Bcr-Abl–induced GCKR activation requires Ras and Bcr-Abl–triggered SAPK activation requires GCKR. (A) A dominant negative Ras inhibits Bcr-Abl–induced GCKR activation. HEK 293T cells (1 × 10⁶) were transfected with HA-GCKR (1 μg) and Ras-V12 (2 μg), v-Abl (4 μg), or Bcr-Abl a (4 μg). The v-Abl and Bcr-Abl transfections were performed in the presence of a control vector (4 μg) or Ras-N17 (4 μg), indicated by bracket. HA-GCKR immunoprecipitates were assayed by an in vitro kinase assay. HA-GCKR and Abl immunoblotting were performed by ECL. These experiments were performed two times with similar results. (B) GCKR antisense expression impairs Ras- and Bcr-Abl–induced SAPK activation. HA-SAPK immunoprecipitates from HEK 293T cells (1 × 10⁶) transfected with Bcr-Abl (4 μg) or Ras-V12 (2 μg) in the presence of pCRIII-GCKR-AS (4 or 8 μg) and HA-SAPK (1 μg) were assayed for kinase activity. A HA-immunoblot verified equivalent levels of HA-SAPK. Ras and Bcr-Abl levels were verified by immunoblotting. Data are reported as increase in SAPK activity compared with basal levels in control transfections. These experiments were performed four times with similar results. (C) A GCKR mutant impairs Bcr-Abl–induced SAPK activation. HA-SAPK immunoprecipitates from HEK 293 cells (1 × 10⁶) transfected with Bcr-Abl (4 μg) and HA-SAPK (1 μg) in the presence or absence of GCKR-T178A (2, 4, or 6 μg) were assessed for kinase activity. Data are reported as in part B. A HA-immunoblot verified equivalent levels of HA-SAPK. Bcr-Abl levels are shown. GCKR (endogenous and the transfected mutant) were detected by immunoblotting with the GCKR-specific antiserum. This experiment was performed twice with similar results.