Abstract 5287: Identification of metadherin in a complex with RACK1/PP2A in mammary epithelial cells

Abstract

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 has a critical role to play in homeostasis where it functions 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. Our understanding is limited because of the complex nature of PP2A holoenzyme assembly, and because it acts through a wide variety of signalling pathways. We have identified the scaffolding protein RACK1 as a PP2A interacting protein and we showed that RACK1 regulates PP2A activity to mediate crosstalk between growth factor and adhesion signalling by an IGF-I-independent mechanism(1, 2) . Our hypothesis is that RACK1 plays a critical role in determining how PP2A functions in signalling pathways. Using a series of truncation mutations and peptide array technology, we have identified and mapped the binding sites between RACK1 and the catalytic subunit of PP2A. Using a series of specific point mutations of these PP2A sites, we show that the RACK1/PP2A interaction is critical to maintain PP2A activity. Disruption of the RACK1/PP2A interaction has dramatic consequences for cell adhesion, proliferation and migration in MCF-7 cells. Our hypothesis is that RACK1 regulates the cellular distribution of PP2A and scaffolds PP2A to a specific subset of cellular targets. Using 3D in vitro breast cancer models and mass spectroscopy approaches, we have identified several proteins that interact with the RACK1/PP2A complex. One of these proteins, Metadherin has been shown to play a role in drug resistance(3). It has also been linked to the progression of many cancers(4). Using a series of novel approaches we have mapped the Metadherin interaction site on RACK1 and PP2A and confirmed that RACK1 and Metadherin colocalise at the cell membrane. Overexpression of Metadherin promotes IGF-I-mediated cell migration and invasion. Taken together, our data suggest that RACK1 scaffolds PP2A to Metadherin where it regulates Metadherin activity and cell migration in breast cancer. 1. Kiely, PA., et al. “Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and β1 integrin and for tumor cell proliferation and migration.” Journal of Biological Chemistry 283.34 (2008): 22952-22961. 2. Kiely, PA., et al. “Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and β1 integrin to promote cell migration.” Molecular and cellular biology 26.11 (2006): 4041-4051. 3. Wei, Yong, Guohong Hu, and Yibin Kang. “Metadherin as a link between metastasis and chemoresistance.” Cell Cycle 8.14 (2009): 2131-2137. 4. Brown, DM., and Erkki R. “Metadherin, a cell surface protein in breast tumors that mediates lung metastasis.” Cancer cell 5.4 (2004): 365-374. Citation Format: Maeve L. Kiely, Rosemary O'Connor, Patrick A. Kiely. Identification of metadherin in a complex with RACK1/PP2A in mammary epithelial cells. [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 5287. doi:10.1158/1538-7445.AM2014-5287