Src Homology Region 2 Domain Research Articles

SHIP1 associates with DAP12 and negatively regulates TREM2/DAP12 signaling

TREM2‐DAP12 stimulation of macrophages and pre‐osteoclasts (OC) leads to enhanced in vitro osteoclastogenesis. To further define the regulation of TREM2‐DAP12 signals, we studied proteins that associated with DAP12 following TREM2 stimulation. We found that TREM2 crosslinking of RAW264.7 cells leads to a novel association of Src homology region 2(SH2)‐containing inositol phosphatase (SHIP1) with DAP12 at 1 and 5 min, which was not seen after stimulation with isotype control Ab. Recombinant GST‐SHIP1‐SH2 protein, but not mutated GST‐SH2(R34G) protein, could bind phosphorylated DAP12 indicating that the SHIP1‐DAP12 interaction is mediated via the SH2 domain of SHIP1. Biotinylated‐DAP12 ITAM phosphopeptide, but not un‐phosphorylated or Y65/76F mutated peptides, interacted with SHIP1. SHIP1 co‐immunoprecipitated with DAP12 in OC generated from C57BL6 bone marrow macrophages (BMM) treated with RANKL and M‐CSF. To determine the functional significance of this interaction, we evaluated TREM2 stimulation of SHIP1 deficient OC precursors during osteoclastogenesis. Crosslinking with TREM2 monoclonal Ab but not control Ab lead to a 4 fold increase in OC generated from SHIP1−/− BMM compared to wild type BMM. Together these data reveal a novel association of SHIP1 with DAP12 that is dependent on the DAP12 ITAM and the SH2 domain of SHIP1 and this association inhibits DAP12 signaling during osteoclastogenesis.

Prediction of the structure of human Janus kinase 2 (JAK2) comprising JAK homology domains 1 through 7.

A theoretical model of human Janus kinase 2 (JAK2) comprising all seven Janus homology domains is presented. The model was generated by application of homology modelling approaches. The three-dimensional structure contains, starting from the N-terminus, FERM (4.1, ezrin, radixin, moesin), SH2 (Src homology region 2), tyrosine kinase-like, and tyrosine kinase domains. The predicted inter-domain orientation in JAK2 is discussed and the currently existing mutational data for Janus kinases are evaluated. Structural details of the SH2 and the FERM domains are presented. The predictions indicate that the SH2 domain is not fully functional. A number of hydrophobic amino acids of the FERM domain that are predicted to be involved in the constitutive association with the cytokine receptors are highlighted. The model gives new insights into the structure-function relationship of this important protein, and areas that could be investigated by mutation studies are highlighted.

Src homology region 2 (SH2) domain-containing phosphatase-1 dephosphorylates B cell linker protein/SH2 domain leukocyte protein of 65 kDa and selectively regulates c-Jun NH2-terminal kinase activation in B cells.

Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk. Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.

The three-dimensional solution structure of the SRC homology domain-2 of the growth factor receptor-bound protein-2.

A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 A for all backbone atoms and 0.40 A for the backbone atoms of the central beta-sheet. The core structure of the SH2 domain contains an antiparallel beta-sheet flanked by two parallel alpha-helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions.

Recruitment and phosphorylation of SH2-containing inositol phosphatase and Shc to the B-cell Fc gamma immunoreceptor tyrosine-based inhibition motif peptide motif.

Recently, we and others have demonstrated that negative signaling in B cells selectively induces the tyrosine phosphorylation of a novel inositol polyphosphate phosphatase, p145SHIP. In this study, we present data indicating that p145SHIP binds directly a phosphorylated motif, immunoreceptor tyrosine-based inhibition motif (ITIM), present in the cytoplasmic domain of Fc gammaRIIB1. Using recombinant SH2 domains, we show that binding is mediated via the Src homology region 2 (SH2)-containing inositol phosphatase (SHIP) SH2 domain. SHIP also bound to a phosphopeptide derived from CD22, raising the possibility that SHIP contributes to negative signaling by this receptor as well as Fc gammaRIIB1. The association of SHIP with the ITIM phosphopeptide was activation independent, while coassociation with Shc was activation dependent. Furthermore, experiments with Fc gammaRIIB1-deficient B cells demonstrated a genetic requirement for expression of Fc gammaRIIB1 in the induction of SHIP phosphorylation and its interaction with Shc. Based on these results, we propose a model of negative signaling in which co-cross-linking of surface immunoglobulin and Fc gammaRIIB1 results in sequential tyrosine phosphorylation of the ITIM, recruitment and phosphorylation of p145SHIP, and subsequent binding of Shc.

