RAPID AND EFFICIENT PURIFICATION OF SRC HOMOLOGY-2 DOMAIN-CONTAINING PROTEINS - FYN, CSK AND PHOSPHATIDYLINOSITOL 3-KINASE P85

To analyse the regulation of Src family tyrosine kinases in vitro, we have purified Fyn and Csk, a kinase capable of regulating Fyn activity by phosphorylation, from baculovirus-infected insect cells. The prote ins were purified by affinity purification over a phosphotyrosine colu mn. Highly purified proteins were eluted from the resin by a salt grad ient and further purified by ion-exchange chromatography. This purific ation scheme was successfully applied to a third, unrelated protein th at also contains the Src homology 2 (SH2) domain, namely the 85 kDa su bunit of phosphatidylinositol 3-kinase, indicating that this method is versatile and should prove applicable to any protein with an accessib le SH2 domain. The binding of Csk to different phosphopeptides was tes ted, and specificity for the autophosphorylation site of Fyn was demon strated. Pure Csk was used to phosphorylate Fyn and down-regulate its kinase activity, and the kinetic parameters of both the active and the repressed forms of Fyn were determined. Repression of Fyn activity by Csk reduced binding of Fyn to phosphopeptides to undetectable levels, supporting the model that predicts an intramolecular interaction of t he Fyn SH2 domain with a C-terminal phosphotyrosine residue.