Abstract 1193: A single phosphorylation site mutant of the Retinoblastoma protein blocks apoptosis induced by UV

Abstract

Abstract The Retinoblastoma protein (Rb) is important in the control of cell proliferation and apoptosis. Its activity is controlled by reversible phosphorylation on several serine and threonine residues. When Rb is hypophosphorylated, it inhibits proliferation by preventing passage through the G1- S phase transition. Hyperphosphorylated Rb promotes cell cycle progression. The role of Rb phosphorylation in the control of apoptosis is largely unknown, although several apoptotic stimuli result in dephosphorylation of Rb. It may be the case that dephosphorylation of specific amino acids may signal apoptosis versus cell cycle arrest. Using glutamic acid mutagenesis, we have generated 15 single phosphorylation site mutants from serine/threonine to glutamic acid, to mimic the phosphorylated state. By calcium phosphate transfection mutant plasmids were introduced into C33A Rb-null cells and apoptosis was induced using UV. Apoptosis was measured by ELISA detection of degraded DNA and by immunoblotting to assess proteolytic cleavage of PARP. We found that only threonine-821 when mutated to glutamic acid blocked apoptosis by 50% whereas other sites tested had little effect. In addition, endogenous Rb is dephosphorylated on threonine-821 when cells are undergoing apoptosis. Experiments to determine the effect of this mutation in response to additional apoptotic stimuli are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1193. doi:1538-7445.AM2012-1193