MOLECULAR-INTERACTIONS OF THE SRC HOMOLOGY-2 DOMAIN PROTEIN SHB WITH PHOSPHOTYROSINE RESIDUES, TYROSINE KINASE RECEPTORS AND SRC HOMOLOGY-3DOMAIN PROTEINS

The molecular interactions of the Src homology 2 (SH2) domain and the N-terminal proline-rich sequence motifs (pro-1 to pro-5) of the SH2 pr otein Shb with other components were presently characterised, Using a degenerate phosphopeptide library the preferred binding site for the S hb SH2 domain was determined to pTyr-Thr/Val/Ile-X-Leu at positions +1 to +3 relative the phosphotyrosine residue, Experiments with competin g peptides and platelet-derived growth factor (PDGF) beta-receptor mut ants with Y to F substitutions in autophosphorylation sites revealed m ultiple binding sites for the Shb SH2 domain in the receptor, The Shb SH2 domain also binds to in vitro phosphorylated fibroblast growth fac tor receptor-1 (FGFR-1) mainly through position Y776, The receptor exp eriments suggest that other residues besides the +1 to +3 positions ma y also be of significance for Shb binding, The pro-4/pro-5 motif of Sh b binds in vitro particularly well to the Src, p85 alpha PI3-kinase an d Eps8 SH3 domains expressed as GST fusion proteins, However, the GST- SH3 domain fusion proteins tested bind in vitro to peptides correspond ing to the pro-1 to pro-5 motifs of Shb with low affinity and selectiv ity, suggesting that sequences outside the core proline motif may also be important for Shb-SH3 domain interactions, In vivo association bet ween Shb-SH3 domain proteins v-Src and Eps8 was detected by coimmunopr ecipitation. PDGF treatment did not affect the association between Eps 8 and Shb. The data suggest that Shb is an adaptor protein linking SH3 domain proteins to tyrosine kinases or other tyrosine phosphorylated proteins.