouse SHPS-1 show identities of 65 to 81 %. In addition to the SH2 domain binding sites, a proline-rich putative SH3 domain binding site was detected in the cytoplasmic region of SHPS-1. Northern blot analysis revealed that human SHPS-1 mRNA is most abundant in brain and that the mouse mRNA is present in embryos as early as day 7. Fluorescence in situ hybridization localized the SHPS-1 gene to human chromosome 20p13 and the F3 band of mouse chromosome 2. Furthermore, interspecific backcross analysis placed the mouse SHPS-1 locus 5.0 centimorgans distal and 1.4 centimorgans proximal to the microsatellite markersD2Mit63andD2Mit19,respectively, in a region associated with the mutations coloboma (Cm), lethal milk (lm), and wellhaarig (we).

Tyrosine Phosphorylation of the Juxtamembrane Domain of the v-Fms Oncogene Product Is Required for Its Association with a 55-kDa Protein

Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high affinity binding sites for cellular proteins with Src homology 2 (SH2) domains that are involved in various signal cascades. Tryptic digestion of the autophosphorylated v-Fms and of its cellular counterpart, the feline c-Fms polypeptide, gave rise to at least six common major phosphopeptides, four of which have been characterized previously. Employing site-directed mutagenesis and phosphopeptide mapping of in vitro phosphorylated glutathione S-transferase v-Fms fusion proteins as well as full-length v-Fms molecules expressed in various cells, we show here that Tyr543 of the juxtamembrane domain and Tyr696 of the kinase insert domain constitute major autophosphorylation sites. Recombinant fusion proteins containing the tyrosine-phosphorylated kinase insert domain bind the growth factor receptor bound protein 2 and the p85 and p110 subunits of phosphatidylinositol 3'-kinase. In contrast, fusion proteins containing the juxtamembrane domain phosphorylated on Tyr543 fail to bind any of the known SH2 domain-containing cellular proteins but associate specifically with an as yet undefined 55-kDa cellular protein that by itself is phosphorylated on tyrosine.

Characterization of the Roles of SH2 Domain-Containing Proteins in T-Lymphocyte Activation by Using Dominant Negative SH2 Domains

Activation of the T-cell antigen receptor initiates a complex signaling cascade leading to changes in cytokine gene transcription. Several proteins containing Src homology 2 (SH2) domains, capable of interacting with phosphotyrosine-containing sequences within other proteins, are involved in the activation of signaling intermediates such as p2l(ras) and phospholipase Cgamma1. In this study, we used dominant negative SH2 domains to determine the importance of SH2 domain-containing proteins for T-cell activation. We show that tandem SH2 domains of either Zap70 or Syk tyrosine kinase are potent inhibitors of signaling initiated by the T-cell receptor zeta chain in vivo while individual SH2 domains are ineffective. Thus, while only the C-terminal SH2 domains appear to have significant affinity for immunoreceptor tyrosine-based activation motifs in vitro, the N-terminal SH2 domains are necessary in vivo. We find the spacing between the tandem SH2 domains of Zap70 to be critical for in vivo interactions. The SH2 domain of the adapter protein Grb2 is an effective inhibitor in our dominant negative assay, although it has little affinity for immunoreceptor tyrosine-based activation motifs. A single point mutation that abolishes phosphotyrosine binding renders the Grb2 SH2 domain incapable of this inhibition. In contrast, the SH2 domain of Shc does not inhibit this signaling cascade. We conclude that Grb2, but not Shc, is involved in T-cell receptor signaling.

