Abstract 2967: Nucleolin phosphorylation by CK2 modulates its role in cell cycle checkpoint activation

Abstract

Abstract Nucleolin is a multifunctional abundant nucleolar protein and its gene expression is transactivated by the proto-oncogene c-myc during exponential cellular growth. This non-ribosomal protein is a major nucleolar phosphoprotein which is involved in ribosomal RNA processing, RNA polymerase I-catalyzed transcription, and the regulation of apoptosis, demonstrating both positive and negative effects on cell proliferation. During an unperturbed cell-cycle, interphase (CK2) and mitotic (Cdc2) kinases regulate nucleolin phosphorylation, modulating its function and nucleolar localization. Following cellular stress, nucleolin has been shown to inhibit chromosomal replication and to relay nucleolar stress signals mediated by its re-localization within the nucleus. Nucleolin also interacts with prominent components of the ARF (Alternate Reading Frame)/p53 checkpoint pathway that controls growth upon oncogenic stress signals. We have previously demonstrated that nucleolin binds to the p53-antagonist Hdm2, inhibits Hdm2-mediated degradation of p53 and causes p53-mediated apoptosis in an ARF-independent manner. Here we further dissect role/s of nucleolin phosphorylation in regulating the ARF/p53 checkpoint. We have generated a novel system by engineering human osteocarcinoma (NARF6) cells to induce expression of nucleolin-wt (wild-type) or -S6A (a phospho-mutant with six consensus site serines mutated to alanine, impairing phosphorylation by CK2). This novel system can control expression of both nucleolin and ARF by different promoters, allowing us to dissect the downstream events that are solely nucleolin- or ARF- dependent, or regulated by both. We found that increased levels of nucleolin-S6A significantly decreased ARF protein levels as compared to nucleolin-wt. Nucleolin-S6A was significantly more nucleoplasmic and, interestingly, cells that express S6A lacked nucleolar ARF expression. Although a dramatic loss in both ARF and p21 protein levels is evident with nucleolin-S6A expression, there was a net increase in p53 levels. Significantly, nucleolin-S6A expression caused an increase in the fraction of G1 cells in an ARF-independent manner. Thus, reduction of nucleolin phosphorylation by CK2 acts as a molecular switch causing the sub-nuclear re-localization of both nucleolin and ARF. These events lead to p53 activation, and the control of growth in both normal and stressed cells. These results also provide validation of our unique cellular system that allows us to manipulate nucleolin wt and mutant levels, facilitating the study of its physiological functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2967. doi:10.1158/1538-7445.AM2011-2967