CHARACTERIZATION OF THE KINASE-ACTIVITY ESSENTIAL FOR TYROSINE PHOSPHORYLATION OF P130(CAS) IN FIBROBLASTS

The cellular transformation by v-Src or v-Crk induces tyrosine phospho rylation of a common substrate molecule, p130(Cas) (Cas), which tightl y binds these oncoproteins ill vivo. From its structure, Cas is deduce d to be an ideal substrate for Src family kinases and Abl kinase. The tyrosine kinase activity associated with Cas was analysed using mouse variant fibroblasts lacking at least one of tyrosine kinases. In norma l fibroblasts, Cas is associated with a significant level of tyrosine kinase activity which efficiently phosphorylates Cas in vitro. The Cas -associated tyrosine kinase activity was remarkably elevated in Csk(-/ -) cells, which resulted in hyperphosphorylation of cellular Cas. The associated kinase activity was slightly increased in Src(-/-) cells wh ereas not significantly changed in Abl(-/-) nor Fak(-/-) cells. On the contrary, the Cas-associated kinase activity was remarkably decreased in Fyn(-/-) cells. At the same time, association of Cas with Fyn kina se ill vitro was most obviously detected in normal fibroblasts as well as Csk(-/-) cells. Transient expression of v-Crk induced elevation of the Cas-associated kinase activity in all of these cell lines except the primary culture of Fyn(-/-) fibroblasts. These results indicate th at Fyn kinase plays an essential role in v-Crk-mediated phosphorylatio n of Cas.