Conditional and Dissociation Mutants of pp60v-src: Tools for Dissecting Transformation and Mitogenesis

Biochemistry, Genetics

Catling, Andrew David

University:University of Glasgow

关键词: Biochemistry, Genetics;   

Others  :  http://theses.gla.ac.uk/78382/1/11011449.pdf

来源: University of Glasgow

【 摘 要 】

The biological and biochemical properties of ts LA32 Src were examined in established rat fibroblasts. A cell clone expressing this protein (Rati f32) was found to be markedly temperature sensitive for morphological transformation and anchorage-independent growth. Ratl f32 are rendered quiescent by serum deprivation at restrictive temperature; temperature shift in the absence of added serum factors results in cell cycle transit. The conditional nature of the biological phenotypes was not reflected in any examined biochemical parameter: tsLA32 Src elevated cellular phosphotyrosine at both restrictive and permissive temperatures, and was non-conditionally autophosphorylated. tsLA32 Src in vitro kinase activity was not measurable by immune-complex assay under conditions where thermolabile and wild-type controls behaved as expected, perhaps through lability of the mutant protein. Published data indicate that tsLA32 Src remains in the particulate fraction of CEF at restrictive temperature. These data suggest that in vivo enzyme activity, autophosphorylation and membrane localization are together insufficient for transformation of Ratl cells. Non-catalytic domains of pp60v-src and their possible roles in transformation are discussed. These properties suggest tsLA32 Src would be useful in defining early Src-responsive events. Preliminary results indicate that very few proteins become differentially labelled with 35S-methionine after temperature shift of quiescent Ratl f32. Dissociation of transformation parameters would allow dissection of these and other Src-responsive events. Dissociation was attempted using a thermolabile kinase defective for morphological transformation through point mutation of the N-terminal glycine. This non-myristylated protein, tsLA29A2 Src, retained thermolabile in vitro kinase properties but was somewhat toxic in Ratl cells. tsLA29A2 was non-mitogenic, and did not induce AP-1 binding activity in temperature-shifted quiescent CEF. Furthermore, tsLA29A2 Src was non-mitogenic in quiescent CEF under conditions where AP-1 activity was artificially elevated. These data suggest a need for membrane association in the induction of both AP-1 binding activity and other v-src-induced events required for mitogenesis.