Crk proteins activate GCKR kinase activity.

(A) Coexpression of Crk proteins enhances GCKR kinase activity. HEK 293T was transfected with a control or with constructs that direct the expression of Crk-I, Crk-II, or CrkL and HA-GCKR. HA-immunoprecipitates were subjected to an in vitro kinase assay using myelin basic protein (MBP) as a substrate (autoradiograph, labeled as kinase assay). The SDS-PAGE fractionated in vitro kinase assay was analyzed by immunoblotting for phosphotyrosine (pY blot). In addition, cell lysates prepared from the same transfections were analyzed by immunoblotting for HA-GCKR (HA blot), CrkL (CrkL blot), and Crk-I and Crk-II (Crk blot). (B) Coexpression of Crk proteins triggers GCKR phosphorylation. HA-GCKR immunoprecipitates from cells transfected as in A were subjected to an in vitro kinase assay in the absence of an exogenous substrate (autoradiograph, labeled as kinase assay). Cell lysate analyzed for HA-GCKR expression by immunoblotting. Each of these experiments was performed at least twice.