CONSTITUTIVE MUTANT AND PUTATIVE REGULATORY SERINE PHOSPHORYLATION SITE OF MAMMALIAN MAP KINASE KINASE (MEK1)

In response to various external stimuli, MAP kinases are activated by phosphorylation on tyrosine and threonine by MAP kinase kinase (MAPKK) , a dual specificity kinase. This kinase is in turn activated via Raf- 1 and MAPKK kinase (MAPKKK). To determine regulatory phosphorylation s ites of MAPKK, we isolated a Chinese hamster cDNA, that we epitope-tag ged and expressed in fibroblasts. This hamster MAPKK (MEK1 isoform) ca n reactivate recombinant p44(mapk) when immunoprecipitated from growth factor-stimulated cells or when incubated with an active form of MAPK KK. Mutations at either of two residues that are conserved among kinas es, D208N or S222A, abolished MAPKK activity. However, only S222A/MAPK K showed a reduction in phosphorylation in response to active MAPKKK a nd exerted a dominant negative effect on the serum-stimulated endogeno us MAPKK. Finally, replacing Ser222 with Asp, a negatively charged res idue, restored MAPKK activity independently of the upstream kinase. Th ese results strongly suggest that Ser222 represents one key MAPKKK-dep endent phosphorylation site switching on and off the activity of MAPKK , an event crucial for growth control.