Evidence for an important role of serine 16 and its phosphorylation in thestabilization of c-Mos

The c-Mos serine/threonine protein kinase is an essential component of cyto static factor (CSF), which is required for metaphase Il arrest of eggs in v ertebrates. Previously, we showed that c-Mos residue Ser-16 is phosphorylat ed in the ts110 Mo-MuSV-encoded Gag-Mos fusion protein, Here we provide evi dence that Mos is phosphorylated at Ser-16 in transfected COS-1 cells. To i nvestigate the role of this phosphorylation, Ser-16 was substituted with al anine or glutamic acid in full-length v-Mos (an Env-Mos fusion protein that contains 31 additional amino acids at the amino terminus of c-Mos), its mo use c-Mos equivalent version (v-Mos residues 32-374, hereafter referred to as Mos), and mouse c-Mos, Constructs expressing mutant versions of Mos,were transfected into COS-l and NIH3T3 cells in a transient and stable manner, respectively, Synthesis and proteolysis of Mos were evaluated by pulse-chas e analysis of S-35-methionine-labeled proteins. Our findings indicate that the S16A mutant of Mos was highly unstable. It accumulated to approximately 10% of the level of wild-type Mos or its S16E mutant. In addition, the S16 A mutation but not the S16E mutation inhibited Mos interaction with a cellu lar protein, p35, suggesting that phosphorylation at Ser-16 may promote Ril es interaction with p35, As expected from its destabilizing effect, the S16 A mutation caused a dramatic decrease in the cellular transforming activity of Mos (determined by soft-agar colony-formation assay with the stably tra nsfected NIH3T3 cells), which is known to correlate with its CSF function. Efficient ubiquitin-mediated proteolysis of c-Mos requires proline as the s econd residue from the amino-terminus. In contrast to Mos, neither the stab ility nor protein kinase activity of v-Mos (in which c-Mos residue Pro-2 be comes Pro-33) was affected by the S16A mutation. To provide further proof t hat, similar to c-Mos, the S16A mutant is recognized by the proteolysis sys tem through Pro-2, we show that the effect of the S16A mutation is reversed by the Pro-2-Ala mutation. Thus, our results indicate that Ser-16 has an i mportant role in the regulation of c-Mos and that phosphorylation at Ser-16 mag inhibit proteolysis of c-Mos.