EVIDENCE OF A FUNCTIONAL INTERACTION BETWEEN SERINE-3 AND SERINE-25 MOS PHOSPHORYLATION SITES - A DOMINANT INHIBITORY ROLE OF SERINE-25 PHOSPHORYLATION ON MOS PROTEIN

Recently, we identified the major in vivo phosphorylation site on v-Mo s as Ser-56, which is phosphorylated by cyclic AMP dependent protein k inase (PKA). Others have shown that c-Mos phosphorylation at Ser-3 (eq uivalent to Ser-34 in v-Mos) is important for the interaction of c-Mos with its substrate MEK and for its stability and cytostatic factor ac tivity in eggs. To investigate the role of Ser-56 phosphorylation, we generated site-directed mutants of v-Mos that would mimic phosphorylat ion in terms of charge at positions 56 and 34. After mutating serine ( S) residues with alanine (A) or glutamic acid (E) in different combina tions, various v-Mos mutants were expressed in a rabbit reticulocyte l ysate in vitro translation system and in COS-1 or MIH/3T3 cells. The e ffect of mutations on Mos function was evaluated by in vitro protein k inase assays and by the ability of Mos to cause neoplastic transformat ion of NIH/3T3 cells. The S56E but not the S56A mutation inhibited v-M os kinase activity suggesting that Ser-56 phosphorylation has an inhib itory role. As predicted from Xenopus c-Mos studies, S34A but not S34E mutation inhibited v-Mos activity. Studies with the double mutants sh owed that the S56E mutation but not S56A mutation inhibited v-Mos kina se activity of both S34A and S34E mutants. Interestingly, the S56A mut ation blocked the inhibitory effect of the S34A mutation on v-Mos kina se suggesting that in c-Mos the corresponding serine (Ser-25) can infl uence the regulation of c-Mos by Ser-3. Results showing inhibition of v Mos kinase activity of the S34E mutant by the S56E mutation is signi ficant as it suggests that doubly phosphorylated Mos at these residues would be inactive. Because residues corresponding to both v-Mos Ser-3 4 and Ser-56 are evolutionarily conserved in c-Mos, the kinase activit y of c-Mos during meiosis may also be regulated in the same manner as v-Mos kinase activity.