Overall our results indicate that the cutaneous HPV

Overall, our results indicate that the cutaneous HPV8 E7 oncoprotein has a leucine rich NES (76IRTFQELLF84) within the zinc-binding domain that interacts with CRM1 export receptor and mediates its nuclear export. We have previously shown that the mucosal low risk HPV11 E7 has a leucine-rich NES (76IRQLQDLLL84) within the zinc-binding domain that interacts with CRM1 (McKee et al., 2013). We also found that the zinc-binding domain of HPV16 E7 containing the leucine-rich NES interacts with CRM1 (data not shown). Another cutaneous beta genus HPV5 E7 protein has also a potential leucine-rich NES (IRAFQQLL) within its zinc-binding domain that interacts with CRM1 export receptor (data not shown). This suggests that both the mucosal and cutaneous HPV E7 oncoproteins have evolved leucine-rich NESs that would mediate their nuclear export via a CRM1 pathway. Overall, this work and a previous study (Onder and Moroianu, 2014) show that cutaneous HPV8 E7 shuttles between the nucleus and the Alda 1 and localizes predominantly in the nucleus with low levels in the cytoplasm, reflecting the contribution to the HPV8 E7 localization of the NLS and NES, respectively. The mucosal HPV16 E7 and HPV11 E7 oncoproteins also shuttle between the nucleus and the cytoplasm and this is in agreement with the fact that these HPV E7 oncoproteins have cellular targets in both cellular compartments.
Materials and methods
Acknowledgments We thank Dr. Jorgen Kjems for the generous gift of GST-CRM1 plasmid. We thank Paul Boucher for help with purification of GST-fusion proteins. This work was supported by a grant from the National Institutes of Health (R01 CA94898) to Junona Moroianu.
Introduction Ovarian cancer (OC) is one of the leading causes of death from gynecological malignancies [1], and its incidence has recently increased in China. Over 80% of ovarian cancers are of epithelial origin (EOC), are highly invasive, respond poorly to therapies, and are usually detected at advanced stages, resulting in poor prognoses [2]. Early EOC usually has no obvious symptoms. In general, EOC is diagnosed when it is already in the late or metastatic stages. At present, no sufficiently accurate screening test has been proven effective in the early detection of EOC. An understanding of the biological mechanisms that regulate the progression of EOC is therefore critical for devising new treatment options and improving patient survival. The importance of G1–S phase progression to the formation of malignant tumors has been highlighted by the high incidence of aberrations in the genes involved in this progression in a wide variety of tumors. p27Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors (CKI). Its role is to bind to and inhibit cyclin/cyclin-dependent kinase complexes, thereby negatively regulating cell cycle progression. Numerous studies have shown that p27Kip1 is a tumor suppressor gene, the loss of which in tandem with mutations in several oncogenes and tumor suppressor genes facilitates tumor growth [3]. p27Kip1 is predominantly regulated by posttranslational modifications (primarily phosphorylation), which determine protein stability and subcellular localization [4]. Some controversy also exists regarding the importance of the cytoplasmic expression of p27Kip1, which was originally thought to represent a mechanism for inactivating p27Kip1 by sequestering it away from its site of action within the nucleus [5]. The quantity and function of p27Kip1 are regulated by changes in its protein levels and localization in the cell. In quiescent cells, p27Kip1 turnover is dependent upon its dephosphorylation at Ser10 and its interaction with cyclin-CDK complexes. However, the enzyme that mediates this degradation remains elusive [6].