Reconstitution of interactions between the Src tyrosine kinases and Ras GTPase-activating protein using a baculovirus expression system.

摘要: Ras GTPase-activating protein (GAP) has been implicated in mitogenic signal transduction downstream of oncogenic and receptor tyrosine kinases. Previous studies have suggested that GAP is phosphorylated by viral Src (v-Src) associated with a complex containing normal cellular (c-Src) vertebrate fibroblasts. To investigate molecular interactions between the kinases GAP, we developed an vitro system for reconstituting Src-GAP complexes. For this purpose, constructed recombinant baculovirus vectors direct expression Rous sarcoma virus v-Src, chicken c-Src, bovine infected Sf9 insect cells. In reconstitution experiments using baculovirus-expressed proteins demonstrate both v-Src c-Src associate complexes GAP. addition, vivo phosphorylation analyses indicate serves as substrate determine which structural features are involved kinases, baculoviruses encode deletion mutants Deletion amino-terminal portion homology 2 regions, highly conserved motifs postulated to mediate among proteins, diminishes association Src. This should facilitate further