Figure 3.

Ser-621 in the 14-3-3 binding motif of CRAF is redundantly phosphorylated by AMPK and CRAF itself, but not by PKA, which is essential for 14-3-3 association. The WT or the kinase-dead mutant (A373F) of CRAF (aa 322–648) was expressed in 293T cells and treated with the indicated inhibitors (10 μm H89, 5 μm Compound C) for 4 h before lysis for immunoprecipitation. A and B, the phospho–Ser-621 of CRAF and its mutants was detected by immunoblot (A) and quantified using ImageJ (B) (n = 3; ****, p < 0.0001). The activity of PKA and AMPK was probed as phospho-CREB or phospho-ACC in whole-cell lysates, respectively (A, bottom panel). C, the association of CRAF or its mutants with 14-3-3 in A was examined by immunoprecipitation and immunoblot. D, the signals of 14-3-3 and CRAF in immunoprecipitants were quantified by ImageJ, and their ration was calculated (D) (n = 3; ****, p < 0.0001). All images are representative of at least three independent experiments.