Figure 4: CRAF pS338 regulates the formation of a complex between CRAF and the DNA repair kinase CHK2.

(a) Stably transfected U87 cells expressing wild-type CRAF (WT) or the CRAF kinase-dead, phospho-mimetic double mutant (S338D/K375M) were exposed to 6 Gy and immunoblotted with indicated antibodies. Data are representative of three independent experiments. (b) HCT-116 cells were exposed to 6 Gy. Lysates were immunoprecipitated for CHK2 pT68 and blotted for CRAF. For reciprocal pull down, lysates were immunoprecipitated and CRAF blotted for CHK2. Total cell lysates were immunoblotted with indicated antibodies. Data are representative of three independent experiments. (c) HCT-116 cells were treated with KG5 and lysates were immunoprecipitated for CRAF or CHK2 and immunoblotted with indicated antibodies. Total cell lysates were immunoblotted with indicated antibodies. Data are representative of three independent experiments. (d) Expression of CHK2 was silenced using siRNA in HCT-116 cells expressing wild-type (WT) CRAF or the phospho-mimetic CRAF S338D mutant. Cells were stained for γH2AX, and the number of γH2AX foci per cell was counted as a measure of DNA double strand breaks. Mean±s.e.m, *P=0.02 from two-sided t-test comparing S338D+si-CTRL versus S338D+si-CHK2 (n=3 fields each group). Data shown are representative of two different siRNAs for CHK2, for two independent experiments. (e) Schematic represents PAK1-mediated activation of CRAF pS338 in response to ionizing radiation and its recruitment of CHK2 leading to CHK2 activation that supports DNA repair and radioresistance.