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Vol. 19, Issue 6, 2609-2619, June 2008

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Endothelin-1 Couples βPix to p66Shc: Role of βPix in Cell Proliferation through FOXO3a Phosphorylation and p27^kip1 Down-Regulation Independently of Akt

Kidney Disease Center, Department of Medicine, Division of Nephrology, Medical College of Wisconsin, Milwaukee, WI 53226

Submitted May 9, 2007; Revised March 18, 2008; Accepted March 25, 2008
Monitoring Editor: Carl-Henrik Heldin

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 βPix and p66Shc up-regulation, resulting in the formation of the βPix/p66Shc complex. In transformed HMCs, ET-1 induces a biphasic phosphorylation of p66Shc and FOXO3a. The second phase leads to p27^kip1 down-regulation independently of Akt. Depletion of βPix blocks the second phase of p66Shc and FOXO3a phosphorylation and prevents p27^kip1 down-regulation induced by ET-1. Depletion of either βPix or p66Shc inhibits ET-1–induced cell proliferation. The expression of β₁Pix induces FOXO3a phosphorylation through activation of Rac1, ERK1/2, and p66Shc. Using either p66Shc- or Akt-depleted cells; we show that β₁Pix-induced FOXO3a phosphorylation requires p66Shc but not Akt. β₁Pix-induced p27^kip1 down-regulation was blocked by U0126 but not by wortmannin. Endogenous βPix and FOXO3a are constitutively associated with endogenous p66Shc. FOXO3a and p66Shc binding requires β₁Pix homodimerization. Expression of β₁Pix homodimerization deficient mutant abrogates β₁Pix-induced p27^kip1 down-regulation and cell proliferation. Our results identify p66Shc and FOXO3a as novel partners of β₁Pix and represent the first direct evidence of β₁Pix in cell proliferation via Erk/p66Shc-dependent and Akt-independent mechanisms.

Address correspondence to: Andrey Sorokin (sorokin{at}mcw.edu)

Abbreviations used: AFX, All 1 Fused gene chromosome X; ET-1, endothelin-1; DH, Dbl homology; FOXO3a, forkhead box class O3A; GEF, guanine nucleotide exchange factor; GIT1, G protein–coupled receptor kinase (GRK)-interacting targets; LZ, leucine zipper; Pak, p21-activated kinase; β₁Pix, Pak-interacting exchange factor; PH, pleckstrin homology; PKL, paxillin kinase linker; p27^kip1, cyclin-dependent kinase inhibitor; SH3, Src homology 3.