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 translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs which are translationally dormant or masked until meiotic maturation. Fertilisation of the oocyte leads to rapid polysomal 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 polyadenylation element binding protein) family, functions as a translational masking factor in oocytes and as a polyadenylation factor in fertilised eggs. In meiotically maturing clam oocytes, p82/CPEB is rapidly phosphorylated on multiple residues to a 92-kDa apparent size, prior to its degradation during the first cell cleavage. Here we examine the protein kinase(s) that phosphorylates clam p82/CPEB using a clam oocyte activation cell-free system that responds to elevated pH, mirroring the pH rise that accompanies fertilisation. We show that p82/CPEB phosphorylation requires Ca2+ (<100 microM) in addition to raised pH. Examination of the calcium dependency combined with the use 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 transient in vitro, as in vivo. While cdc2 kinase plays a role in the maintenance 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 kinase initially phosphorylates clam p82/CPEB, at a minor subset of sites that 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 clam p82/CPEB. The possible roles of phosphorylation for the function and regulation of p82/CPEB are discussed.
Katsu
Y
Y
Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, England.
Minshall
N
N
Nagahama
Y
Y
Standart
N
N
D000818:Animals; D049872:Bivalvia / Q000378:metabolism*; D016203:CDC2 Protein Kinase / Q000502:physiology*; D002122:Calcium Chloride / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002474:Cell-Free System; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D066298:In Vitro Techniques; D015636:Magnesium Chloride / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D011233:Precipitin Tests; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013997:Time Factors
D016601:RNA-Binding Proteins; C117068:p82 protein, clam; D015636:Magnesium Chloride; D004533:Egtazic Acid; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016203:CDC2 Protein Kinase; D002122:Calcium Chloride