Hematopoietic cell phosphatase, SHP-1, is constitutively associated with the SH2 domain-containing leukocyte protein, SLP-76, in B cells.

Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1; previously named HCP, PTP1C, SH-PTP1, and SHP) is a cytosolic protein tyrosine phosphatase that contains two SH2 domains. Recent data have demonstrated that the gene encoding SHP-1 is mutated in motheaten (mc) and viable motheaten (mc') mice resulting in autoimmune disease. More recently, SHP-1 has been shown to negatively regulate B cell antigen receptor (BCR)-initiated signaling. To elucidate potential mechanisms of SHP-1 action in BCR signal transduction, we studied proteins that interact with SHP-1 in B cells. Both anti-SHP-1 antibody and the two SH2 domains of SHP-1 expressed as glutathione S-transferase fusion proteins precipitated at least three phosphoproteins of approximately 75, 110, and 150 kD upon anti-immunoglobulin M stimulation of the WEHI-231 immature B cell line. Binding of SHP-1 to the 75- and 110-kD proteins appeared to be mediated mainly by the NH2-terminal SH2 domain of SHP-1, whereas both the NH2- and COOH-terminal SH2 domains are required for maximal binding to the 150-kD protein. Immunoprecipitation and Western blot analysis revealed that the SHP-1-associated 75-kD protein is the hematopoietic cell-specific, SH2-containing protein SLP-76. Further, this protein-protein association was constitutively observed and stable during the early phase of BCR signaling. However, significant tyrosine phosphorylation of SLP-76 as well as of SHP-1 was observed after BCR ligation. Constitutive association of SHP-1 with SLP-76 could also be detected in normal splenic B cells. Collectively, these results suggest possible mechanisms by which SHP-1 may modulate signals delivered by BCR engagement.

Nerve Growth Factor Stimulates the Tyrosine Phosphorylation of Endogenous Crk-II and Augments Its Association with p130Cas in PC-12 Cells

The cellular homologs of the v-Crk oncogene product consist primarily of Src homology region 2 (SH2) and 3 (SH3) domains. v-Crk overexpression causes cell transformation and elevation of tyrosine phosphorylation in fibroblasts and accelerates differentiation of PC-12 cells in response to nerve growth factor (NGF). To further explore the role of Crk in NGF-induced PC-12 cell differentiation, we found that both NGF and epidermal growth factor stimulate the tyrosine phosphorylation of endogenous Crk II. Moreover, hormone stimulation enhanced the specific association of Crk proteins with the tyrosine-phosphorylated p130Cas, the major phosphotyrosine-containing protein in cells transformed with v-Crk. This interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. Furthermore, the Crk-SH2 domain binds tyrosine-phosphorylated paxillin, a cytoskeletal protein, following treatment of PC-12 cells with NGF or epidermal growth factor. These data suggest that Crk functions in a number of signaling processes in PC-12 cells.

The product of the cbl oncogene forms stable complexes in vivo with endogenous Crk in a tyrosine phosphorylation-dependent manner.

The cellular homologs of the v-Crk oncogene product are composed exclusively of Src homology region 2 (SH2) and SH3 domains. v-Crk overexpression in fibroblasts causes cell transformation and elevated tyrosine phosphorylation of specific cellular proteins. Among these proteins is a 130-kDa protein, identified as p130cas, that forms a stable complex in vivo with v-Crk. We have explored the role of endogenous Crk proteins in Bcr-Abl-transformed cells. In the K562 human chronic myelogenous leukemia cell line, p130cas is not tyrosine phosphorylated or bound to Crk. Instead, Crk proteins predominantly associate with the tyrosine-phosphorylated proto-oncogene product of Cbl. In vitro analysis showed that this interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. In NIH 3T3 cells transformed by Bcr-Abl, c-Cbl becomes strongly tyrosine phosphorylated and associates with c-Crk. The complex between c-Crk and c-Cbl is also seen upon T-cell receptor cross-linking or with the transforming, tyrosine-phosphorylated c-Cbl. These results indicate that Crk binds to c-Cbl in a tyrosine phosphorylation-dependent manner, suggesting a physiological role for the Crk-c-Cbl complex in Bcr-Abl tyrosine phosphorylation-mediated transformation.

