Fig. 3. Phosphorylation of CPEB. (A) A selected alignment of vertebrate CPEB proteins containing the two conserved LDS/TR motifs. Asterisks denote consensus IAK1/Eg2 phosphorylation sites. (B) Sequences of wild-type and mutant IAK1/Eg2 substrate peptides derived from mouse CPEB. The C-terminal cysteine is not found in CPEB, but was added to the peptide so it could be coupled to ovalbumin. (C) Phosphorylation of substrate peptide during mouse oocyte maturation. The peptides noted above were coupled to ovalbumin and added to extracts prepared from GV, MI and MII stage mouse oocytes that were supplemented with [-³²P]ATP. After incubation, the radioactive products were analyzed by SDS-PAGE. (D) 2D phosphopeptide mapping of CPEB. Escherichia coli-expressed Xenopus CPEB was added to extracts prepared from GV or MI stage mouse oocytes that were supplemented with [-³²P]ATP. After incubation, the products were resolved by SDS-PAGE and CPEB was electroblotted onto PVDF membrane. CPEB was then digested with TPCK-treated trypsin and the resulting phosphopeptides were resolved in two dimensions. The asterisk denotes the origin. In the top panel, ‘X’ denotes an area of the chromatogram that is unoccupied by a phosphopeptide. A new phosphopeptide appears in this region when MI oocytes are used as the kinase source. Purified recombinant CPEB was also phosphorylated in vitro by pure baculovirus-expressed Eg2. The resulting phosphopeptide map reveals a single predominant spot (arrow) corresponding to the first LDSR motif in CPEB, and was identified by amino acid sequencing (Mendez et al., 2000a). A mix of the two tryptic digests (i.e. kinase sources were MI oocytes and recombinant Eg2) before the 2D mapping reveals co-migration of the LDSR phosphopeptides (arrow).