DETERMINATION OF V-MOS-CATALYZED PHOSPHORYLATION SITES AND AUTOPHOSPHORYLATION SITES ON MAP KINASE KINASE BY ESI MS/

MAP kinase kinase (MAPKK), a key component of the MAP kinase cascade, is activated through phosphorylation by several protein kinases, inclu ding the oncogene v-Mos and its cellular counterpart, c-Mos. The v-Mos -catalyzed phosphorylation sites on recombinant MAPKK1 were identified by electrospray ionization mass spectrometry as S-218 and S-222, loca ted within a sequence that aligns with the T loop structure of cAMP-de pendent protein kinase; these are the same as the Raf-1 phosphorylatio n sites identified previously [Alessi, D. R., et al. (1994) EMBO J. 13 , 1610-1619]. Phosphorylation of these sites was kinetically ordered, with S-222 preferred over S-218. Intramolecular autophosphorylation of MAPKK occurred at several residues and was increased upon the stimula tion of MAPKK activity by v-Mos. Major autophosphorylation sites were residues S-298 and Y-300. Minor autophosphorylation sites included T-2 3, S-299, S-218, and either S-24 or S-25. Sequence similarities were n oted between MAPKK autophosphorylation sites and exogenous phosphoryla tion sites on MAP kinase. Phosphorylation of either S-218 or S-222 was sufficient for partial MAPKK activation by Mos, and phosphorylation o f S-222 alone was sufficient for autophosphorylation at S-298 and Y-30 0. Mass spectral analysis was also performed on MAPKK1 purified from r abbit skeletal muscle. The peptide containing S-218 and S-222 was obse rved in only a singly phosphorylated form, and the peptide containing S-298, S-299, and Y-300 was observed in multiply phosphorylated forms, suggesting that MAPKK is only partially phosphorylated within the T l oop but significantly modified in the autophosphorylation loop under p hysiological conditions.