The SH2 Domain of P210BCR/ABL Is Not Required for the Transformation of Hematopoietic Factor-Dependent Cells

Src-homology region 2 (SH2) domains, by binding to tyrosine-phosphorylated sequences, mediate specific protein-protein interactions important in diverse signal transduction pathways. Previous studies have shown that activated forms of the Abl tyrosine kinase, including P210BCR/ABL of human chronic myelogenous leukemia, require the SH2 domain for the transformation of fibroblasts. To determine whether SH2 is also required for Bcr/Abl to transform hematopoietic cells, we have studied two SH2 domain mutations in P210BCR/ABL: a point mutation in the conserved FLVRES motif (P210/R1033K), which interferes with phosphotyrosine-binding by SH2, and a complete deletion of SH2 (P210/delta SH2). Despite a negative effect on intrinsic Abl kinase activity, both P210 SH2 mutants were still able to transform the hematopoietic factor-dependent cell lines Ba/F3 and FDC-P1 to growth factor independence. Unexpectedly, both mutants showed greater transforming activity than wild-type P210 in a quantitative transformation assay, probably as a consequence of increased stability of the SH2 mutant proteins in vivo. Cells transformed by both P210 SH2 mutants were leukemogenic in synaptic mice and P210/r1053K mice exhibited a distinct disease phenotype, reminiscent of that induced by v-Abl. These results demonstrate that while the Abl SH2 domain is essential for BCR/ABL transformation of fibroblasts, it is dispensable for the transformation of hematopoietic factor-dependent cell lines.

Comparison of calcium-dependent conformational changes in the N-terminal SH2 domains of p85 and gap defines distinct properties for SH2 domains

Src-homology region 2 (SH2) domains are stretches of about 100 amino acids which are found to be structurally conserved in a number of signaling molecules. These regions have been shown to bind with high affinity to phosphotyrosine residues within activated receptor tyrosine kinases. Here we report the bacterial expression and purification of individual N-terminal SH2 (NSH2) domains of phosphatidylinositol 3-kinase (PI-3K) binding subunit (p85) and Ras GTPase activating protein (GAP) in amounts suitable for structure-function studies. The p85NSH2 domain stains dark purple and absorbs around 620-640 nm with Stains-all, a dye known to bind to calcium binding proteins. This effect was not observed for the GAPNSH2 domain. Circular dichroism analysis of the N-terminal SH2 domain of these proteins shows that p85NSH2, but not GAPNSH2, undergoes a significant dose-dependent change in conformation in the presence of increasing calcium concentrations. Moreover, the conformational change of p85NSH2 induced by calcium could be replicated by addition of a phosphorylated hexapeptide (DYpMDMK) representing the alpha-PDGFR binding site for p85. Limited proteolysis studies showed a significant calcium-dependent increase in protection of p85NSH2 but not GAPNSH2 from degradation by subtilisin. Our results further indicate that holmium, a trivalent lanthanide ion, which has been previously shown to substitute for calcium, could also protect the p85NSH2 domain from proteolysis even at 10-fold lower concentrations. In vitro binding studies using purified preparations of activated alpha-PDGFR show that calcium did not affect the binding of GAPNSH2 domains to activated alpha-PDGFR.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of fibroblast growth factor receptor kinase signal transduction by heterodimerization of combinatorial splice variants.

