Regulated CPEB phosphorylation during meiotic progression suggests a mechanism for temporal control of maternal mRNA translation - PubMed (original) (raw)

Regulated CPEB phosphorylation during meiotic progression suggests a mechanism for temporal control of maternal mRNA translation

Joyce Tay et al. Genes Dev. 2003.

Abstract

CPEB is an mRNA-binding protein that stimulates polyadenylation-induced translation of maternal mRNA once it is phosphorylated on Ser 174 or Thr 171 (species-dependent). Disruption of the CPEB gene in mice causes an arrest of oogenesis at embryonic day 16.5 (E16.5), when most oocytes are in pachytene of prophase I. Here, we show that CPEB undergoes Thr 171 phosphorylation at E16.5, but dephosphorylation at the E18.5, when most oocytes are entering diplotene. Although phosphorylation is mediated by the kinase aurora, the dephosphorylation is due to the phosphatase PP1. The temporal control of CPEB phosphorylation suggests a mechanism in which CPE-containing mRNA translation is stimulated at pachytene and metaphase I.

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Figures

Figure 1.

Figure 1.

CPEB activity during prophase I. (A) E16.5 oocyte chromatin from wild-type and CPEB knockout animals was immunostained for SCP1 and SCP3. The filaments in the wild-type oocytes are synaptonemal complexes, which are absent in the knockout oocytes. (B) Extracts prepared from wild-type E16.5 and E18.5 ovaries were supplemented with recombinant CPEB containing a wild-type or mutated aurora phosphorylation site, together with γ32P-ATP. After incubation, CPEB was gel isolated, digested with trypsin, and analyzed by two-dimensional phospho-peptide mapping. The horizontal arrow denotes a reference peptide that is present in all panels. The white vertical arrow denotes the phospho-peptide containing the aurora phosphorylation site, which, although present when the E16.5 ovary extract was used as the kinase source (cf. wild-type and mutant CPEB proteins), was absent when the E18.5 ovary extract was the kinase source.

Figure 2.

Figure 2.

CPEB phosphorylation is regulated during prophase I progression. (A) CPEB mRNA was translated in a reticulocyte lysate, some of which was then supplemented with recombinant aurora. The proteins were then Western blotted and probed with general CPEB antibody or phospho-specific antibody. Some lysate that was not primed with any RNA was supplemented with aurora and then probed with both CPEB antibodies. GV and GVBD oocytes were also probed with the general and phospho-specific CPEB antibodies. Although only nonphosphorylated CPEB is present in GV stage oocytes (Tay et al. 2000; Hodgman et al. 2001), aurora-phosphorylated CPEB is present after GVBD. Some CPEB is additionally phosphorylated by cdc2 at this time, which slows its electrophoretic mobility (Mendez et al. 2000; Tay et al. 2000). (B) Ovaries from E16.5 wild-type and CPEB knockout animals were fixed, embedded, sectioned, and immunostained with general CPEB antibody or the phospho-specific antibody. The sections were also immunostained for GCNA1, a marker for oocyte nuclei. Note that although both the general and phospho-specific antibodies were immunoreactive with the wild-type ovaries, there was no signal with the knockout ovaries. Ovaries from wild-type E14.5, E16.5, and E18.5 ovaries were immunostained with the same three antibodies noted above. Although general CPEB immunoreactivity was detected at all three stages, the phospho-specific antibody was immunoreactive only with the E16.5 ovary section.

Figure 3.

Figure 3.

Aurora is present in oocytes during prophase I progression. Wild-type E14.5, E16.5, and E18.5 ovary sections were probed with aurora antibody or preimmune serum, as well as GNCA1 antibody. Compared with preimmune serum, the aurora antibody yielded an immunoreactive signal significantly above background at all stages. To confirm this result, protein wild-type E16.5 and E18.5 ovaries, as well as GV stage oocytes, were Western blotted and probed with aurora antibody.

Figure 4.

Figure 4.

Phosphatase activity in E18.5 ovaries. (A) Recombinant histidine-tagged CPEB, containing a wild-type or mutated (LDAR) aurora phosphorylation site, was incubated in a Xenopus egg extract together with γ32P-ATP. The CPEB proteins were then isolated on a nickel-agarose column and incubated with buffer alone, phosphatase PP1, phosphatase PP2A, or an extract derived from E18.5 wild-type ovaries. When analyzed by quantitative SDS-PAGE, only PP1 and the E18.5 ovary extract had the capability of dephosphorylating CPEB. All phosphorylated CPEB proteins were subsequently analyzed by qualitative two-dimensional phospho-peptide mapping. In each panel, the vertical arrow denotes the aurora-catalyzed phospho-peptide, which was absent from the mutant CPEB (white arrow). The signals from the panels denoted E18.5 ovary and PP1 were enhanced to show the individual phospho-peptides, which otherwise would not be detected at the same exposure level as in the other three panels. (B) Phospho-CPEB was added to E16.5 and E18.5 ovary extracts, as well as E18.5 ovary extracts that were preincubated with I-2, a specific inhibitor of PP1. The degree of CPEB dephosphorylation was then monitored by SDS-PAGE and autoradiography, as well as two-dimensional phospho-peptide mapping (arrows are as noted above). Some of the extract was also Western blotted and probed for CPEB.

Figure 5.

Figure 5.

Model for CPEB activity during meiosis. Primordial germ cells (PGCs), which divide mitotically, enter meiosis in the genital ridge and progress to pachytene, at which time CPEB is phosphorylated by aurora and activated. Active CPEB then induces the polyadenylation and translation of synaptonemal complex proteins 1 and 3 (SCPs 1 and 3) mRNAs, which are necessary for the formation of the synaptonemal complex. As meiosis progresses to diplotene, PP1 dephosphorylates and inactivates CPEB, thereby preventing the translation of CPE-containing mRNAs during this period. As oocytes subsequently mature, CPEB is again phosphorylated and activated by aurora, which stimulates the translation of mos, cyclin B, and other mRNAs.

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