FIG. 12.

Phosphorylation of c-Raf kinase BXB mutant protein by G-kinase. The BXB mutant protein, which is lacking amino acids 26 to 303 of c-Raf kinase, was purified from bacteria as a GST fusion protein and incubated with G-kinase and [γ-³²PO₄]ATP as described in the legend to Fig. 10. Reaction products were analyzed by SDS-PAGE, and the gel was stained with Coomassie blue R250 (left panels) and exposed to X-ray film (right panels); the BXB protein has an approximate mass of 60 kDa. (A) Lane 1, BXB protein; lane 2, BXB protein with Ser 619 mutated to alanine [BXB(S619-A)]; lane 3, BXB protein with Ser 621 mutated to alanine [BSB(S621-A)]. (B) For direct comparison, similar amounts of GST(1-149)Raf (lane 1) and BXB protein (lane 3) were incubated with G-kinase as described above. Lane 2, G-kinase (GK) only. As in Fig. 10, G-kinase added to the reaction can be seen both on the Coomassie blue stain and on the autoradiograph.