It may seem counterintuitive that CPEB destruction during oocyte maturation is necessary for stimulating the polyadenylation and translation of mRNAs. However, the polyadenylation of CPE-containing RNAs is hierarchical; that is, the polyadenylation of different mRNAs occurs in a temporal-specific manner that is governed by the levels of CPEB. For example, the single-CPE-containing c-mos mRNA is among the first to be polyadenylated while polyadenylation of the two-CPE-containing cyclin B1 mRNA takes place at a later time after CPEB has been partially destroyed (10
). There are now thought to be a number of mRNAs whose time-dependent polyadenylation may be regulated by the number of CPEs they contain (31
). Thus, the partial destruction of CPEB, which is necessary for oocyte development, is complex and involves multiple posttranslational modifications and recognition factors. Several phosphorylation events and two distinct pools of CPEB are involved in the regulation of its destruction. During oocyte maturation, CPEB becomes phosphorylated on 6 S/T-P pairs by cdk1, although the one on S210 is particularly important for protein destruction (10
). CPEB also contains a PEST (proline, glutamic acid, serine, and threonine) domain, which often mediates the protein half-life. Within the PEST domain is a TSG (threonine, serine, and glycine) motif, which resembles the binding site for β-TrCP, the F-box protein of the SCF complex (28
). Plx1, a polo-like kinase, recognizes a CPEB cdk1 phosphorylation site and in turn phosphorylates the TSG motif, which is recognized by β-TrCP that then ubiquitinates CPEB, resulting in proteasome-mediated destruction (28
). Recent evidence also demonstrates that CPEB resides in two distinct pools in oocytes: one pool is bound to RNA while the second forms a homodimer through its RNA binding regions and thus does not bind RNA. The homodimers are destroyed very rapidly upon oocyte maturation, perhaps releasing essential factors to associate with the cytoplasmic polyadenylation complex (32
In this study, we show that Pin1 is a new CPEB-interacting protein that mediates CPEB degradation. It was surprising to find that Pin1, which selectively recognizes and isomerizes phosphorylated S/T-P bonds, associates with CPEB prior to detectable CPEB phosphorylation. However, this finding is consistent with previous observations, indicating that Pin1 might have two different modes of protein interaction: a preferred one that is phosphorylation dependent and a secondary one that is phosphorylation independent (33
). Abrahamsen et al. (33
) showed that, upon Pin1 interaction with and subsequent isomerization of protein kinase C (PKC), the conventional PKC isozymes are converted into species that are rapidly ubiquitinated following phorbol ester stimulation. Interestingly, Abrahamsen et al. also showed that the Pin1-PKC interaction is based on a hydrophobic motif in the C-terminal segment of the substrate and does not require phosphorylation, even though the interaction is strengthened when the Pin1 target sites are phosphorylated after phorbol ester treatment. This observation is consistent with our findings, indicating a two-step mode of Pin1-substrate interaction. Using a number of CPEB deletion mutants, we identified a site on CPEB corresponding to residues 48 to 183 that is necessary and sufficient for Pin1 interaction. It is worth noting that this domain (48 to 183) contains ~30% hydrophobic amino acids, although they are not organized in any detectably conserved motif. Because the three-dimensional structure of CPEB is not known, we do not know which of these amino acids are solvent exposed and might serve as a platform for Pin1 interaction. An alternative hypothesis might be that several hydrophobic amino acids are needed to build a charged region for this interaction to occur. However, two additional regions of CPEB, the RRMs and residues 211 to 290, abrogate Pin1 interaction when deleted, which suggests that the tertiary structure of CPEB or multiple contact points are important for Pin1 binding prior to CPEB phosphorylation. Furthermore, we show that, at least in vitro
, this first phase interaction is dependent on the WW domain of Pin1 (). Although surprising, this first phosphorylation-independent interaction that does not induce substrate destruction may serve another purpose. Consider that Xenopus
oocytes are exceptionally large (volume of 1 μl) and that CPEB must be rapidly destroyed after progesterone treatment. Therefore, to facilitate this rapid destruction, CPEB is “preloaded” with Pin1, where it may function as a molecular switch to coordinate subsequent cellular processes like maturation. Indeed, other proteins in the CPEB complex are subjected to cdk1-mediated phosphorylation upon progesterone stimulation and also might be subjected to Pin1-mediated regulation. Maskin, for example, is an eIF4E binding protein that prevents the assembly of the initiation complex (eIF4E-eIF4G) on the 5′ end of the mRNA, thus inhibiting translation (7
). Upon progesterone stimulation, maskin is phosphorylated by cdk1, inducing its dissociation from eIF4E, thereby ensuring the translation initiation complex assembly. CPEB destruction and maskin dissociation need to occur simultaneously to activate the translation of these mRNAs. Therefore, Pin1 assembly into this complex before progesterone treatment will ensure rapid isomerization of both CPEB and maskin for this manner.
Following the induction of oocyte maturation, nearly simultaneous CPEB phosphorylation on S/T-P pairs and Pin1 dephosphorylation of serine 71 cause the Pin1 interaction with CPEB to become CPEB phosphorylation dependent. We suspect that the affinity of Pin1 for CPEB might be higher when the substrate is phosphorylated because the ratio of immunoprecipitated CPEB to coprecipitated Pin1 is greater after maturation than before. However, this inference is somewhat speculative because we cannot perform an in vitro kinetic analysis of purified components due to the fact that it is difficult to isolate soluble recombinant CPEB.
