IDENTIFICATION OF 2 SERINE RESIDUES OF MEK-1 THAT ARE DIFFERENTIALLY PHOSPHORYLATED DURING ACTIVATION BY RAF AND MEK KINASE

The signal transduction kinase MEK (mitogen-activated protein (MAP) or extracellular signal-regulated (Erk) kinase)-1 is activated via phosp horylation by MEKK (MEK kinase) and rafkinases. We show here that thes e two kinases phosphorylate rat MEK-1 exclusively on two serine codons , Ser(318) and Ser(222). Phosphorylation of MEK-1 on serines 218 and 2 22 is both necessary and sufficient for MEK-1 to be activated and able to phosphorylate MAP kinase. A mutant form of MEK-1 that replaces the se two codons with alanine cannot be activated, and one that substitut es glutamic acid residues in place of these 2 serines is active indepe ndent of activation by phosphorylation. These sites of activation occu r in a region of MEK-1 that is similar to sites of activating phosphor ylation in several other serine/threonine kinases, suggesting that thi s region may represent a conserved ''activating domain'' of many kinas es. MEKK and raf display differences in site preference between these two codons, with MEKK showing preference for the amino acid at codon 2 18 and raf phosphorylating each residue approximately equally. This si te preference might result in differences in the temporal or subsequen t substrate patterns of MEK activation that result from these two acti vation pathways.