The phosphoprotein nucleolin integrates several critical cellular processes, such as cell growth, proliferation, cell cycle arrest, apoptosis as well as DDR. Elevated levels of nucleolin are found in highly proliferative cells including a variety of tumors. Nucleolin is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase (Cdk). Earlier we have demonstrated that the N-terminus of nucleolin associates with Hdm2 to stabilize p53 protein and causes p53-mediated apoptosis. Additionally, nucleolin via its RNA-binding properties has been demonstrated to regulate the stability of several mRNAs and enhance translation. Besides, nucleolin post-translational modifications are linked to its role as an RNA-binding stress-responsive protein. Based on these studies, we hypothesize a role for nucleolin phosphorylation in regulating mRNA stability of different target genes during DDR.
To test this we have engineered a novel system with tet-off promoter in human osteosarcoma cells to express 3xFlag-tagged nucleolin-wt or phospho-mutant [6/S*A, defective in undergoing phosphorylation at six consensus CK2 sites] that allow us to control expression of nucleolin using doxycycline. Cell proliferation assays with these inducible cells indicated that although nucleolin phosphorylation by CK2 was required for cell proliferation in unstressed cells, a hypo-phosphorylated nucleolin mutant caused higher cell viability upon genotoxic stress. Further, nucleolin phospho-variants differentially elevated p53 protein levels and hypo-phosphorylated nucleolin associated with p53 even under non-stressed conditions. Our co-immunoprecipitation (co-IP) assays also showed that nucleolin associated with poly(A)-specific ribonuclease (PARN), an mRNA decay enzyme that controls mRNA stability of different genes during DDR. Importantly, hypo-phosphorylated nucleolin mutant decreased PARN deadenylase activity in vitro. Interestingly, nucleolin also interacted with cellular p53-mRNA as detected by RNA immunoprecipitation assays with nucleolin antibodies followed by qRT-PCR. Our data for the first time provides evidential support that nucleolin phosphorylation can play a regulatory role in driving the cell cycle and controlling gene expression during DDR. Direct associations of nucleolin with p53 and PARN probably affect p53-signaling as well as PARN-deadenylase activity. Understanding the control of gene expression that is regulated by nucleolin phosphorylation during DDR, will provide valuable insights into the mechanism(s) behind the cellular decisions of cell survival or death upon DNA damage.
Citation Format: Xiaokan Zhang, Shu Xiao, Emral Devany, Zaineb Nadeem, Elif Caglar ýþýn, Frida Kleiman, Anjana D. Saxena. Nucleolin phosphorylation mediated regulation of gene expression in determining cellular fate during the DNA damage response (DDR). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3188. doi:10.1158/1538-7445.AM2013-3188