N-terminal Src Homology 2 Domain Research Articles

Structure of the N-terminal SH3 domain of GRB2 complexed with a peptide from the guanine nucleotide releasing factor Sos

Src-homology 3 (SH3) domains mediate signal transduction by binding to proline-rich motifs in target proteins. We have determined the high-resolution NMR structure of the complex between the amino-terminal SH3 domain of GRB2 and a ten amino acid peptide derived from the guanine nucleotide releasing factor Sos. The NMR data show that the peptide adopts the conformation of a left-handed polyproline type II helix and interacts with three major sites on the SH3 domain. The orientation of the bound peptide is opposite to that of proline-rich peptides bound to the SH3 domains of Abl, Fyn and p85.

Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor.

Aggregation of the high affinity IgE receptor (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in activation of the protein tyrosine kinases Syk and Lyn. Here, we report that fusion proteins containing the Src homology 2 (SH2) domains of Syk and Lyn precipitated tyrosine-phosphorylated proteins from RBL-2H3 cell lysates. There was no detectable precipitation with the N-terminal Syk SH2 domain, minimal with the C-terminal Syk SH2, and strong when the two SH2 domains were expressed in tandem. The Syk SH2 domains showed pronounced selectivity in the binding of tyrosine-phosphorylated proteins. Thus, Syk SH2 precipitated only three phosphoproteins, two of which were the beta and gamma subunits of Fc epsilon RI. In contrast, the fusion protein with Lyn SH2, in addition to precipitating the same Fc epsilon RI components, bound to many other phosphoproteins. Tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI was essential for their precipitation with the SH2 fusion proteins. By direct binding studies, there was more binding of Syk SH2 to Fc epsilon RI gamma than to Fc epsilon RI beta, whereas Lyn SH2 bound only to Fc epsilon RI beta. The Syk SH2 fusion protein competitively inhibited the association of gamma and beta with Syk in lysates of activated cells. The SH2-mediated association of these two protein tyrosine kinases with Fc epsilon RI could play an important role in receptor signaling.

Genetic Analysis of a Phosphatidylinositol 3-Kinase SH2 Domain Reveals Determinants of Specificity

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.

Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity.

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.

In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases, from the adaptor protein SHC and from GTPase-activating protein in Ramos B cells.

Antigen receptors of B lymphocytes transmit their activation signal to the cell interior by associating with and activation of specific non-receptor tyrosine kinases. Most of these kinases as well as other cytoplasmic effectors contain at least one Src homology 2 (SH2) domain, known to bind tyrosine-phosphorylated proteins. We examined the binding specificity of SH2 domains from different signaling molecules in B cells and found that each of the SH2 domains tested bound distinct subsets of stimulation-dependent phosphoproteins in vitro. SH2 domains from Src-like tyrosine kinases bound predominantly to the HS1 phosphoprotein. The tandem SH2 domains of the ZAP-70 tyrosine kinase bound to phosphorylated Ig-beta but only weakly to Ig-alpha. Also the SHC-derived SH2 domain formed complexes with the tyrosine-phosphorylated Ig-alpha/beta heterodimer, while the C- and N-terminal SH2 domains of GTPase-activating protein displayed completely different binding preferences. These results suggest that cytoplasmic effector molecules can be recruited to the activated B cell receptor in an SH2-phosphotyrosine-mediated manner. The data also provide a possible explanation for the notion that Ig-alpha and Ig-beta might couple to different biochemical pathways.

