Endothelin-1 Couples βPix to p66Shc: Role of βPix in Cell Proliferation through FOXO3a Phosphorylation and p27^kip1Down-Regulation Independently of Akt

Abstract

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 betaPix and p66Shc up-regulation, resulting in the formation of the betaPix/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 betaPix blocks the second phase of p66Shc and FOXO3a phosphorylation and prevents p27(kip1) down-regulation induced by ET-1. Depletion of either betaPix 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 p27(kip1) down-regulation was blocked by U0126 but not by wortmannin. Endogenous betaPix 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 p27(kip1) 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.

Endothelin-1 Inhibits the Epithelial Na+ Channel through βPix/14-3-3/Nedd4-2

“…For example, we have recently demonstrated that ET-1 stimulation of primary human mesangial cells induces ␤Pix and p66Shc up-regulation, resulting in the formation of the ␤Pix/p66Shc complex. 29 In our current and previous studies, 32 we have further explored the interaction between 14-3-3␤ and ␤ 1 Pix using coimmunoprecipitation studies. Moreover, we have shown that there is less interaction of 14-3-3 with Nedd4-2 when coexpressed with ␤Pix.…”

“…27,29 Thus, we were interested in testing the hypothesis that ␤Pix is involved in ET-1-mediated decrease in ENaC activity. Immunohistochemistry analysis was performed in Sprague-Dawley rat kidneys to identify a distribution of ␤Pix protein.…”

“…The expression vectors encoding wild-type ␤ 1 Pix, ␤ 1 Pix (L238R, L239R), and ␤ 1 Pix (⌬602 to 611) were described previously. 27,29 The expression vectors encoding ET B R, wild-type Rac1, and wildtype and constitutively active Cdc42 (G12V) were from the UMR cDNA Resource Center (http://www.cdna.org). The expression vector encoding constitutively active Rac1 (QL) was from Dr. A. Chan (Medical College of Wisconsin).…”

“…28 Moreover, we have reported up-regulation of ␤Pix expression by ET-1 in primary human mesangial cells and identified a number of signaling molecules that form a multiunit signaling complex with ␤Pix. 29 Consequently, ␤Pix plays an important role in ET-1 signal transduction and possesses both GEF and scaffolding activity.…”

P66 Shc regulates renal vascular tone in hypertension-induced nephropathy

“…ET-1 is an important regulator of the renal microcirculation that affects renal function through actions at multiple cell types in kidney, including vascular SMCs (26). We have shown that ET-1 induces p66Shc Ser36 phosphorylation and mediates protein-protein interactions of p66Shc in renal glomerular cells (7,41,42). Our recent experiments using 2-photon imaging also revealed that treatment of isolated rat renal resistance arteries with ET-1 caused a rapid increase of [Ca 2+ ] i in smooth muscle vasculature of these vessels (43).…”