Association of Polyomavirus Middle Tumor Antigen with Phospholipase C-γ1

Middle tumor antigen (MT) is the primary transforming protein of murine Polyomavirus. MT transforms by associating with and modulating the activities of cellular proteins involved in control of cell proliferation. MT binds to and is phosphorylated by cellular tyrosine kinases. The phosphorylated tyrosines become docking sites for SH2 (Src homology 2) domain-containing molecules. Tyrosine 322 of MT is known to be phosphorylated but has no known binding protein. We have found that phospholipase C-gamma 1 (PLC-gamma 1), a SH2 domain-containing protein, coimmunoprecipitates with MT. Tyrosine phosphorylation of PLC-gamma 1 is elevated in cells expressing MT, suggesting activation of this enzyme by MT. A Tyr-322-->Phe mutation in MT renders it defective in MT-PLC-gamma 1 interaction and in transformation. From the correlation between transformation and MT-PLC-gamma 1 interaction, we suggest that PLC-gamma 1 may play a role in transformation.

Molecular Cloning of SLP-76, a 76-kDa Tyrosine Phosphoprotein Associated with Grb2 in T Cells

The activation of protein tyrosine kinases is a critical event in T cell antigen receptor (TCR)-mediated signaling. One substrate of the TCR-activated protein tyrosine kinase pathway is a 76-kDa protein (pp76) that associates with the adaptor protein Grb2. In this report we describe the purification of pp76 and the molecular cloning of its cDNA, which encodes a novel 533-amino acid protein with a single carboxyl-terminal Src homology 2 (SH2) domain. Although no recognizable motifs related to tyrosine, serine/threonine, or lipid kinase domains are present in the predicted amino acid sequence, it contains several potential motifs recognized by SH2 and SH3 domains. A cDNA encoding the murine homologue of pp76 was also isolated and predicts a protein with 84% amino acid identity to human pp76. Northern analysis demonstrates that pp76 mRNA is expressed solely in peripheral blood leukocytes, thymus, and spleen; and in human T cell, B cell and monocytic cell lines. In vitro translation of pp76 cDNA gives rise to a single product of 76 kDa that associates with a GST/Grb2 fusion protein, demonstrating a direct association between these two molecules. Additionally, a GST fusion protein consisting of the predicted SH2 domain of pp76 precipitates two tyrosine phosphoproteins from Jurkat cell lysates, and antiserum directed against phospholipase C-gamma 1 coprecipitates a tyrosine phosphoprotein with an electrophoretic mobility identical to that of pp76. These results demonstrate that this novel protein, which we term SLP-76 (SH2 domain-containing Leukocyte Protein of 76 kDa), is likely to play an important role in TCR-mediated intracellular signal transduction.

Direct interaction between Shc and the platelet-derived growth factor beta-receptor.

The Src homology 2 (SH2) domain-containing Shc proteins p52shc and p46shc become phosphorylated upon activation of several tyrosine kinases and are implicated in mitogenic signal transduction. Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to autophosphorylation of tyrosine residues, which is known to mediate interactions with several SH2 domain-containing signaling molecules. In this study, we have characterized the interaction between the PDGF beta-receptor and Shc. PDGF beta-receptor coprecipitation in Shc immunoprecipitates was dependent on stimulation with PDGF-BB. The Shc SH2 domain expressed as a bacterial fusion protein bound the autophosphorylated PDGF beta-receptor. Moreover, the Shc SH2 domain could bind the autophosphorylated purified baculovirus-expressed PDGF beta-receptor intracellular domain, which indicates a direct association of Shc with the PDGF beta-receptor. Activation of the PDGF beta-receptor induced the preferential phosphorylation of p52shc. Tyrosine-phosphorylated Shc, in turn, formed a complex with the signaling molecule Grb2. Synthetic peptide analysis revealed that certain autophosphorylation sites in the PDGF beta-receptor (Tyr-579, Tyr-740, Tyr-751, and Tyr-771) were able to mediate the specific binding of the Shc SH2 domain as well as intact Shc proteins. A mutant PDGF beta-receptor in which Tyr-579 was replaced with phenylalanine showed 40% impaired association of Shc in vivo, but phosphorylation of Shc proteins was not affected. We conclude that multiple autophosphorylation sites in the PDGF beta-receptor are responsible for the binding of Shc. This is in contrast to previously characterized interactions between the PDGF beta-receptor and SH2 domain-containing proteins, which generally involve one high affinity binding site in the receptor.

Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

Individual epidermal growth factor receptor autophosphorylation sites do not stringently define association motifs for several SH2-containing proteins.

To determine whether individual autophosphorylation sites in the epidermal growth factor (EGF) receptor define specific interaction sites for the in vivo association of signal transduction proteins that contain src homology 2 (SH2) domains, the capacity of wild-type and mutant EGF receptors to associate with several SH2 domain-containing proteins has been assayed. Mutants included receptors with single autophosphorylation site mutations at each of five autophosphorylation sites and receptors in which multiple autophosphorylation sites were removed by point mutation or deletion of carboxyl-terminal residues. Receptor association, as measured by coimmunoprecipitation, has been determined for phospholipase C-gamma 1, the ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3-kinase, and the src homology and collagen protein. In contrast to data obtained with single autophosphorylation site mutants of other receptor tyrosine kinases, none of the EGF receptor single site mutants was dramatically impaired in its capacity to associate with any of these SH2-containing proteins. However, association was completely abrogated when all five autophosphorylation sites were mutated or removed by deletion. These results indicate that individual autophosphorylation sites in the EGF receptor are not stringently required for the recognition and association of different SH2-containing substrates. Thus, EGF receptor autophosphorylation sites seem to be flexible and/or compensatory in their capacity to mediate association with these four SH2-containing substrates.

RNA splicing regulates the activity of a SH2 domain-containing protein tyrosine phosphatase.

A cDNA which encodes a protein tyrosine phosphatase with two src homology 2 (SH2) domains was isolated from a rat brain cDNA library. This phosphatase appears to be a rat homologue of PTP1D based on its amino acid sequence. The gene is expressed in a variety of tissues, and its mRNA is enriched in the brain, skeletal muscle, and lung. An RNA splice variant (PTP1Di) was also isolated which has four additional amino acid residues (Ala-Leu-Leu-Gln) in the catalytic domain. The catalytic domains of PTP1D and PTP1Di were expressed in Escherichia coli as glutathione S-transferase fusion proteins and purified to near homogeneity. Whereas both PTP1D and PTP1Di had catalytic activity, the Vmax of PTP1Di relative to that of PTP1D was 8-fold lower for para-nitrophenylphosphate, 20-fold lower for nicotinic acetylcholine receptor, and 14-fold lower for myelin basic protein. The Km values of PTP1Di were lower than those of PTP1D for both nicotinic acetylcholine receptor and myelin basic protein, suggesting a higher affinity of PTP1Di for a protein substrate. These two forms also differed in optimum pH for para-nitrophenylphosphate and sensitivity to the inhibitory effects of vanadate, molybdate, and spermidine. In order to see if this insert would affect the catalytic activity of other related phosphatases, the 4-amino acids were inserted in the corresponding region of the catalytic domain of PTP1C. Whereas both the wild type and PTP1Ci which contained the 4-amino acid insert dephosphorylated para-nitrophenylphosphate, nicotinic receptor, and myelin basic protein, the enzyme activity of PTP1Ci was only 11-24% of that of PTP1C wild type. These results demonstrate that the 4-amino acid insert in the catalytic domains of PTP1D down-regulates its phosphatase activity and suggests that RNA splicing may serve as a regulatory mechanism of protein tyrosine phosphatase activity.

Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.

Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.