A differentiated liver cell (HepG2), which exhibits a dose-dependent growth-stimulatory and growth-inhibitory response to heparin-binding fibroblast growth factor type 1 (FGF-1), displays high- and low-affinity receptor phenotypes and expresses specific combinatorial splice variants alpha 1, beta 1, and alpha 2 of the FGF receptor (FGF-R) gene (flg). The extracellular domains of the alpha and beta variants consist of three and two immunoglobulin loops, respectively, while the intracellular variants consist of a tyrosine kinase (type 1) isoform and a kinase-defective (type 2) isoform. The type 2 isoform is also devoid of the two major intracellular tyrosine autophosphorylation sites (Tyr-653 and Tyr-766) in the type 1 kinase. An analysis of ligand affinity, dimerization, autophosphorylation, and interaction with src homology region 2 (SH2) substrates of the recombinant alpha 1, beta 1, and alpha 2 isoforms was carried out to determine whether dimerization of the combinatorial splice variants might explain the dose-dependent opposite mitogenic effects of FGF. Scatchard analysis indicated that the alpha and beta isoforms exhibit low and high affinity for ligand, respectively. The three combinatorial splice variants dimerized in all combinations. FGF enhanced dimerization and kinase activity, as assessed by receptor autophosphorylation. Phosphopeptide analysis revealed that phosphorylation of Tyr-653 was reduced relative to phosphorylation of Tyr-766 in the type 1 kinase component of heterodimers of the type 1 and type 2 isoforms. The SH2 domain substrate, phospholipase C gamma 1 (PLC gamma 1), associated with the phosphorylated type 1-type 2 heterodimers but was phosphorylated only in preparations containing the type 1 kinase homodimer. The results suggest that phosphorylation of Tyr-653 within the kinase catalytic domain, but not Tyr-766 in the COOH-terminal domain, may be stringently dependent on a trans intermolecular mechanism within FGF-R kinase homodimers. Although phosphotyrosine 766 is sufficient for interaction of PLC gamma 1 and other SH2 substrates with the FGF-R kinase, phosphorylation and presumably activation of substrates require the kinase homodimer and phosphorylation of Tyr-653. We propose that complexes of phosphotyrosine 766 kinase monomers and SH2 domain signal transducers may constitute unactivated presignal complexes whose active or inactive fate depends on homodimerization with a kinase or heterodimerization with a kinase-defective monomer, respectively. The results suggest a mechanism for control of signal transduction by different concentrations of ligand through heterodimerization of combinatorial splice variants from the same receptor gene.

Control of Fibroblast Growth Factor Receptor Kinase Signal Transduction by Heterodimerization of Combinatorial Splice Variants

A differentiated liver cell (HepG2), which exhibits a dose-dependent growth-stimulatory and growth-inhibitory response to heparin-binding fibroblast growth factor type 1 (FGF-1), displays high- and low-affinity receptor phenotypes and expresses specific combinatorial splice variants alpha 1, beta 1, and alpha 2 of the FGF receptor (FGF-R) gene (flg). The extracellular domains of the alpha and beta variants consist of three and two immunoglobulin loops, respectively, while the intracellular variants consist of a tyrosine kinase (type 1) isoform and a kinase-defective (type 2) isoform. The type 2 isoform is also devoid of the two major intracellular tyrosine autophosphorylation sites (Tyr-653 and Tyr-766) in the type 1 kinase. An analysis of ligand affinity, dimerization, autophosphorylation, and interaction with src homology region 2 (SH2) substrates of the recombinant alpha 1, beta 1, and alpha 2 isoforms was carried out to determine whether dimerization of the combinatorial splice variants might explain the dose-dependent opposite mitogenic effects of FGF. Scatchard analysis indicated that the alpha and beta isoforms exhibit low and high affinity for ligand, respectively. The three combinatorial splice variants dimerized in all combinations. FGF enhanced dimerization and kinase activity, as assessed by receptor autophosphorylation. Phosphopeptide analysis revealed that phosphorylation of Tyr-653 was reduced relative to phosphorylation of Tyr-766 in the type 1 kinase component of heterodimers of the type 1 and type 2 isoforms. The SH2 domain substrate, phospholipase C gamma 1 (PLC gamma 1), associated with the phosphorylated type 1-type 2 heterodimers but was phosphorylated only in preparations containing the type 1 kinase homodimer. The results suggest that phosphorylation of Tyr-653 within the kinase catalytic domain, but not Tyr-766 in the COOH-terminal domain, may be stringently dependent on a trans intermolecular mechanism within FGF-R kinase homodimers. Although phosphotyrosine 766 is sufficient for interaction of PLC gamma 1 and other SH2 substrates with the FGF-R kinase, phosphorylation and presumably activation of substrates require the kinase homodimer and phosphorylation of Tyr-653. We propose that complexes of phosphotyrosine 766 kinase monomers and SH2 domain signal transducers may constitute unactivated presignal complexes whose active or inactive fate depends on homodimerization with a kinase or heterodimerization with a kinase-defective monomer, respectively. The results suggest a mechanism for control of signal transduction by different concentrations of ligand through heterodimerization of combinatorial splice variants from the same receptor gene.

