Evidence that GCKR is required for CrkL-induced SAPK activation.

(A) A GCKR antisense construct inhibits CrkL-induced SAPK activation. HEK 293 cells were cotransfected with constructs that direct the expression of HA-SAPK (lanes 1 to 5), CrkL (lanes 2 to 5), and a GCKR antisense RNA (lanes 4, 5). HA-SAPK immunoprecipitates were subjected to an in vitro kinase assay using GST-jun^1–79 as a substrate. The fold induction compared to the control is shown below the in vitro kinase assay result. Levels of HA-SAPK and CrkL expression are shown as detected by immunoblotting. (B) GCKRT178A inhibits CrkL-induced SAPK activation. HEK 293 cells were cotransfected with constructs that direct the expression of HA-SAPK (lanes 1 to 5), CrkL (lanes 2 to 5), and increasing concentrations of GCKRT178A (1, 2, and 3 μg/mL, lanes 3 to 5, respectively). HA-SAPK immunoprecipitates were subjected to an in vitro kinase assay using GST-jun^1–79 as a substrate. The fold induction compared to cells transfected only with the construct directing HA-SAPK expression is indicated below the autoradiograph. Levels of HA-SAPK, CrkL, and GCKRT178A as detected by immunoblotting are shown. Low levels of endogenous GCKR are detected (lanes 1, 2, bottom). These experiments were performed twice with similar results.