PHOSPHORYLATION OF CONSERVED SERINE RESIDUES DOES NOT REGULATE THE ABILITY OF MOSXE PROTEIN-KINASE TO INDUCE OOCYTE MATURATION OR FUNCTION AS CYTOSTATIC FACTOR

Expression of the mos(xe) protein kinase is required for the normal meiotic maturation of Xenopus oocytes and overexpression induces maturation in the absence of other stimuli. In addition, mos(xe) functions as a component of cytostatic factor (CSF), an activity responsible for arrest of the mature egg at metaphase II. After microinjection of Xenopus oocytes with in vitro synthesized RNA encoding either wild-type mos(xe) or kinase-inactive mos(xe(R9O), both proteins are phosphorylated exclusively on serine residues and exhibit essentially identical chymotryptic maps. Since the phosphorylated kinase-inactive mos(Xe(R90) protein was recovered from resting oocytes that have not yet begun to translate endogenous mos(xe), this indicates that the major phosphopeptides of mos(xe(R90) are phosphorylated by a preexisting protein kinase present in resting oocytes, and are not the result of autophosphorylation. The results presented here also indicate that the mos(xe) protein does not undergo significant phosphorylation at unique sites during oocyte maturation. If the biological activity of mos(xe) were regulated by phosphorylation, a site of regulatory phosphorylation would most likely be conserved among mos proteins of different species. Site-directed mutagenesis was used to construct 13 individual serine --> alanine mutations at conserved residues (3, 16, 18, 25, 26, 57, 71, 76, 102, 105, 127, 211, and 258). These 13 mutants were analyzed for their abilities to induce oocyte maturation and to function as CSF. Results obtained with the mos(xe(A105) mutant revealed that serine-105 is required for both maturation induction and CSF activity, even though serine-105 does not represent a major site of phosphorylation. All of the remaining serine --> alanine mos(xe) mutants induced oocyte maturation and exhibited CSF activity comparable with the wild type. These results demonstrate that none of the conserved serines examined in this study function as regulatory phosphorylation sites for these biological activities. Peptide mapping of the remaining mos(xe) mutants identified serine-3 as a major phosphorylation site in vivo, which is contained within the chymotryptic peptide MPSPIPVERF.