Role of cdc2 kinase phosphorylation and conserved N-terminal proteolysis motifs in cytoplasmic polyadenylation-element-binding protein (CPEB) complex dissociation and degradation.
Cytoplasmic polyadenylation-element-binding protein (CPEB) is a well-characterized and important regulator of translation of maternal mRNA in early development in organisms ranging from worms, flies and clams to frogs and mice. Previous studies provided evidence that clam and Xenopus CPEB are hyperphosphorylated at germinal vesicle breakdown (GVBD) by cdc2 kinase, and degraded shortly after. To examine the conserved features of CPEB that mediate its modification during meiotic maturation, we microinjected mRNA encoding wild-type and mutated clam CPEB into Xenopus oocytes that were subsequently allowed to mature with progesterone. We observed that (i) ectopically expressed clam CPEB is phosphorylated at GVBD and subsequently degraded, mirroring the fate of the endogenous Xenopus CPEB protein, (ii) mutation of nine Ser/Thr Pro-directed kinase sites prevents phosphorylation and degradation and (iii) deletion of the PEST box, and to a lesser extent of the putative cyclin destruction box, generates a stable and phosphorylated version of CPEB. We conclude that phosphorylation of both consensus and non-consensus sites by cdc2 kinase targets clam CPEB for PEST-mediated destruction. We also show that phosphorylation of CPEB mediates its dissociation from ribonucleoprotein complexes, prior to degradation. Our findings reinforce results obtained in Xenopus, and have implications for CPEB from other invertebrates including Drosophila, Caenorhabditis elegans and Aplysia, which lack PEST boxes.
Thom
George
G
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Minshall
Nicola
N
Git
Anna
A
Argasinska
Joanna
J
Standart
Nancy
N
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase; D006868:Hydrolysis; D008845:Microinjections; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins; D039221:mRNA Cleavage and Polyadenylation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C490388:Cpeb1 protein, Xenopus; D014157:Transcription Factors; D029867:Xenopus Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D016203:CDC2 Protein Kinase