Abstract 3955: Identification of RACK1 as a novel regulatory subunit of PP2A

Abstract

Abstract Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase with a key role to play in the regulation of cell spreading and cell migration in tumour cells. We have identified PP2A as a RACK1 interacting protein downstream of the IGF-IR (1). In tumour cells PP2A and β1 integrin compete for binding to the C-terminus of RACK1. The IGF-I-mediated release of PP2A from RACK1 results in a decrease in PP2A activity, and promotes cell migration (2). This is coupled with the IGF-I-mediated dephosphorylation of FAK and focal adhesion turnover in MCF-7 cells (3). PP2A enzymes exist as heterotrimers comprising of the catalytic C-, the structural A- and the regulatory/variable B-type subunits. The B-type subunits have a critical role in targeting the PP2A protein to specific areas of the cell and also in regulating the overall activity and substrate specificity of PP2A. To date, at least 15 known PP2A B-type subunits have been identified but their detection has proven difficult because of the transient nature of their interaction with the PP2A holoenzyme. We used in vitro PP2A activity assays to determine that RACK1 stabilizes PP2A activity. This led us to predict that RACK1 may function as a regulatory subunit of PP2A. A comparative study between RACK1 and the well studied B56 family of PP2A regulatory subunits reveals strong homology. Both of these proteins are characterized by the presence of 7 WD repeat sequences which in the case of B56 gamma facilitates PP2A holoenzyme assembly. We used immobilized peptide arrays representing the entire PP2A-catalytic protein to identify a series of amino acids on the catalytic subunit of PP2A that are required for the binding of RACK1. Mutation analysis of these residues reveals the binding site for RACK1 on PP2A. Suppression of RACK1 expression in MCF-7 and DU145 cells results in a decrease in the activity and an altered location of PP2A-C suggesting that RACK1 has an essential role to play in the overall activity and substrate specificity of PP2A. Taken together the data supports our hypothesis that RACK1 functions as a B-type subunit of PP2A which is required for the regulation of PP2A activity. Our data also indicate that RACK1 targets PP2A to specific locations to promote IGF-I-mediated cell migration and proliferation in tumour cells. 1. Kiely, P. A., O'Gorman, D., Luong, K., Ron, D., and O'Connor, R. (2006) Mol Cell Biol 26, 4041-4051 2. Kiely, P. A., Baillie, G. S., Lynch, M. J., Houslay, M. D., and O'Connor, R. (2008) J Biol Chem 3. Kiely, P. A., Baillie, G. S., Barrett, R., Buckley, D. A., Adams, D. R., Houslay, M. D., and O'Connor, R. (2009) J Biol Chem 284, 20263-20274 Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3955.