IDENTIFICATION OF THE SITES IN MAP KINASE KINASE-1 PHOSPHORYLATED BY P74(RAF-1)

Many growth factors whose receptors are protein tyrosine kinases stimu late the MAP kinase pathway by activating first the GTP-binding protei n Ras and then the protein kinase p74(raf-1). p74(raf-1) phosphorylate s andactivates MAP kinase kinase (MAPKK). To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74(raf-1). This represents the first characterization ofsites phosphorylated by this proto-oncogene product . Ser217 and Ser221 lie ina region of the catalytic domain where the a ctivating phosphorylation sites of several other protein kinases are l ocated. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosph orylation of these sites. A 'kinase-dead' MAPKK1 mutant was phosphoryl ated at the same residues as the wild-type enzyme, establishing that b oth sites are phosphorylated directly by p74(raf-1), and not by autoph osphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylate d at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation of one of these sites is rate limiting, phosphoryl ation of the second then occurring extremely rapidly. Ser217 and Ser22 1 were both phosphorylatedin vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in whi ch either Ser217 or Ser221 were changed to glutamic acid, and the find ing that inactivation of maximally activated MAPKK1 required the depho sphorylation of both serines, shows that phosphorylation of either res idue is sufficient for maximal activation.