A putative modular domain present in diverse signaling proteins.
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Amino Acid Sequence↗
Biomedical subjects
B J Mayer
Publications and source records attributed to B J Mayer.
Identification of a ten-amino acid proline-rich SH3 binding site.
The Src homology 3 (SH3) region is a small protein domain present in a very large group of proteins, including cytoskeletal elements and signaling proteins. It is believed that SH3 domains serve as modules that mediate protein-protein associations and, along with Src homology 2 (SH2) domains, regulate cytoplasmic signaling. The SH3 binding sites of two SH3 binding proteins were localized to a nine- or ten-amino acid stretch very rich in proline residues. Similar SH3 binding motifs exist in the formins, proteins that function in pattern formation in embryonic limbs of the mouse, and one subtype of the muscarinic acetylcholine receptor. Identification of the SH3 binding site provides a basis for understanding the interaction between the SH3 domains and their targets.
Amino Acid Sequence↗
Direct analysis of the binding of the abl Src homology 2 domain to the activated epidermal growth factor receptor.
Src homology regions 2 (SH2) and 3 (SH3) are noncatalytic domains that are conserved among several proteins implicated in the regulation of cell proliferation. Using bacterially expressed fusion proteins containing the SH2 domain of the abl tyrosine kinase, we have quantitated the binding of these domains to the activated epidermal growth factor (EGF) receptor (EGFR). A 35S-labeled abl SH2 fusion protein binds to the human EGFR immunoprecipitated from EGF-treated NIH3T3 cells that overexpress the receptor. This binding is totally dependent on the pretreatment of cells with EGF. The interaction is rapid, reaching 50% of maximum within 1 min, and attaining apparent equilibrium by 10 min. Dissociation of the complex is biphasic with a rapidly dissociating component (t1/2 of less than 1 min), as well as a slowly dissociable component. The 35S-labeled abl SH2 fusion protein specifically binds to the EGFR in a saturable manner and is differentially inhibited by unlabeled fusion proteins containing SH2 domains from phospholipase C, the p85 subunit of phosphatidylinositol-3 kinase, and the GTPase activation protein of ras. To identify residues critical for abl SH2-EGFR binding, six point mutants were constructed in the highly conserved FLVRES motif. Three mutants (V170L, E172Q, and E174Q) display binding affinities similar to that of wild type. However, three other mutants (R171K, S173C, and S175C) have greatly reduced affinity. Interestingly, the binding affinity to the EGFR determined by the in vitro assay directly correlates with the transforming ability of the corresponding v-abl constructs in vivo (Mayer, B. J., Jackson, P. K., Etten, R. A. V., and Baltimore, D. (1992) Mol. Cell. Biol. 12, 609-618). These data indicate that the Arg-171, Ser-173, and Ser-175 are critical for both transformation and abl SH2 domain binding to phosphotyrosine-containing proteins.
Amino Acid Sequence↗
Signalling through SH2 and SH3 domains.
In 1986, Pawson's group recognized a region of homology between two oncogenic tyrosine kinases that lay outside the catalytic domain. They termed this the Src homology 2, or SH2, domain. In the ensuing years, SH2 domains have been found in an impressive variety of proteins, as has a second region of homology, inevitably termed SH3. These domains appear to mediate controlled protein-protein interactions. Many proteins that contain SH2 and SH3 domains are involved in signal transduction, suggesting a new paradigm for regulation of intracellular signalling pathways.
Journal Article↗
Three-dimensional solution structure of the src homology 2 domain of c-abl.
SH2 regions are protein motifs capable of binding target protein sequences that contain a phosphotyrosine. The solution structure of the abl SH2 product, a protein of 109 residues and 12.1 kd, has been determined by multidimensional nuclear magnetic resonance spectroscopy. It is a compact spherical domain with a pair of three-stranded antiparallel beta sheets and a C-terminal alpha helix enclosing the hydrophobic core. Three arginines project from a short N-terminal alpha helix and one beta sheet into the putative phosphotyrosine-binding site, which lies on a face distal from the termini. Comparison with other SH2 sequences supports a common global fold and mode of phosphotyrosine binding for this family.
Amino Acid Sequence↗
Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.
Three-dimensional structures of complexes of the SH2 domain of the v-src oncogene product with two phosphotyrosyl peptides have been determined by X-ray crystallography at resolutions of 1.5 and 2.0 A, respectively. A central antiparallel beta-sheet in the structure is flanked by two alpha-helices, with peptide binding mediated by the sheet, intervening loops and one of the helices. The specific recognition of phosphotyrosine involves amino-aromatic interactions between lysine and arginine side chains and the ring system in addition to hydrogen-bonding interactions with the phosphate.
Amino Acid Sequence↗
Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho.