Cerebellar beta 2-chimaerin, a GTPase-activating protein for p21 ras-related rac is specifically expressed in granule cells and has a unique N-terminal SH2 domain.

beta-Chimaerin, a 30-kDa GTPase-activating protein (GAP) for the p21 Ras-related Rac, is expressed specifically in late stage spermatocytes (Leung, T., How, B.-E., Manser, E., and Lim, L. (1993) J. Biol. Chem. 268, 3813-3816). Using antibodies raised against beta-chimaerin, we detected a major 46-kDa RacGAP in the rat cerebellum. With beta-chimaerin cDNA as a probe and using polymerase chain reaction, cDNAs from both human and rat cerebellum were isolated. The human and rat cDNAs encoded sequences containing cysteine-rich and GAP domains identical to those of testis beta-chimaerin. The cDNAs also encoded an additional N-terminal SH2 (Src homology 2) domain, probably derived from the beta-chimaerin gene by alternate splicing. This SH2 domain of the predicted 54-kDa protein was strikingly similar to that of alpha 2-chimaerin, including replacement by glutamic acid of the invariant tryptophan present at the start of other SH2 domains. The SH2 domains of alpha- and beta-chimaerin thus represent a subset of SH2 domains. The cerebellar beta-chimaerin (beta 2-) is expressed mainly in granule cells and exhibits postnatal developmental increases. beta 2-Chimaerin was enriched in particulate/synaptosomal fractions. In the mouse weaver mutant lacking mature granule cells, there is a corresponding decrease in beta 2-chimaerin, which could well serve as a marker of granule cell differentiation.

SH-PTP2/Syp SH2 domain binding specificity is defined by direct interactions with platelet-derived growth factor beta-receptor, epidermal growth factor receptor, and insulin receptor substrate-1-derived phosphopeptides.

Signaling by tyrosine kinases involves direct associations between proteins with Src homology 2 (SH2) domains and sites of tyrosine phosphorylation. Specificity in signaling pathways results in part from inherent selectivity in interactions between particular SH2 domains and phosphopeptide sequences. The cytoplasmic phosphotyrosine phosphatase SH-PTP2 (Syp, PTP 1D, PTP-2C) contains two SH2 domains (N and C) which mediate its association with and activation by the platelet-derived growth factor (PDGF) and epidermal growth factor receptors and IRS-1. We have developed a competitive phosphopeptide binding assay to analyze specificity of the SH-PTP2 N-SH2 domain for phosphorylation sites of these phosphoproteins. The sequence surrounding Tyr1009 bound with greatest affinity (ID50 = 14 microM) of eight PDGF receptor-derived phosphopeptides tested. No peptides corresponding to known epidermal growth factor receptor phosphorylation sites bound with high affinity. However, an alternative sequence surrounding Tyr954 bound tightly (ID50 = 21 microM). Of the 13 IRS-1-related peptides analyzed, sequences surrounding Tyr546, Tyr895, and Tyr1172 bound with highest affinity (ID50 = 11, 4, and 1 microM, respectively). Alternative phosphopeptides generally bound with much weaker affinity (ID50 > 150 microM). These findings are consistent with recent mutational analyses of the PDGF receptor and predict site-specific interactions between SH-PTP2 and each of these phosphoproteins. Comparisons between peptide sequences suggest that the N-terminal SH2 domain of SH-PTP2 binds with highest affinity to phosphotyrosine (pY) followed by a beta-branched residue (Val, Ile, Thr) at pY+1 and a hydrophobic residue (Val, Leu, Ile) at pY+3 positions. Peptide truncation studies also indicate that residues outside of the pY-1 to pY+4 motif are required for high affinity interactions.

T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif.

