Mutations in v-Src SH3 and catalytic domains that jointly confer temperature-sensitive transformation with minimal temperature-dependent changes in cellular tyrosine phosphorylation.
We have analyzed two functionally significant amino acid alterations encoded by the temperature-sensitive (ts) v-src mutant of Rous sarcoma virus, LA32. The G-to-V change at residue 300 in the catalytic domain nonconditionally impairs morphological transformation, in vitro kinase activity, in vivo tyrosine phosphorylation, and the cytoskeletal association of v-Src while rendering anchorage- and serum-independent growth ts. The R-to-P mutation in the SH3 domain subtly enhances morphological transformation but has no phenotype if the catalytic domain is inactivated. In the presence of the G-300-to-V mutation, this SH3 domain lesion does not affect v-Src in vitro kinase activity and cytoskeletal association, but it nonconditionally enhances cellular tyrosine phosphorylation and restores morphological transformation at the permissive temperature only. This ability to induce a ts transformed morphology, in concert with nonconditional elevations of cellular phosphotyrosine, suggest that a subset of v-Src targets that are crucial to transformation may be affected in ts fashion by the SH3 mutation. Consistent with this, we find that the R-107-to-P mutation confers ts activity and tyrosine phosphorylation on the SH3-binding enzyme phosphatidylinositol 3'-kinase. Thus, both the SH3 and catalytic domain mutations in LA32 have some ts attributes and they cooperate in determining the mutant's behavior. The ts SH3 mutation is unique and offers the potential for deeper understanding of the function of this domain.
Catling
A D
AD
CRC Beatson Laboratories, Beatson Institute for Cancer Research, Bearsden, Glasgow, United Kingdom.
Fincham
V J
VJ
Frame
M C
MC
Haefner
B
B
Wyke
J A
JA
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002384:Catalysis; D002472:Cell Transformation, Viral / Q000235:genetics*; D002478:Cells, Cultured; D002642:Chick Embryo; D009154:Mutation; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D013696:Temperature; D014443:Tyrosine / Q000378:metabolism*
D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D015688:Oncogene Protein pp60(v-src)