CHARACTERIZATION OF MEK1 PHOSPHORYLATION BY THE V-MOS PROTEIN

Activation of MAP kinase/Erk Kinase (MEK) via direct phosphorylation b y Mos may be crucial for cellular transformation by the activated c-mo s or v-mos gene, Recent studies on a number of different protein kinas es showed that phosphorylation within a subdomain of the catalytic dom ain may represent a common mode of activation, In this regard, activat ion of MEK1 by Raf involves phosphorylation of serine residues 218 and 222, Here we show that recombinant kinase-inactive MEK1 is phosphoryl ated by v-Mos with equal efficiency at both Ser 218 and Ser 222 in vit ro, Tryptic phosphopeptide analysis of glutathione-S-transferase (GST) -MEK1 K97R and its alanine-for-serine mutants indicated that Ser 222 i s the preferred phosphorylation site. Wild-type GST-MEK1 was phosphory lated at the same sites but contained a significantly lower amount of doubly phosphorylated species then its K97R kinase-inactive mutant, Th e ratio of GST-MEK1 species phosphorylated at two serines to those pho sphorylated at one serine was similar in auto-phosphorylated and v-Mos -phosphorylated GST-MEK1, Consistent with the in vitro data, phosphope ptide mapping of MEK1 immunoprecipitated from mos transformed cells sh owed an increased amount of singly phosphorylated phosphopeptide compa red to nontransformed cells, MEK1 was found to be more highly activate d in NM3T3 cells transformed by an activated c-mos or v-mos gene than in cells growing normally in medium containing serum, Our data indicat e that Mos activates MEK1 in vitro as well as in vivo by phosphorylati ng Ser 222.