FIG. 1.

PKB-independent phosphorylation of AFX by the Ras-Ral pathway. (A) In vivo-phosphorylated HA-AFX was immunoprecipitated from ³²P-labeled A14 cells which were left untreated, stimulated with insulin for 30 min, pretreated with LY294002 for 10 min prior to insulin stimulation, or cotransfected with 2 μg of pMT2-HA-Ral-N28. Following exposure to film, bands were cut out of the blot and processed for two-dimensional phosphopeptide mapping using trypsin as the digestive enzyme. Positions of peptides 1, 2, and 4 are indicated by number. (B) ³²P-labeled HA-AFX was isolated from transfected A14 cells coexpressing either pSVE-Ras-V12 or pcDNA3-Myc-Rlf-CAAX and analyzed as described for panel A.