Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and beta1 integrin and for tumor cell proliferation and migration.
Insulin-like growth factor (IGF)-I regulates a mutually exclusive interaction of PP2A and beta1 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 beta1 integrin in RACK1. A WD7 deletion mutant of RACK1 did not associate with PP2A but retained some interaction with beta1 integrin, whereas a WD6/WD7 mutant lost the ability to bind to both PP2A and beta1 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 beta1 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 beta1 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 beta1 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 beta1 integrin, for regulation of PP2A activity, and for IGF-I-mediated cell migration and proliferation.
Department of Biochemistry, BioSciences Institute, University College Cork, Ireland.
Patrick A
PA
Kiely
George S
GS
Baillie
Martin J
MJ
Lynch
Miles D
MD
Houslay
Rosemary
R
O'Connor
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D019204:GTP-Binding Proteins / Q000737:chemistry*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D019012:Integrin beta1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry*; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
D019012:Integrin beta1; D009363:Neoplasm Proteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; C517189:PPP2CA protein, human; D054648:Protein Phosphatase 2; D019204:GTP-Binding Proteins