Abstract 5093: Nucleolin phosphorylation profiling under normal and cellular stress conditions

Abstract

Abstract Nucleolin (NCL) is a ubiquitous nucleolar phosphoprotein that integrates critical cellular processes, from cell growth (ribosome biogenesis) and cell proliferation to cell cycle arrest and cell death. Deregulation of these processes often lead to different pathological conditions including cancer and heightened levels of nucleolin are often found in 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 1 (Cdk1). Although increased nucleolin phosphorylation by CK2 is finely regulated in exponentially dividing cells, the role of nucleolin phosphorylation in regulating cell cycle remains largely unexplored. The high content of acidic stretches in the N-terminus of nucleolin protein limits the effective use of mass-spectrophotometry as well as our ability to generate various phosphorylation-specific nucleolin antibodies. In this study, we used novel phosphate-binding tag to analyze nucleolin phosphorylation in human osteosarcoma cells. With phosphate affinity gel electrophoresis and subsequent western analyses, we have enabled the detection and quantification of different phosphorylation states of nucleolin during interphase as well as in mitosis. CK2 phosphorylated nucleolin (as in interphase) has multiple bands that migrate slower when bound to phos-tag as compared to hypo-phosphorylated nucleolin mutant (6/S*A, where six consensus CK2 sites were modified from serine to alanine and hence defective in undergoing phosphorylation). In contrast, a single hyper-phosphorylated nuclolin band was predominant in mitotic arrested cells. Furthermore, we elucidated nucleolin phosphorylation patterns under various cellular stresses e.g. DNA damage (UV, CPT) as well as kinase stimulation (e.g. arachidonic acid) or inhibition by CK2-specific inhibitor CX4945. Our data provide for the first time informative nucleolin phosphorylation patterns during different cellular conditions. This profiling of nucleolin phosphoprotein is essential in understanding the role of nucleolin phosphorylation in various cellular processes as well as during cellular response to stress. This work is supported by grants PSC/CUNY 66740-00 44 and CA175794. Citation Format: Kenneth Ng, Allana Rodriguez, Anjana D. Saxena. Nucleolin phosphorylation profiling under normal and cellular stress conditions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5093. doi:10.1158/1538-7445.AM2014-5093