A Src homology 3 (SH3) region is a sequence of approximately 50 amino acids found in many nonreceptor tyrosine kinases and other proteins. Deletion of the SH3 region from the protein encoded by the c-abl proto-oncogene activates the protein's transforming capacity, thereby suggesting the participation of the SH3 region in the negative regulation of transformation. A complementary DNA was isolated that encoded a protein, 3BP-1, to which the SH3 region of Abl bound with high specificity and to which SH3 regions from other proteins bound differentially. The sequence of the 3BP-1 protein is similar to that of a COOH-terminal segment of Bcr and to guanosine triphosphatase-activating protein (GAP)-rho, which suggests that it might have GAP activity for Ras-related proteins. The 3BP-1 protein may therefore be a mediator of SH3 function in transformation inhibition and may link tyrosine kinases to Ras-related proteins.
3T3 Cells↗
Tyrosine-phosphorylated epidermal growth factor receptor and cellular p130 provide high affinity binding substrates to analyze Crk-phosphotyrosine-dependent interactions in vitro.
The genome of CT10 avian sarcoma virus encodes a 47-kDa fusion protein that consists of viral gag sequences fused to a cell-derived sequence containing SH2 and SH3 domains (v-crk). Genetic and biochemical evidence suggests that v-Crk can induce transformation of chicken embryo fibroblasts by influencing the activity of cellular proteins involved in growth regulation. In this report, we have developed an in vitro microtiter assay to study the binding of bacterially expressed glutathione S-transferase-fusion proteins of v-Crk and its cellular homolog, c-Crk, to the phosphorylated epidermal growth factor receptor (EGFR). Competitive binding data are presented that compare the abilities of heterologous glutathione S-transferase-fusion proteins containing GAPSH2[N], AblSH2, SrcSH2, and PLC-gamma SH2[N] sequences to inhibit Crk binding. Results indicate that both full-length Crk and GAPSH2[N] bind the phosphorylated EGFR with high affinity and can quantitatively compete the binding of each other by competitive enzyme-linked immunosorbent assay. Binding of full-length Crk or the isolated SH2 domains of GAP or Abl resulted in a significant protection of phosphorylated EGFR against dephosphorylation by cellular phosphatase activity, but did not appear to stimulate the intrinsic tyrosine kinase activity of the EGFR. To extend these findings to p130, the major phosphotyrosine-containing protein in CT10-transformed cells, we utilized a nitrocellulose filter binding assay. Results demonstrate high affinity binding of Crk toward denatured p130 and, as is the case for phosphorylated EGFR, Crk binding can partially protect p130 from phosphatase activity. However, no apparent competition of Crk binding was noted with heterologous SH2-containing proteins including GAPSH2[N], suggesting a possible specificity of Crk-p130 binding. These data are consistent with a direct role of SH2 in the modulation of cellular phosphotyrosine status in vivo.
Animals↗
A novel oncogene, v-ryk, encoding a truncated receptor tyrosine kinase is transduced into the RPL30 virus without loss of viral sequences.
The RPL viruses are acute oncogenic avian retroviruses isolated from chicken tumors. We carried out a genetic analysis of three of the viruses, RPL25, RPL28, and RPL30. While RPL25 and RPL28 were shown to contain the erbB oncogene, RPL30 appeared to contain a novel protein tyrosine kinase oncogene. This gene, v-ryk, was cloned and sequenced. The v-ryk oncogene contains a 1.39-kb nonretroviral sequence that includes a tyrosine kinase domain which was inserted into the viral envelope protein gp37-coding region and fused in frame with upstream gp37 to generate a P69gp37-ryk fusion oncoprotein. Unlike that of other acutely transforming retroviruses, transduction of the v-ryk gene into RPL30 did not result in deletion of viral sequences. Sequence analysis suggested that v-Ryk is more homologous to receptor-type tyrosine kinases than to nonreceptor-type kinases. By reconstitution of a virus from its cDNA, the v-ryk oncogene has been shown to be fully responsible for the transforming activity of the RPL30 virus. Antibodies specific to v-Ryk immunoprecipitated the v-Ryk oncoprotein from cells transformed by the RPL30 virus. The v-Ryk protein was shown to be first synthesized as a 150-kDa precursor and then cleaved into the mature 69-kDa gp37-Ryk fusion protein, both parts of which were found to be localized to the membrane fraction. As expected from the sequence of v-Ryk, immunoprecipitates of v-Ryk from RPL30-transformed cells were found to display a protein tyrosine kinase activity in vitro, and the levels of tyrosine-phosphorylated proteins are elevated in v-ryk-transformed cells.
Amino Acid Sequence↗
Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo.
We have constructed a series of point mutations in the highly conserved FLVRES motif of the src homology 2 (SH2) domain of the abl tyrosine kinase. Mutant SH2 domains were expressed in bacteria, and their ability to bind to tyrosine-phosphorylated proteins was examined in vitro. Three mutants were greatly reduced in their ability to bind both phosphotyrosine itself and tyrosine-phosphorylated cellular proteins. All of the mutants that retained activity bound to the same set of tyrosine-phosphorylated proteins as did the wild type, suggesting that binding specificity was unaffected. These results implicate the FLVRES motif in direct binding to phosphotyrosine. When the mutant SH2 domains were inserted into an activated abl kinase and expressed in murine fibroblasts, decreased in vitro phosphotyrosine binding correlated with decreased transforming ability. This finding implies that SH2-phosphotyrosine interactions are involved in transmission of positive growth signals by the nonreceptor tyrosine kinases, most likely via the assembly of multiprotein complexes with other tyrosine-phosphorylated proteins.
