Ca2+ is required for phosphorylation of clam p82/CPEB in vitro: Implications for dual and independent roles of MAP and Cdc2 kinases

During early development gene expression is controlled principally at the t ranslational level. Oocytes of the surf clam Spisula solidissima contain la rge stockpiles of maternal mRNAs which are translationally dormant or maske d until meiotic maturation. Fertilisation of the oocyte leads to rapid poly somal recruitment of the abundant cyclin and ribonucleotide reductase mRNAs at about the time they undergo cytoplasmic polyadenylation. Clam p82, a 3' UTR RNA-binding protein, and a member of the CPEB (cytoplasmic polyadenyla tion element binding protein) family, functions as a translational masking factor in oocytes and as a polyadenylation factor in fertilised eggs. In me iotically maturing clam oocytes, p82/CPEB is rapidly phosphorylated on mult iple residues to a 92-kDa apparent size, prior to its degradation during th e first cell cleavage. Here we examine the protein kinase(s) that phosphory lates clam p82/CPEB using a clam oocyte activation cell-free system that re sponds to elevated pH, mirroring the pH rise that accompanies fertilisation . We show that p82/CPEB phosphorylation requires Ca2+ (<100 mu M) in additi on to raised pH. Examination of the calcium dependency combined with the us e of specific inhibitors implicates the combined and independent actions of cdc2 and MAP kinases in p82/CPEB phosphorylation. Calcium is necessary for both the activation and the maintenance of MAP kinase, whose activity is t ransient in vitro, as in vivo. While cdc2 kinase plays a role in the mainte nance of MAP kinase activity, it is not required for the activation of MAP kinase. We propose a model of clam p82/CPEB phosphorylation in which MAP ki nase initially phosphorylates clam p82/CPEB, at a minor subset of sites tha t does not alter its migration, and cdc2 kinase is necessary for the second wave of phosphorylation that results in the large mobility size shift of c lam p82/CPEB. The possible roles of phosphorylation for the function and re gulation of p82/CPEB are discussed. (C) 1999 Academic Press.