The insulin receptor substrate 1 associates with the SH2-containing phosphotyrosine phosphatase Syp

The insulin receptor substrate 1 (IRS1) is a protein that is rapidly phosphorylated on tyrosine by the activated insulin receptor. Syp is a recently discovered, broadly expressed phosphotyrosine (Tyr(P)) phosphatase that contains two Src homology 2 (SH2) domains. We have found that insulin treatment of 3T3-L1 adipocytes leads to complex formation between IRS1 and Syp. Syp was detected in immunoadsorbates of IRS1 from extracts of insulin-treated but not basal cells by both immunoblotting and Tyr(P) phosphatase activity. The association of Syp with IRS1 apparently occurs between the SH2 domains of Syp and Tyr(P)-containing sequences of IRS1, since a fusion protein containing only the SH2 domains of Syp bound the Tyr(P) form of IRS1. Unlike the receptors for epidermal and platelet-derived growth factors, which in their activated state bind to the SH2 domains of Syp and elicit phosphorylation of Syp on tyrosine in intact cells, the Tyr(P) form of the insulin receptor did not bind to the SH2 domains of Syp, and no phosphorylation of Syp on tyrosine was detected in insulin-treated 3T3-L1 adipocytes. In combination with other findings these results indicate that IRS1 functions as a docking protein for SH2 domain-containing proteins participating in signaling from the insulin receptor.

The new elements of insulin signaling. Insulin receptor substrate-1 and proteins with SH2 domains.

Since the discovery of insulin and its receptor, the downstream elements responsible for the pleiotropic insulin signal have been difficult to define. The recently discovered insulin receptor substrate, IRS-1, provides an innovative and simple way to think about this problem: IRS-1 may mediate the control of various cellular processes by insulin. Overexpression of IRS-1 enhances insulin-stimulated DNA synthesis in Chinese hamster ovary cells, and microinjection of IRS-1 protein potentiates the maturation of Xenopus oocytes. We suspect that insulin signals are enabled when the activated insulin receptor kinase phosphorylates specific tyrosine residues in IRS-1. These phosphorylated sites associate with high affinity to cellular proteins that contain SH2 (src homology-2) domains. This association is specific and depends on the amino acid sequence surrounding the phosphotyrosine residue and the isoform of the SH2 domain. A growing number of SH2 domain-containing proteins have been identified, and we suspect that IRS-1 has the potential to simultaneously regulate many of them. We have only begun to identify the specific proteins that associate with phosphorylated IRS-1. One of them, the phosphatidylinositol 3'-kinase, is activated when the SH2 domains in its 85,000-M(r) regulatory subunit bind to phosphorylated IRS-1. IRS-1 also interacts with other proteins such as SHPTP2, a novel SH2 domain-containing Tyr phosphatase, and GRB-2/sem-5, a protein that is implicated in p21ras signaling. The interaction between phosphorylated IRS-1 and multiple SH2 domain-containing proteins may ultimately explain the pleiotropic effects of insulin.

Phosphatidylinositol 3-kinase p85 SH2 domain specificity defined by direct phosphopeptide/SH2 domain binding.

We have developed a competition binding assay to quantify relative affinities of isolated Src-homology 2 (SH2) domains for phosphopeptide sequences. Eleven synthetic 11-12-amino acid phosphopeptides containing YMXM or YVXM recognition motifs bound to a PI 3-kinase p85 SH2 domain with highest affinities, including sequences surrounding phosphorylated tyrosines of the PDGF, CSF-1/c-Fms, and kit-encoded receptors, IRS-1, and polyoma middle T antigens; matched, unphosphorylated sequences did not bind. A scrambled YMXM phosphopeptide or sequences corresponding to the GAP or PLC-gamma SH2 domain binding motifs of the PDGF, FGF, and EGF receptors bound to the p85 SH2 domain with 30-100-fold reduced affinity, indicating that this affinity range confers specificity. Binding specificity was appropriately reversed with an SH2 domain from PLC-gamma: a phosphopeptide corresponding to the site surrounding PDGF receptor Tyr1021 binds with approximately 40-fold higher affinity than a YMXM-phosphopeptide. We conclude that essential features of specific phosphoprotein/SH2 domain interactions can be reconstituted using truncated versions of both the phosphoprotein (a phosphopeptide) and cognate SH2 domain-containing protein (the SH2 domain). SH2 domain binding specificity results from differences in affinity conferred by the linear sequence surrounding phosphotyrosine.

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.

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.

The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 ( ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.