To investigate these possibilities, we addressed whether CPEB is phosphorylated on Thr 171, the aurora phosphorylation site, during pachytene. 2002), a kinase that phosphorylates Ser 174 of CPEB, a sequence-specific RNA binding protein (Mendez et al. 2000a, 2002). Phospho-CPEB, which is bound to the 3 untranslated region (UTR) cytoplasmic polyadenylation element (CPE), then associates with CPSF (cleavage and polyadenylation specificity factor), possibly helping it stably bind the AAUAAA, a nearby element that is also necessary for polyadenylation (Dickson et al. 1999; Mendez et al. 2000b). CPSF in turn attracts poly(A) polymerase to the end of the mRNA. Another factor, maskin, mediates polyadenylation and translational activation. Maskin interacts with both CPEB and the cap binding factor eIF4E (Stebbins-Boaz et al. 1999), a configuration that excludes eIF4G from interacting with eIF4E, which is necessary to form an initiation complex on CPE-containing mRNAs. Translational repression is usually alleviated when the newly elongated poly(A) tail associates with poly(A) binding protein (PABP), a factor that helps eIF4G displace maskin from and itself bind to eIF4E, thereby initiating translation (Cao and Richter 2002). Because many of the events explained above also occur in mouse oocytes, disruption of the CPEB gene would be expected to inhibit meiotic maturation. Surprisingly, meiotic progression in CPEB knockout (KO) mice was prevented not during access into metaphase I but during the earlier pachytene to diplotene transition in prophase I (Tay and Richter 2001). Oocytes of CPEB KO animals fail to polyadenylate and translate the CPE-containing synaptonemal complex proteins (SCPs) 1 and 3 mRNAs. Consequently, synaptonemal complexes are not formed, and possibly as a result, the oocytes and ovaries are resorbed (Tay and Richter 2001). Aurora-catalyzed phosphorylation is necessary for the polyadenylation-inducing activity of CPEB during oocyte maturation (Mendez et al. 2000a; Hodgman et K114 al. 2001), and thus, this posttranslational modification would be expected to also occur during pachytene. However, the inactivity of CPEB during the prolonged diplotene stage at the end of prophase I suggests that it is silenced during this time, perhaps by dephosphorylation. To investigate these possibilities, we resolved whether CPEB is usually phosphorylated on Thr 171, the aurora phosphorylation site, during pachytene. By using a phospho-specific antibody, we show that CPEB is usually phosphorylated on this site at embryonic day 16.5 (E16.5; when most oocytes are in pachytene), but not at E14.5 (most oocytes in leptotene-zygotene) or E18.5 (most oocytes in diplotene). Even though kinase aurora is present at E16.5 and E18.5, it catalyzes CPEB phosphorylation at the earlier time period. At E18.5, the phosphatase PP1 dephosphorylates CPEB, thereby rendering it and CPE-mediated mRNA translation inactive until oocyte maturation. These data suggest a mechanism whereby polyadenylation-induced translation can be stimulated and subsequently inactivated at different phases of K114 meiosis. The results also suggest that the activities of both aurora and PP1 are tightly regulated during prophase I progression. Results and Conversation Disruption of the CPEB gene in mice results in the cessation of oocyte meiosis at pachytene and female sterility. At pachytene, homologous chromosome synapsis is usually maintained by the SC, a large multiprotein structure that is regulated at least in part at the translational level. mRNAs encoding two components of the SC, SCPs 1 and 3, contain CPEs in their 3 UTRs and are not polyadenylated or translated in CPEB KO mice (Tay and Richter 2001). The requirement of CPEB for SC formation is shown in Physique Mouse monoclonal to CD64.CT101 reacts with high affinity receptor for IgG (FcyRI), a 75 kDa type 1 trasmembrane glycoprotein. CD64 is expressed on monocytes and macrophages but not on lymphocytes or resting granulocytes. CD64 play a role in phagocytosis, and dependent cellular cytotoxicity ( ADCC). It also participates in cytokine and superoxide release 1A, in which E16.5 oocytes from wild-type and CPEB KO mice were immunostained for SCP1 and SCP3. Although both proteins were clearly obvious K114 in wild-type oocytes, neither was detected in the KO oocytes, thus establishing the necessity of CPEB for SC formation. During oocyte maturation (MI), CPEB activity is usually controlled by aurora-mediated phosphorylation (Ser 174 in egg extract, which phosphorylates not only the aurora site (Mendez et al. 2000a) but also a few other sites (by cdc2) as well (Mendez et al. 2002). For comparison, an LDAR mutant CPEB was also phosphorylated with the egg extract. The phosphorylated CPEB proteins were then incubated with buffer only, the E18.5 ovary extract, or commercial phosphatases PP1 or PP2A. When analyzed by quantitative SDS-PAGE and autoradiography, treatment only with the E18.5 ovary extract and PP1 resulted in a significant decrease in phosphorylated CPEB (Fig. 4A; CPEB stability was unaffected, cf. Fig. 4B). To determine whether specific sites of CPEB phosphorylation were affected by the phosphatases, we performed two-dimensional phospho-peptide mapping (Fig. 4A). A comparison of wild-type CPEB and the LDAR mutant CPEB phospho-peptide maps denotes the aurora-induced phospho-peptide (Fig. 4A, arrow). Although PP2A experienced no quantitative or qualitative impact on CPEB phosphorylation, treatment with either PP1 or the E18.5 ovary extract resulted in substantially reduced phosphorylation of CPEB at all sites (note that the signals.