Reduced phosphotyrosine binding by the v-Src SH2 domain is compatible with wild-type transformation.
The SH2 domain of v-Src binds phosphotyrosyl-proteins in vivo and in vitro. The function of this domain is necessary for transformation of Rat-2 cells and for morphologically wild-type transformation of chicken embryo fibroblasts (CEF). The phosphate group of phosphotyrosine interacts directly with a conserved arginine residue in the FLVRES motif of the SH2 domain, R175 in v-Src. To examine the role of phosphotyrosine binding in transformation by v-Src, we have characterized the effects of R175 mutations on the transforming ability of v-Src and on the interaction of the v-Src SH2 domain with phosphotyrosyl-proteins. The R175H mutation, and to a lesser extent the R175K mutation, reduced but did not eliminate the binding of phosphotyrosyl-proteins to the v-Src SH2 domain. However neither mutation affected transformation of CEF or Rat-2 cells by v-Src and neither mutation resulted in major changes in the level or pattern of protein-tyrosine phosphorylation in transformed CEF. In contrast, the R175E mutant of v-Src induced fusiform transformation of CEF and failed to transform Rat-2 cells; the mutant SH2 domain was insoluble when expressed in bacteria, suggesting that the R175E mutation disrupts the structure of the v-Src SH2 domain. We conclude that, although the Arg residue in the FLVRES motif is invariant in most if not all SH2 domains, at position 175 in the v-Src SH2 domain residues other than arginine can support the binding of phosphotyrosyl-proteins, albeit at reduced levels. Furthermore under the expression conditions normally used, that is when v-Src is expressed under the control of a retroviral LTR, the reduced binding of phosphotyrosyl-proteins is compatible with wild-type transformation.
Tian
M
M
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3204, USA.
Martin
G S
GS
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002642:Chick Embryo; D017124:Conserved Sequence; D017931:DNA Primers; D005347:Fibroblasts; D016391:Genes, src; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015688:Oncogene Protein pp60(v-src) / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D018909:src Homology Domains
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D015688:Oncogene Protein pp60(v-src)