title: Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins.
abstract: 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 protein Shb with other components were presently characterised. Using a degenerate phosphopeptide library the preferred binding site for the Shb SH2 domain was determined to pTyr-Thr/Val/Ile-X-Leu at positions +1 to +3 relative the phosphotyrosine residue. Experiments with competing peptides and platelet-derived growth factor (PDGF) beta-receptor mutants with Y to F substitutions in autophosphorylation sites revealed multiple binding sites for the Shb SH2 domain in the receptor. The Shb SH2 domain also binds to in vitro phosphorylated fibroblast growth factor receptor-1 (FGFR-1) mainly through position Y776. The receptor experiments suggest that other residues besides the +1 to +3 positions may also be of significance for Shb binding. The pro-4/pro-5 motif of Shb binds in vitro particularly well to the Src, p85 alpha PI3-kinase and Eps8 SH3 domains expressed as GST fusion proteins. However, the GST-SH3 domain fusion proteins tested bind in vitro to peptides corresponding to the pro-1 to pro-5 motifs of Shb with low affinity and selectivity, suggesting that sequences outside the core proline motif may also be important for Shb-SH3 domain interactions. In vivo association between Shb-SH3 domain proteins v-Src and Eps8 was detected by coimmunoprecipitation. PDGF treatment did not affect the association between Eps8 and Shb. The data suggest that Shb is an adaptor protein linking SH3 domain proteins to tyrosine kinases or other tyrosine phosphorylated proteins.
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Sweden.
authors.forename: T
authors.initials: T
authors.lastname: Karlsson
authors.forename: Z
authors.initials: Z
authors.lastname: Songyang
authors.forename: E
authors.initials: E
authors.lastname: Landgren
authors.forename: C
authors.initials: C
authors.lastname: Lavergne
authors.forename: P P
authors.initials: PP
authors.lastname: Di Fiore
authors.forename: M
authors.initials: M
authors.lastname: Anafi
authors.forename: T
authors.initials: T
authors.lastname: Pawson
authors.forename: L C
authors.initials: LC
authors.lastname: Cantley
authors.forename: L
authors.initials: L
authors.lastname: Claesson-Welsh
authors.forename: M
authors.initials: M
authors.lastname: Welsh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011817:Rabbits; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D017468:Receptors, Fibroblast Growth Factor; D019000:Phosphotyrosine; D014443:Tyrosine; D020794:Receptor Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D015688:Oncogene Protein pp60(v-src)