Elsevier

Cellular Signalling

Volume 22, Issue 2, February 2010, Pages 325-329
Cellular Signalling

Endothelin-1 induces p66Shc activation through EGF receptor transactivation: Role of β1Pix/Gαi3 interaction

https://doi.org/10.1016/j.cellsig.2009.09.039Get rights and content

Abstract

Endothelin-1 (ET-1) is a vasoconstrictor peptide known to be a potent mitogen for glomerular mesangial cells. We have shown that ET-1 stimulates the adaptor protein p66Shc through Rac/Cdc42 guanine nucleotide exchange factor β1Pix. In this study, we demonstrate that ET-1-induced serine phosphorylation of p66Shc is mediated through Gαi3. Pertussis toxin treatment of cells induced a significant decrease in the interaction between β1Pix and ETA-R, and an increase in the binding of Gαi3 and Gβ1 to β1Pix. Activation of heterotrimeric G proteins by AlF4 resulted in an increase of Gαi3 binding to β1Pix, which was significantly disrupted in cells expressing β1Pix dimerization deficient mutant, β1PixΔ (602-611). In cells expressing β1PixΔ (602-611), ET-1-induced p66Shc activation was also significantly decreased. Specific inhibition of EGF receptor by AG1478 blocked ET-1-induced p66Shc activation and the binding of p66Shc and Gαi3 to β1Pix. Inhibition of Erk1/2 blocked p66Shc activation induced by ET-1. Altogether, our results indicate that ET-1 activates p66Shc through EGF receptor transactivation, leading to the activation of Gαi3, β1Pix and Erk1/2.

Introduction

Endothelin-1 (ET-1) is the best characterized member of the endothelin family of vasoactive peptides. ET-1 binds to specific G protein-coupled receptors and evokes a wide variety of cellular responses via activation of tyrosine and serine–threonine protein kinases. ET-1-derived intracellular signals are also transduced by channel and transport proteins, as well as by various phospholipases, all of which cooperate in amplifying the biological properties of this peptide [1], [2].

β1Pix (Pak-interacting exchange factor) is a guanine nucleotide exchange factor (GEF) for Rac 1 and Cdc42 [3], [4]. GEF proteins contain a pleckstrin homology (PH) domain and the catalytic Dbl homology (DH) domain. In addition to these domains, β1Pix contains a Src homology 3 (SH3) domain that binds with high affinity to a polyproline stretch in Pak1 [3]. β1Pix also binds GIT1 through a GIT1-binding domain [5]. The leucine zipper (LZ) domain at the C-terminus mediates β1Pix homodimerization [6]. There is mounting evidence showing that Pix functions as an integrator of signaling pathways controlling adhesion and cytoskeletal organization. Recently we have shown that endothelin-1 (ET-1) induces β1Pix translocation to focal adhesions through a PKA-dependent pathway [7]. Pix was found to form a complex with paxillin, Pak, and p95PKL, which regulates cytoskeletal remodeling [8]. Moreover, the Pak-Pix-GIT complex has been shown to target and regulate focal adhesions [5]. We have shown recently that β1Pix binds 14-3-3β resulting in the inhibition of β1Pix GEF activity toward Rac1 [9]. Moreover, ET-1 induces β1Pix and p66Shc up-regulation through Erk1/2 activation resulting in cell proliferation independently of Akt [10]. β1Pix was also found to enhance mast cell secretion through Gi-dependent mechanism [11].

The adaptor protein Shc exists in three isoforms with relative molecular masses (Kd) of 46, 52, and 66 [12]. They consist of a phosphotyrosine binding domain (PTB), a collagen homology domain (CH1) and a C-terminal Src homology 2 domain (SH2). In addition, p66Shc has a collagen homology domain 2 (CH2) at the N-terminus in which Ser36 is located. The serine 36 is phosphorylated in response to oxidative stress and appears to be critical for coupling Shc to stress response leading to apoptosis [13]. p66Shc knockout mice had a 30% increase in average lifespan when compared with control animals [14]. This increased longevity has been linked to enhanced resistance to oxidative stress. We have previously demonstrated the crucial role of p52Shc tyrosine phosphorylation in ET-1-mediated Ras activation in mesangial cells [15]. This phosphorylation enables and stabilizes the formation of a Shc-Grb2 complex and its subsequent association with Ras exchange factor Sos (Son of sevenless) facilitates the biphasic activation of Ras [15].

Section snippets

Materials

Cell culture media and supplements were obtained from Invitrogen (Carlsbad, CA). Endothelin-1 was purchased from Calbiochem (La Jolla, CA). Anti-ETA-R was from Millipore (Billerica, MD). Anti-Myc, anti-Gαi3, and anti-Gβ1 antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). Anti-phospho-Erk1/2 was purchased from Cell Signaling (Danvers, MA). Anti-p66Shc was from Invitrogen and anti-phosphoSer36p66Shc was purchased from Calbiochem. Anti-SHC was from BD transduction Laboratories (San

Endothelin-1 induces serine phosphorylation of p66Shc through Gαi3

We have previously shown that exposure of rat mesangial cells to ET-1 stimulation induces ERK1/2 and p66Shc activation [16]. In this study, we have used lysates of human mesangial cells (HMCs) expressing Myc-tagged β1Pix for immunoprecipitation with anti-p66Shc antibody. Results showed that β1Pix alone induced sizeable p66Shc activation as revealed by anti-phospho-p66ShcSer36 antibody and this activation was further increased by ET-1 stimulation (Fig. 1). Treatment of the cells with pertussis

Discussion

These studies have demonstrated that serine 36 phosphorylation of p66Shc induced by ET-1 is mediated through PTx-sensitive Gαi3 protein of Gi/o family. We provide evidence that both β1Pix and ET-1 induced p66Shc activation through Gαi3-dependent mechanism. We show that PTx treatment decreased the binding of Gαi3 to ETA-R and at the same time increased the binding of Gαi3 and Gβ1 to β1Pix, strongly suggesting that β1Pix interacts with ETA-R through Gαi3 since PTx is known to block the

Acknowledgment

This work was supported in part by the National Institutes of Health Grant HL22563.

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