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J. Biol. Chem., Vol. 283, Issue 34, 22952-22961, August 22, 2008

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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^*

Patrick A. Kiely, George S. Baillie, Martin J. Lynch, Miles D. Houslay, and Rosemary O'Connor¹

From the Cell Biology Laboratory, Department of Biochemistry, BioSciences Institute, University College Cork, Ireland and the Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom

Insulin-like growth factor (IGF)-I regulates a mutually exclusive interaction of PP2A and β1 integrin with the WD repeat scaffolding protein RACK1. This interaction is required for the integration of IGF-I receptor (IGF-IR) and adhesion signaling. Here we investigated the nature of the binding site for PP2A and β1 integrin in RACK1. A WD7 deletion mutant of RACK1 did not associate with PP2A but retained some interaction with β1 integrin, whereas a WD6/WD7 mutant lost the ability to bind to both PP2A and β1 integrin. Using immobilized peptide arrays representing the entire RACK1 protein, we identified a common cluster of amino acids (FAGY) at positions 299–302 within WD7 of RACK1 which were essential for binding of both PP2A and β1 integrin to RACK1. PP2A showed a higher level of association with a peptide in which Tyr-302 was phosphorylated compared with an unphosphorylated peptide, whereas β1 integrin binding was not affected by phosphorylation. RACK1 mutants in which either the FAGY cluster or Tyr-302 were mutated to AAAF, or Phe, respectively, did not interact with either PP2A or β1 integrin. These mutants were unable to rescue the decrease in PP2A activity caused by suppression of RACK1 in MCF-7 cells with small interfering RNA. MCF-7 cells and R+ (IGF-IR-overexpressing fibroblasts) expressing these mutants exhibited decreased proliferation and migration, whereas R– cells (IGF-IR null fibroblasts) were unaffected. Taken together, the data demonstrate that Tyr-302 in RACK1 is required for interaction with PP2A and β1 integrin, for regulation of PP2A activity, and for IGF-I-mediated cell migration and proliferation.

Received for publication, January 30, 2008 , and in revised form, June 11, 2008.

^* This work is supported by the Health Research Board of Ireland, Cancer Research Ireland, and Science Foundation Ireland. Work in the laboratories of G. S. B. and M. D. H. Laboratory was supported by Medical Research Council Grants G8604010 and G0400053, the European Union Grant 037189, and the Foundation Leducq (Paris). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 353-21-4901312; Fax: 353-21-4901382; E-mail: r.oconnor{at}ucc.ie.

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