En bloc substitution of the Src homology region 2 domain activates the transforming potential of the c-Abl protein tyrosine kinase.

Src homology region 2 (SH2) domains are present in many proteins involved in signal transduction. In nonreceptor protein tyrosine kinases the SH2 domain has been implicated in regulation of tyrosine kinase activity and in mediating interactions involved in downstream signaling. Different SH2 domains exhibit distinct binding specificities for both phosphotyrosine- and phosphoserine/phosphothreonine-containing proteins. We show that different SH2 domains are not functionally equivalent within the context of the c-ABL1b protooncogene. c-ABL1b, altered by replacement of its SH2 domain with the N-terminal SH2 domain of Ras GTPase-activating protein, exhibited activated transforming capability, caused intracellular tyrosine phosphorylation of p62, and was relocalized from nucleus to cytoplasm. This en bloc substitution apparently uncouples two distinct functions of the SH2 domain so that c-ABL escapes normal regulatory control while it retains the capability to elicit signals that promote transformation. The SH2 domain of the ARG protein tyrosine kinase, which shares high amino acid-sequence homology with the SH2 domain of ABL, was less effective in activating the oncogenic potential of c-ABL. The effects that the N-terminal SH2 domain of Ras GTPase-activating protein has in the context of c-ABL resemble the effects of deleting the SH3 domain. Thus, the SH2 and SH3 domains may have coordinate roles as regulatory control elements within the context of c-ABL.

A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction.

SH2 (src homology region 2) domains are implicated in protein-protein interactions involved in signal transduction pathways. Isolated SH2 domains bind proteins that are tyrosine phosphorylated. A novel, phosphotyrosine-independent binding interaction between BCR, the Philadelphia chromosome breakpoint cluster region gene product, and the SH2 domain of its translocation partner c-ABL has recently been reported. We have examined the ability of additional SH2 domains to bind phosphotyrosine-free BCR and compared this with their ability to bind tyrosine-phosphorylated c-ABL 1b. Of 11 individual SH2 domains examined, 8 exhibited relatively high affinity for c-ABL 1b, whereas only 4 exhibited relatively high affinity for BCR. Binding of tyrosine-phosphorylated c-ABL 1b by the relatively high-affinity ABL and ARG SH2 domains was quantitatively analyzed, and equilibrium dissociation constants for both interactions were estimated to be in the range of 5 x 10(-7) M. The ABL SH2 domain exhibited relatively high affinity for phosphotyrosine-free BCR as well; however, this interaction appears to be about two orders of magnitude weaker than binding of tyrosine-phosphorylated c-ABL 1b. The ARG SH2 domain exhibited relatively weak affinity for BCR and was determined to bind about 10-fold less strongly than the ABL SH2 domain. The ABL and ARG SH2 domains differ by only 10 of 91 amino acids, and the substitution of ABL-specific amino acids into either the amino- or carboxy-terminal half of the ARG SH2 domain was found to increase its affinity for BCR. We discuss these results in terms of a model which has been proposed for peptide binding by class I histocompatibility glycoproteins.

A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction.

SH2 (src homology region 2) domains are implicated in protein-protein interactions involved in signal transduction pathways. Isolated SH2 domains bind proteins that are tyrosine phosphorylated. A novel, phosphotyrosine-independent binding interaction between BCR, the Philadelphia chromosome breakpoint cluster region gene product, and the SH2 domain of its translocation partner c-ABL has recently been reported. We have examined the ability of additional SH2 domains to bind phosphotyrosine-free BCR and compared this with their ability to bind tyrosine-phosphorylated c-ABL 1b. Of 11 individual SH2 domains examined, 8 exhibited relatively high affinity for c-ABL 1b, whereas only 4 exhibited relatively high affinity for BCR. Binding of tyrosine-phosphorylated c-ABL 1b by the relatively high-affinity ABL and ARG SH2 domains was quantitatively analyzed, and equilibrium dissociation constants for both interactions were estimated to be in the range of 5 x 10(-7) M. The ABL SH2 domain exhibited relatively high affinity for phosphotyrosine-free BCR as well; however, this interaction appears to be about two orders of magnitude weaker than binding of tyrosine-phosphorylated c-ABL 1b. The ARG SH2 domain exhibited relatively weak affinity for BCR and was determined to bind about 10-fold less strongly than the ABL SH2 domain. The ABL and ARG SH2 domains differ by only 10 of 91 amino acids, and the substitution of ABL-specific amino acids into either the amino- or carboxy-terminal half of the ARG SH2 domain was found to increase its affinity for BCR. We discuss these results in terms of a model which has been proposed for peptide binding by class I histocompatibility glycoproteins.

Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.

Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.

Highly conserved eight amino acid sequence in SH2 is important for recognition of phosphotyrosine site

Src homology region 2(SH2) has been demonstrated to recognize phosphotyrosine site. To clarify the precise mechanism of the recognition, we developed in vitro binding assay system using EGF receptor and SH2/SH3 region of phospholipase C(PLC) gamma 1. Phosphorylated EGF receptor bound to immobilized SH2/SH3 of PLC gamma 1 in Sepharose beads, while nonphosphorylated EGF receptor did not bind. In SH2 domain of PLC gamma 1, there are several highly conserved amino acid sequences that are common in a variety of SH2-containing proteins. Especially the eight amino acid sequence, G(S/T)FLVR(E/D)S is highly conserved in these proteins. We synthesized several peptides related to these sequences and examined the effect of peptides on the binding of EGF receptor to SH2 of PLC gamma 1. P1, GSFLVRES was the most effective inhibitor to suppress the binding. P2, GSFLVAES in which one amino acid, arginine of P1 is substituted by alanine is still effective. But a peptide, P3, SFLVRE in which two amino acids are deleted from P1 did not inhibit markedly. Moreover, P1 peptide immobilized in Sepharose beads also bound phosphorylated EGF receptor. These data suggest that highly conserved amino acid sequence GSFLVRES is the minimum essential unit to recognize tyrosine phosphorylated site.

The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity.

Several proteins implicated in the regulation of cell proliferation contain a common noncatalytic domain, src homology region 2 (SH2). We have used the bacterially expressed SH2 domain of abl protein-tyrosine kinase to evaluate the ability of this domain to bind to cellular proteins. ablSH2 specifically bound to a number of tyrosine-phosphorylated proteins from cells transformed by tyrosine kinase oncogenes in a filter-binding assay and to a subset of those proteins in solution. The SH2 probe bound almost exclusively to tyrosine-phosphorylated proteins, and binding was eliminated by dephosphorylation of cell proteins. Free phosphotyrosine could partially disrupt SH2 binding, suggesting that phosphotyrosine is directly involved in the binding interaction. These results demonstrate that an SH2 domain is sufficient to confer direct, high-affinity phosphotyrosine-dependent binding to proteins and suggest a general role for SH2 domains in cellular signaling pathways.

Src homology region 2 domains direct protein-protein interactions in signal transduction.

Cytoplasmic proteins that regulate signal transduction or induce cellular transformation, including cytoplasmic protein-tyrosine kinases, p21ras GTPase-activating protein (GAP), phospholipase C gamma, and the v-crk oncoprotein, possess one or two copies of a conserved noncatalytic domain, Src homology region 2 (SH2). Here we provide direct evidence that SH2 domains can mediate the interactions of these diverse signaling proteins with a related set of phosphotyrosine ligands, including the epidermal growth factor (EGF) receptor. In src-transformed cells GAP forms heteromeric complexes, notably with a highly tyrosine phosphorylated 62-kDa protein (p62). The stable association between GAP and p62 can be specifically reconstituted in vitro by using a bacterial polypeptide containing only the N-terminal GAP SH2 domain. The efficient phosphorylation of p62 by the v-Src or v-Fps tyrosine kinases depends, in turn, on their SH2 domains and correlates with their transforming activity. In lysates of EGF-stimulated cells, the N-terminal GAP SH2 domain binds to both the EGF receptor and p62. Fusion proteins containing GAP or v-Crk SH2 domains complex with similar phosphotyrosine proteins from src-transformed or EGF-stimulated cells but with different efficiencies. SH2 sequences, therefore, form autonomous domains that direct signaling proteins, such as GAP, to bind specific phosphotyrosine-containing polypeptides. By promoting the formation of these complexes, SH2 domains are ideally suited to regulate the activation of intracellular signaling pathways by growth factors.