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

Abstract

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

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis

“…To identify the regions of FLIP that are important for the FLIP-Ku70 interaction, we used a FLIP peptide array of overlapping 25-mer peptides, each shifted by four amino acids, encompassing the entire FLIP(S) sequence. 9 Duplicate membranes were overlaid with lysates from cells transfected with Flag-Ku70, and the Flag epitope detected by immunoblotting. Strong binding of Ku70 to the array was detected in four regions (Figure 2a and table).…”

ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation

“…53 Phosphorylation is well known to represent a post-translational mechanism for the rapid regulation of protein activity. Herein, we provided the first evidence of the post-translational modification of ATG16L1, suggesting that regulation of ATG16L1 phosphorylation is a critical step in autophagy induction in hypoxic cardiomyocytes.…”

Sex differences in the GSK3β-mediated survival of adherent leukemic progenitors

“…Conversely, PP2A was already found associated with a 5 b 1 -integrin in suspended cells. RACK1 has been shown to bind the cytoplasmic tail of a 5 b 1 and to potentially trigger activation of PP2A when cells are cultured without the serum (Kiely et al, 2008). As it has not been shown that RACK1 is a direct partner of GSK3b, we thought that the integrin engagement might trigger a cytoskeletal reorganization allowing the recruitment of RACK1 and GSK3b in the vicinity of PP2A, thereby allowing its phosphatase activity on a pool of GSK3b.…”

“…For instance, higher production of interferon-a by activated peripheral blood mononuclear cells from women compared with cells from men has been described (Bergho¨fer et al, 2006). Interestingly, interferon-a restores normal b 1 -integrin-mediated inhibition of leukemic progenitor proliferation (Bhatia et al, 1996) and RACK1 has been shown to be a partner of both integrin and interferon-a receptor (Kubota et al, 2002;Kiely et al, 2008). A large study on differential cytokine production by immature leukemic cells from female and male patients should be performed in the future.…”

“…RACK1 has been described to associate and to regulate PP2A (Kiely et al, 2008), and we have previously shown that the a 5 b 1 recruits RACK1 and triggers PP2A activation that controls the ser-9 dephosphorylation of GSK3b in the leukemic cell line U937 (De Toni-Costes et al, 2010). Indeed, inhibition of PP2A by okadaic acid triggered both ser-9 phosphorylation (inhibition) of GSK3b and apoptosis in adherent CD34 þ 38 À 123 þ cells from female patients (Figure 4a).…”