Endothelin-1 couples βPix to p66Shc:: Role of βPix in cell proliferation through FOXO3a phosphorylation and p27kip1 down-regulation independently of akt

The phosphorylation of forkhead transcription factor FOXO3a by Akt is critical regulator of cell proliferation induced by serum. We show that endothelin-1 (ET-1) stimulation of primary human mesangial cells (HMCs) induces beta Pix and p66Shc up-regulation, resulting in the formation of the beta Pix/p66Shc complex. In transformed HMCs, ET-1 induces a biphasic phosphorylation of p66Shc and FOXO3a. The second phase leads to p27kip1 down-regulation independently of Akt. Depletion of beta Pix blocks the second phase of p66Shc and FOXO3a phosphorylation and prevents p27kip1 down-regulation induced by ET-1. Depletion of either beta Pix or p66Shc inhibits ET-1-induced cell proliferation. The expression of beta(1)Pix induces FOXO3a phosphorylation through activation of Rac1, ERK1/2, and p66Shc. Using either p66Shc- or Akt-depleted cells; we show that beta(1)Pix-induced FOXO3a phosphorylation requires p66Shc but not Akt. beta(1)Pix-induced p27kip1 down-regulation was blocked by U0126 but not by wortmannin. Endogenous beta Pix and FOXO3a are constitutively associated with endogenous p66Shc. FOXO3a and p66Shc binding requires beta(1)Pix homodimerization. Expression of beta(1)Pix homodimerization deficient mutant abrogates beta(1)Pix-induced p27kip1 down-regulation and cell proliferation. Our results identify p66Shc and FOXO3a as novel partners of beta(1)Pix and represent the first direct evidence of beta(1)Pix in cell proliferation via Erk/p66Shc-dependent and Akt-independent mechanisms.