Insulin-like growth factor (IGF)-I regulates a mutually exclusive interaction of PP2A and beta 1 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 beta 1 integrin in RACK1. A WD7 deletion mutant of RACK1 did not associate with PP2A but retained some interaction with beta 1 integrin, whereas a WD6/WD7 mutant lost the ability to bind to both PP2A and beta 1 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 beta 1 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 beta 1 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 beta 1 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 beta 1 integrin, for regulation of PP2A activity, and for IGF-I-mediated cell migration and proliferation.