This change in interaction sites and the isomerization of the phosphorylated residues results in CPEB ubiquitination and subsequent destruction. Accordingly, Pin1 dephosphorylation and subsequent enzymatic activation is a crucial step in CPEB ubiquitination. Although the inhibitory serine 71 phosphorylation is catalyzed by DAPK1 in mammalian cells (17
), we do not know whether the same kinase acts in Xenopus
oocytes. In addition, we find that serine 16 is not phosphorylated, but that its mutation abrogates serine 71 dephosphorylation during oocyte maturation. This result might indicate that serine 16 is necessary and acts as a docking site for recruitment of a phosphatase such as PP2A to Pin1 during maturation; such an enzyme could then dephosphorylate serine 71 for Pin1 activation. Irrespective of the kinase/phosphatase involved, an intriguing question is how does the Pin1-induced conformational change in CPEB control ubiquitination? Liou et al. (12
) suggested that cis-trans
isomerization of Pin1 substrates establishes a conformation needed for E3 ligase recognition, and possibly other regulatory proteins such as phosphatases (35
). Our data support this hypothesis and suggest that, upon Pin1 activation and simultaneously CPEB phosphorylation, Pin1 induces a CPEB conformational change that favors its interaction with the E3 ligase β-TrCP.
In maturing mouse oocytes, CPEB undergoes phosphorylation/destruction similar to that in Xenopus
). In mitotically cycling Xenopus
embryonic cells, polyadenylation increases during M phase and decreases during interphase, which also occurs in cyclin mammalian cells (39
). This observation prompted us to investigate whether CPEB undergoes an M phase destruction (like that which occurs during oocyte maturation, an M phase progression) and, if so, whether Pin1 might be involved. Indeed, demonstrates that, in synchronized HEK293T cells, CPEB is partially destroyed at M phase and interacts with Pin1 prior to its destruction (as in Xenopus
oocytes). However, it should not be assumed that maturing Xenopus
oocytes and cycling mammalian cells are equivalent vis-à-vis cell cycle progression or by how mRNA translation is regulated. For example, oocytes are arrested at a G2
-like phase and then enter M phase; there is no S phase in maturing oocytes. Xenopus
CPEB is hyperphosphorylated only during the G2
-to-M phase transition. At the same time, Pin1-CPEB interaction is evident before and after maturation. In mammalian cells, CPEB is phosphorylated at S phase and during other phases of the cell cycle. However, the CPEB-Pin1 interaction is only evident after cell cycle progression into the G2
-to-M phase. The lack of Pin1-CPEB interaction in S phase likely indicates that the phospho-CPEB residues are not involved in recruiting Pin1. Despite the differences between these two systems, in both cases, the CPEB-Pin1 interaction is evident during the G2
-to-M transition and precedes CPEB destruction. Moreover, ectopic Pin1 expression dramatically reduced the CPEB half-life while ectopic expression of a dominant negative Pin1 W34A mutant protein partially prevented CPEB destruction; mutations in the PPIase domain had no effect on Pin1's capability to promote CPEB destruction.
Fujimoto et al. (40
) previously reported that Pin1 may affect the function and degradation of a target protein, peroxisome proliferator-activated receptor γ (PPARγ), solely by the interaction between the N-terminal activation function 1 (AF-1) domain of PPARγ and the WW domain in Pin1. In this case, the proline isomerization by the PPIase domain seemed to be dispensable. This prompted us to investigate the contribution of each Pin1 domain separately on CPEB destruction. We found that, in contrast to full-length Pin1, expression of either the WW or PPIase domain had minor effects on the CPEB half-life, indicating that both domains are needed for full Pin1 activity to promote CPEB destruction. These results may also indicate that other amino acids in the PPIase domain, such as C113, which was shown to be important for Pin1 isomerization activity (40
), are needed for Pin1-mediated CPEB destruction.
Based on the functions of its interacting substrates, Liou et al. (12
) suggest that Pin1 induces the destruction or inactivation of tumor suppressors but enhances the stability of proteins involved in malignant transformation. Although we have focused most of this present work on Xenopus
oocytes, it is important to note that CPEB might be considered to be a tumor suppressor, and thus Pin1 could have important implications for oncogenic transformation via translation. That is, CPEB knockout (KO) mice, while not developing cancer spontaneously, contract papillomas much more readily than wild-type animals when challenged with a DNA damaging agent (41
). Moreover, mouse embryo fibroblasts (MEFs) lacking CPEB do not senesce as do wild-type MEFS, but instead are immortal (42
). Primary human cells depleted of CPEB also bypass senescence (41
). Because the lack of CPEB leads to reduced translation of p53 mRNA, and because reduced p53 immortalizes primary cells, it may be inferred that CPEB can also act as a tumor suppressor. Although we do not know whether Pin1 control of CPEB destruction leads to a bypass of senescence, we speculate that it might and are presently investigating this phenomenon.