Figure 5. CRAF S338 phosphorylation is stimulated by MEK activation.
A. MEK inhibitors inhibit CRAF S338 phosphorylation induced by EGF. Untransfected 293 cells were pre-treated with MEK inhibitor (UO126-20uM or PD98059-1uM), PAK inhibitor (IPA3-20uM), CAMKII inhibitor (KN-93-10uM) or vehicle for 2 hours before addition of EGF (100ng/ml). Cells were lysed after 5 minutes and CRAF S338 and MEK phosphorylation assessed using phosphor-specific antibodies. See also Figure S2. B. Quantification of the effect of kinase inhibitors on CRAF S338 phosphorylation. Results from 4 separate experiments are presented as mean −/+ SEM. The amount of S338 phosphorylation from the EGF treatment alone was set as 1. C. Co-expression of constitutively active MEK with CRAF induces S338 phosphorylation. Wild-type HA-CRAF was co-expressed with constitutively active MEK1 (DD) with and without dominant negative RAS (RasN17). Cells were lysed after 24 hours and immunoprecipitates prepared with antibodies to HA. CRAF S338 phosphorylation was assessed using a phosphor-specific antibody. D. MEK inhibition blocks CRAF S338 phosphorylation induced by co-expression with constitutively active MEK. Cells were prepared as described in B, except that MEK (UO126) or ERK inhibitor (ERK inhibitor II) was added 24 hours after transfection. Cells were lysed 2 hours after addition of the inhibitor. Immunoprecipitates were analyzed as in B.