Fig. 1. Changes in Raf-1 phosphorylation during activation. Serum-starved NIH 3T3 cells were labelled with 0.5 mCi/ml [³²P]ortho phosphoric acid for 6 h and stimulated with 20% FCS for 5 min. Endogenous Raf-1 was immunoprecipitated, and (A) examined for kinase activity. (B) Another Raf-1 aliquot was digested with trypsin and subjected to two-dimensional phosphopeptide mapping. Equal amounts of Raf were loaded, as evident from the western blot shown in (A). Phosphorylation sites were assigned by comparison with the respective Raf-1 point mutants. The peptide containing Ser621 produces two spots due to partial digestion; the minor spot is labelled by an asterisk. Spots were quantified by phosphoimager, and the changes relative to serum-starved cells are shown. (C) Dephosphoryl ation of endogenous Raf-1 during serum and mitogen stimulation. COS cells were treated with 10% FCS or 100 ng/ml TPA for the indicated times. Raf-1 was immunoprecipitated from the cells with an anti-Raf-1 antibody. The immunoprecipitates were then blotted with antibodies against Raf-1 and phosphoserines 259 and 338. (D) Time course of Raf-1 kinase activation. COS cells were transfected with myc-Raf, serum starved and stimulated with 20 ng/ml EGF for the indicated times. myc-Raf-1 was immunoprecipitated with the 9E10 monoclonal antibody, and catalytic activity was measured using a two-step kinase assay. (E) Myc-Raf immunoprecipitates were blotted with phospho specific antisera against phosphoserines 259 and 338. The blots were quantified by laser densitometry shown in the right hand panels.