The T-cell antigen CD28 provides a costimulatory signal that is required for T-cell proliferation. T-cell receptor zeta/CD3 engagement without CD28 ligation leads to a state of nonresponsiveness/anergy, thereby implicating CD28 in the control of peripheral tolerance to foreign antigens or tumors. A key unresolved question has concerned the mechanism by which CD28 generates intracellular signals. Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase with Src-homology 2 (SH2) domain(s) that binds to the platelet-derived growth factor receptor (PDGF-R), an interaction that is essential for signaling by growth factor. In this study, we demonstrate that CD28 binds to PI 3-kinase by means of a Y(P)MXM motif within its cytoplasmic tail. CD28-associated PI 3-kinase was detected by lipid kinase and HPLC analysis as well as by reconstitution experiments with baculoviral-expressed p85 subunit of PI 3-kinase. CD28 bound directly to the p85 subunit without the need for the associated p110 subunit. Site-directed mutagenesis and peptide competition analysis using Y(P)-MXM-containing peptides showed that PI 3-kinase bound to a Y(P)MXM motif within the CD28 cytoplasmic tail (residues 191-194). Mutation of the Y191 within the motif resulted in a complete loss of binding, while mutation of M194 caused partial loss of binding. Binding analysis showed that the CD28 Y(P)-MXM motif bound to the p85 C- and N-terminal SH2 domains with an affinity comparable to that observed for PDGF-R and insulin receptor substrate 1. In terms of signaling, CD28 ligation induced a dramatic increase in the recruitment and association of PI 3-kinase with the receptor. CD28 is likely to use PI 3-kinase as the second signal leading to T-cell proliferation, an event with implications for anergy and peripheral T-cell tolerance.

Insulin Receptor Kinase Phosphorylates Protein Tyrosine Phosphatase Containing Src Homology 2 Regions and Modulates Its PTPase Activity in Vitro

To clarify the role of protein tyrosine phosphatase (PTPase) containing a pair of Src homology 2 (SH2) regions upon insulin signaling, we studied the interactions between the insulin receptor and SH-PTP2 coupled to glutathione-S-transferase. A full length SH-PTP2 was phosphorylated by insulin receptor kinase and associated with the insulin receptor in vitro. The N-terminal SH2 domain was more phosphorylated than the other SH2 domain of SH-PTP2. However, both SH2 domains of SH-PTP2 were necessary for association with insulin receptors. Phosphorylation of the SH2 domains of SH-PTP2 resulted in decreased PTPase activities toward the phosphorylated insulin receptor. These results indicate that the insulin receptor can negatively regulate SH-PTP2 activity by means of phosphorylating the SH2 domains.

Alpha 2-chimerin, an SH2-containing GTPase-activating protein for the ras-related protein p21rac derived by alternate splicing of the human n-chimerin gene, is selectively expressed in brain regions and testes.

n-Chimerin (alpha 1-chimerin) is a brain GTPase-activating protein (GAP) for the ras-related p21rac. We now report the occurrence of another form of chimerin, termed alpha 2-chimerin. This is the product of an alternately spliced transcript of the human n-chimerin gene encoding an N-terminal SH2 (src homology 2) domain in addition to the phorbol ester receptor and GAP domains. alpha 1- and alpha 2-chimerin mRNAs were expressed differently. In the rat brain, only alpha 1-chimerin mRNA was expressed in cerebellar Purkinje cells, although both alpha 1- and alpha 2-chimerin mRNAs occurred in neurons in the cerebral cortex, hippocampus, and thalamus. Only alpha 2-chimerin RNA was expressed in rat testes, in early pachytene spermatocytes. A 45-kDa SH2-containing chimerin corresponding to the alpha 2 form was purified from rat brain. As with Escherichia coli 45-kDa recombinant alpha 2-chimerin, purified brain alpha 2-chimerin exhibited racGAP activity which was stimulated by phosphatidylserine. The recombinant SH2 domain bound several 32P-labelled phosphoproteins of PC12 cells, whose phosphorylation increased in response to trophic factors, including nerve growth factor. To examine the relationships of alpha 1- and alpha 2-chimerin transcripts, human genomic DNA clones were characterized. In alpha 2-chimerin mRNA, a 3' splice acceptor site within exon 1 of alpha 1-chimerin mRNA was used, replacing its 5' untranslated region and N-terminal coding sequence. The single human n-chimerin gene was mapped to chromosome 2q31-q32.1, colocalizing with the CRE-BP1 transcription factor gene (2q32). It contained several splice junctions conserved with the sequence-related protein kinase C and bcr genes. alpha 2-Chimerin is only the second SH2-containing GAP and the first example of an SH2 domain generated by alternate splicing.

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)

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)