p Kip is a critical component of the
p27Kip1 is a critical component of the cell cycle machinery . As an inhibitor of cyclin E-Cdk2, p27Kip1 plays a pivotal role in controlling cell proliferation, S-phase entry, and G1-phase exit during development and tumorigenesis . p27Kip1 is predominantly regulated via posttranslational modifications (primarily phosphorylation), which determine protein stability and subcellular localization. Ser10 is one of the phosphorylation sites on p27Kip1. In contrast to Thr157 and Thr198, this modification is necessary for the CRM1-mediated nuclear export of proteins. Ser10 phosphorylation is required for the binding of p27Kip1 to CRM1. The p27 mutant, S10A, displayed reduced CRM1 association, and p27Kip1 was retained in the nucleus. In contract, the phosphor-mimetic p27Kip1 mutants, in which Ser10 was substituted with aspartic Adox (S10D), displayed markedly enhanced interactions with CRM1, and the expression of p27Kip1 was increased in the cytoplasm . Hence, point mutations in Ser10 and mutations in basic residues within the nuclear export sequence also impair p27Kip1 nuclear export, suggesting that export is mediated by the interaction with CRM1. However, we did not examine the interaction of CRM1 and pSer10p27 in our current observations. We will investigate whether this preferential interaction with S10D occurs in SKOV3 ovarian cancer cells in a future study.
CRM1 mediates the nuclear export of proteins and messenger RNAs, and is thus an important regulator of the subcellular distribution of key molecules. An increasingly large number of cancer-associated proteins that shuttle into and out of the nucleus, such as tumor suppressor and oncogenic proteins, require CRM1 for their nuclear exit . Interestingly, mutations and/or dysregulation of such proteins in cancer cells can lead to aberrantly high levels of expression within the cytosol, which prevents them from performing their normal functions within the nucleus . In addition to studies that suggested a fundamental role for CRM1 in the regulation of mitosis, Noske et al. reported that the expression of this protein is a prognostic factor in human EOC . However, more data should be obtained in other cancers, such as pancreatic cancers , osteosarcomas , gliomas , and ovarian cancers , in order to further evaluate the prognostic value of CRM1 expression.
Recent research has revealed that increasing the levels of CRM1 increases p27Kip1 degradation, indicating that CRM1 controls the activity of p27Kip1 by facilitating its degradation . This finding suggested that CRM1 may act as a negative regulator of important cell cycle control proteins by targeting them for degradation. In a further investigation of the apparent role of CRM1-mediated p27Kip1 down-regulation in ovarian carcinoma, we transfected SKOV3 cells with the pEGFP-N3-WT/S10A/S10D expression vector and found that p27Kip1 levels were significantly reduced after S10D gene transfer. We also observed that the S10D overexpression-mediated downregulation of p27Kip1 was inhibited in cells that had been treated with MG132, indicating that CRM1 promoted p27Kip1 degradation through proteolysis and was sensitive to 26S proteasome inhibitors (Fig. 5B). Furthermore, using subcellular fractionation, we demonstrated that S10D overexpression resulted in p27Kip1 cytoplasmic accumulation, which was consistent with the results of CRM1 knockdown using siRNAs.
In summary, we found CRM1 to be frequently expressed and positively correlated with p27Kip1 subcellular localization in EOC; this association appeared to be due to Ser10 phosphorylation of p27Kip1. Changes in CRM1 and pSer10p27 expression levels may contribute to cell cycle deregulation, and may precede EOC progression.
Conflict of Interest Statement
As an important member of the importin β family, Crm1 has been shown to transport some RNAs and a number of NES containing proteins from the nucleus to the cytoplasm in a RanGTP-regulated way , . Until now, more than 75 proteins have been found to be mediated by Crm1, such as PKI, MAPKK and Cyclin B . All of these proteins contain at least one NES sequence consisting of leucines and other hydrophobic residues which is responsible for the nuclear export process .