Endothelin-1 couples betaPix to p66Shc: role of betaPix 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 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.
Chahdi
Ahmed
A
Kidney Disease Center, Department of Medicine, Division of Nephrology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Sorokin
Andrey
A
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D019281:Dimerization; D015536:Down-Regulation / Q000187:drug effects*; D019332:Endothelin-1 / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors; D055765:Shc Signaling Adaptor Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
C576607:Arhgef7 protein, mouse; D019332:Endothelin-1; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D055765:Shc Signaling Adaptor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D010768:Phosphoserine; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases