GCKR is a downstream target of Bcr-Abl and v-Abl. (A) Bcr-Abl, v-Abl, and Ras-V12 activate GCKR kinase activity. HA-GCKR immunoprecipitates from 1 × 106 HEK 293T cells transfected with control vector (2 μg), Bcr-Abl (2 μg), v-Abl (2 μg), Ras-V12 (2 μg), Cdc42-QL (2 μg), or Rac-QL (2 μg) in the presence of HA-GCKR (1 μg) were assayed for kinase activity using myelin basic protein (MBP) as a substrate. The amount of 32P incorporated into MBP was determined by excising the appropriate band and scintillation counting (results shown in bar graph) and the fold increases compared with control transfections. A portion of the gel was transferred to nitrocellulose and autoradiographed (AR). The same blot was reacted with an anti-HA MoAb (WB), stripped, and then with an antiphosphotyrosine MoAb (pY). Immunoreactivity was detected by enhanced chemiluminescence (ECL). Experiments were performed three times with similar results. (B) Overexpression of GCKR augments the induction of SAPK activity triggered by Bcr-Abl, v-Abl, or Ras-V12. HA-SAPK immunoprecipitates from 1 × 106 HEK 293T cells transfected with Bcr-Abl (2 μg), v-Abl (2 μg), Ras-V12 (2 μg) in the presence of pCR3-GCKR (1 μg) or a control vector (1 μg) along with HA-SAPK (1 μg) were assayed for kinase activity using GST-Jun (79) as a substrate. Bcr-Abl, v-Abl, GCKR, and Ras levels were assessed by immunoblotting (second, third, and fourth panels). The GCKR immunoblot was performed with the GCKR-specific antiserum. HA immunoblotting verified equivalent levels of HA-SAPK (bottom panel). These experiments were performed three times with similar results.
