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CPEB phosphorylation and cytoplasmic polyadenylation are catalyzed by the kinase IAK1/Eg2 in maturing mouse oocytes

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, USA

View larger version (41K): [in a new window]   Fig. 1. Polyadenylation in maturing mouse oocytes. Radiolabeled 3' UTR of cyclin B1 mRNA, which contains a CPE and polyadenylation hexanucleotide (top) was injected into GV stage oocytes that were then allowed to mature for 16.5 hours (center), or for 3 (GV intact) and 6 (GVBD) hours (right). The RNA was extracted and assessed by denaturing PAGE and phosphorimaging. The approximate size of the newly acquired poly(A) tail is indicted. As a control, a mutated cyclin B1 3' UTR that did not contain a CPE was also injected and the oocytes were analyzed after 16.5 hours of culture. For this experiment, the left panel represents 21 oocytes (-CPE), the middle panel 87 oocytes (+CPE), and the right panel 31 (0 hours), 27 (3 hours) and 25 (6 hours) oocytes. All analyses were performed twice.

View larger version (66K): [in a new window]   Fig. 2. Immunohistochemistry of polyadenylation/translation factors in maturing mouse oocytes. Oocytes with an intact GV, or those cultured for 6 (usually at MI), or 16.5 (MII) hours were immunostained with antibodies directed against CPEB, IAK1 (Eg2), the 100 kDa subunit of CPSF, PAP and maskin (the signals for these proteins are red; IAKI is in green). The oocytes were also counterstained with DAPI (blue). Each immunostaining procedure was performed on at least 30 oocytes. Western blots of GV stage oocyte protein were probed with the same antibodies noted above; in each case, only a single immunoreactive species was observed. No signal was observed in either the immunofluorescence experiment or in the western blot experiment when preimmune serum was used as the source for the first antibody (data not shown).

View larger version (50K): [in a new window]   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).

View larger version (10K): [in a new window]   Fig. 4. IAK1/Eg2 blocking peptide inhibits first polar body extrusion. Oocytes were injected with the peptides noted in Fig. 3B, except that in this case they were not coupled to ovalbumin. The rate of polar body extrusion during maturation was assessed. While the wild-type peptide only moderately inhibited polar body extrusion, the mutant, alanine-containing peptide was more effective in doing so. Although both peptides bind Eg2, the binding to the mutant peptide is essentially irreversible, and thus it is a more effective competitive inhibitor of kinase activity. In this analysis, 29 oocytes were assessed for the wild-type peptide, 18 oocytes for the mutant peptide and 47 oocytes that were not injected. The experiment was performed twice.

View larger version (60K): [in a new window]   Fig. 5. CPEB is required for cytoplasmic polyadenylation. GV stage oocytes were injected with a mixture of radiolabeled cyclin B1 3' UTR and mRNA encoding either wild-type CPEB or a mutant CPEB (N) that lacks the N-terminal 146 amino acids. Both proteins contain the two LDSR motifs, as well as the RNA recognition motifs (RRM) and zinc finger (ZF) that are necessary for RNA binding (Hake et al., 1998). The RNA was then extracted from maturing oocytes and the polyadenylation of the cyclin B1 3' UTR was analyzed by denaturing PAGE and phosphorimaging.