The Role of Nucleolin Phosphorylation by CK2 in Regulating Cellular Fate Under Normal and Stress Conditions

Nucleolin (NCL or C23) is an abundant genotoxic stress-responsive RNA binding phosphoprotein. NCL constitutes 10% of total nucleolar protein that has functions in multiple biological processes, including ribosome biogenesis, DNA/RNA metabolism, cellular response to DNA damage, cell growth, proliferation and death. In this dissertation, I elucidate the role of nucleolin phosphorylation by casein kinase 2 (CK2) in controlling cellular fate by regulating p53 checkpoint under normal and stressed conditions. First, I demonstrate that the six consensus CK2 sites on the N-terminus of NCL are important for cell survival and proliferation. Expression of CK2 phosphorylation-deficient NCL mutant leads to dominant negative effects on cell proliferation and triggers p53 checkpoint activation to induce expression of pro-apoptotic markers downstream. Next, I present evidence that lack of NCL phosphorylation by CK2 increases cells’ sensitivity to apoptosis upon genotoxic stress (UV irradiation and topoisomerase II inhibitor etoposide). The substantially upregulated PUMA expression in dephosphorylated-NCL expressing cells may play a pivotal role in the increased cells’ sensitivity to apoptosis. In addition, phosphorylation-deficient NCL mutant expression also induces significant upregulation of AIF. Last but not least, in collaborate with Dr.Kleiman’s lab, we further demonstrate that NCL phosphorylation by CK2 regulates p53 checkpoint post-transcriptionally through affecting NCL interaction with PARN deadenylase and the target gene TP53 mRNA, as well as with ARE-binding protein HuR to mediate HuR availability to their common target TP53 mRNA. Dr. Kleiman’s lab determined that NCL can bind to the 3’UTR ARE of TP53 mRNA under non-stressed conditions. Moreover, in vitro assays show that NCL interacts directly with the N-terminus of PARN, however, while NCL-WT enhances PARN activity, phosphorylation-deficient NCL mutant abolishes PARN deadenylation. In this dissertation, I provide evidence that phosphorylation of NCL by CK2 not only plays a crucial role in NCL associating with TP53 mRNA both under non-stressed and stressed conditions, but also regulates the interaction of NCL with p53 protein, PARN deadenylase and HuR respectively in cell extracts. Post-transcriptional regulation of TP53 mRNA by the effect of functional interplay of NCL-PARN-HuR might ultimately link to the increased p53 protein levels and induction of apoptosis pathways. These data provide new insights into the role of NCL phosphorylation by CK2 in regulating gene expression post-transcriptionally to control cell fate during cellular stress response