Abstract 2325: Identification of RACK1 as a novel regulatory subunit of PP2A.

Abstract Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase in cells. It consists of a catalytic (C), structural (A) and regulatory/variable B-type subunits. PP2A plays a critical role as a tumour suppressor. Changes in the activity of PP2A have a direct role in maintaining the transformed phenotype in cancer. However, the mechanism of action of PP2A during this process is poorly understood. Understanding is limited by the complex nature of PP2A holoenzyme assembly, and because it acts through a wide variety of signalling pathways. The regulatory B-type subunits have a critical role in targeting PP2A 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 is difficult because of the transient nature of their interaction with the PP2A holoenzyme. The scaffolding protein RACK1 is a PP2A interacting protein and we have published a mechanism by which PP2A regulates IGF-IR and adhesion signalling through RACK1 [1, 2]. Our hypothesis is that RACK1 plays a critical role in determining how PP2A functions in signalling pathways and may function as a regulatory subunit of PP2A. We predict it does this by modulating the activity of PP2A and regulating its distribution to a specific subset of targets. We examined how the PP2A holoenzyme is assembled in MCF-7 cells. We have found that both the catalytic subunit and structural subunit of PP2A (PP2A C and PP2A A respectively) are assembled on the RACK1 scaffold in response to IGF-I. Our findings indicate that both the C subunit and the A subunit of PP2A are released from RACK1 in response to IGF-I and reassembled after 30 minutes IGF-I stimulation. We used immobilized peptide arrays representing the entire PP2A-catalytic protein to identify amino acids on the catalytic subunit of PP2A that are required for the binding of RACK1. Analysis of these residues revealed the binding sites for RACK1 on PP2A and supports their role in mediating the interaction with RACK1. When cells are transfected with specific mutations of PP2A C that disrupt the association with RACK1, cells show decreased PP2A activity and deregulated cell adhesion and migration suggesting that RACK1 has an essential role to play in the overall activity and substrate specificity of PP2A. This 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 indicates that RACK1 targets PP2A to specific locations to promote IGF-I-mediated cell migration and proliferation in tumour cells. 1. Kiely, P.A., et al. Molecular and cellular biology, 2006. 26(11): p. 4041-4051. 2. Kiely, P.A., et al. Journal of Biological Chemistry, 2008. 283(34): p. 22952-22961. Citation Format: Maeve L. Kiely, Rosemary O'Connor, Patrick A. Kiely. Identification of RACK1 as a novel regulatory subunit of PP2A. [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 2325. doi:10.1158/1538-7445.AM2013-2325