During the past year we have made considerable progress in identifying downstream effectors of the Raf-1 signalling pathway. We have identified the first Raf-1 substrate, mitogen-activated protein kinase-kinase (MAPKK). We have recapitulated the Raf-induced kinase cascade in vitro using purified components (i.e., activated Raf-1 overexpressed from baculovirus vectors in SF9 cells phosphorylated and activated purified MAPKK which then phosphorylated and activated p44-MAP kinase). We have now extended these experiments and determined the Raf-specific phosphorylation site on MAPKK. To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74raf-1. This represents the first characterization of sites phosphorylated by this proto-oncogene product. Ser217 and Ser221 lie in a region of the catalytic domain where the activating phosphorylation sites of several other protein kinases are located. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosphorylation of these sites. A "kinase-dead" MAPKK1 mutant was phosphorylated at the same residues as the wild-type enzyme, establishing that both sites are phosphorylated directly by p74raf-1 and not by autophosphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylated at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation by p74raf-1 was low, indicating that phosphorylation of one of these sites is rate-limiting, phosphorylation of the second then occurring extremely rapidly. Ser217 and Ser221 were both phosphorylated in vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in which either Ser217 or Ser221 were changed to glutamic acid, and the finding that inactivation of maximally activated MAPKK1 required the dephosphorylation of both serines, shows that phosphorylation of either residue is sufficient for maximal activation.