Amino Acid Sequence↗
The product of the cellular crk gene consists primarily of SH2 and SH3 regions.
We have cloned and sequenced a complementary DNA encoding the cellular homologue of the transforming oncogene v-crk of avian sarcoma virus CT10. This complementary DNA contains an open reading frame of 915 base pairs that encodes a polypeptide of 305 amino acids. The first 205 amino acids of this c-Crk protein were identical to those of the CT10 encoded v-Crk protein, with the exception of 5 amino acids. Like v-Crk, this portion of c-Crk contained one each of the Src homology domains SH2, SH2', and SH3. The 100 carboxy-terminal amino acids of c-Crk protein, which are not coded for in the CT10 viral genome, contain another SH3 region. We found limited sequence homology between c-crk and the avian retrovirus genome, which explains recombination events in the transduction of this protooncogene. Using a polyclonal antiserum made against bacterially expressed v-crk, we identified a 35-kilodalton protein in normal chicken embryo fibroblasts and in all embryonic chicken tissues examined. This 35-kilodalton protein was indistinguishable from a polypeptide made by in vitro translation of c-crk complementary DNA.
Amino Acid Sequence↗
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.
Animals↗
Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins.
The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.
Animals↗
Binding of transforming protein, P47gag-crk, to a broad range of phosphotyrosine-containing proteins.
Although the oncogene product of CT10 virus, P47gag-crk, does not itself phosphorylate proteins at tyrosine residues, it elevates phosphotyrosine in transformed cells. The P47gag-crk oncoprotein contains SH2 and SH3 domains, which are conserved in several proteins involved in signal transduction, including nonreceptor tyrosine kinases. P47gag-crk bound in vitro to phosphotyrosine-containing proteins from crk-transformed cells and from cells transformed by oncogenic tyrosine kinases. The association between P47gag-crk and p60v-src, a phosphotyrosine-containing protein, was abolished by dephosphorylation of p60v-src. This suggests that the SH2 and SH3 regions function to regulate protein interactions in a phosphotyrosine-dependent manner.
Animals↗
Association of the v-crk oncogene product with phosphotyrosine-containing proteins and protein kinase activity.
An antiserum specific for P47gag-crk, the oncogene product of avian sarcoma virus CT10, was used to investigate possible crk-binding proteins. In in vitro kinase assays, four proteins were phosphorylated in anti-crk immunoprecipitates. Tyrosine, serine, and threonine residues were phosphorylated. A number of tyrosine-phosphorylated proteins were identified in anti-crk immunoprecipitates of 32P-labeled cells, including the three major phosphotyrosine-containing proteins of CT10-infected cells. These three proteins also bound to bacterially synthesized crk protein. These results suggest that the crk transforming protein can stably associate with both endogenous kinases and cellular kinase substrates.
Animals↗
Mutagenic analysis of the v-crk oncogene: requirement for SH2 and SH3 domains and correlation between increased cellular phosphotyrosine and transformation.
We have constructed a series of mutants with deletion, linker insertion, and point mutations in the v-crk oncogene of avian sarcoma virus CT10. The v-crk gene contains no apparent catalytic domain, but does contain two blocks of homology to putative regulatory domains, termed SH2 and SH3, found in a variety of proteins implicated in signal transduction. Infection with CT10 causes a dramatic increase in the level of tyrosine phosphorylation of several cellular proteins. We found that mutation of either the SH2 or SH3 domain of v-crk reduced or eliminated transforming activity, whereas mutation of regions outside the conserved domains had no effect. Deletion of amino-terminal gag sequences caused a partial loss of transforming activity and a change in subcellular distribution of the crk protein. In all cases, there was an absolute correlation between increased cellular phosphotyrosine and transformation.
Animals↗
Cell transformation by an oncogene that regulates protein kinases.
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Avian Sarcoma Viruses↗
A novel viral oncogene with structural similarity to phospholipase C.
Numerous oncogenes have been isolated from acutely transforming retroviruses. To date, the products of these viral oncogenes have been protein kinases, nuclear proteins, growth factors, or GTP-binding proteins. We have cloned the previously uncharacterized avian sarcoma virus CT10 and sequenced its genome. This virus encodes a protein, p47gag-crk, that has blocks of sequence similarity to the amino-terminal, non-catalytic region of the non-receptor class of tyrosine kinases. In addition, the structure of p47gag-crk has striking similarity to a 180-amino acid region of bovine brain phospholipase C. Biochemical data suggest that p47gag-crk activates one or several endogenous tyrosine kinases.
Amino Acid Sequence↗