Atrogin-1 inhibits Akt-dependent cardiac hypertrophy in mice via ubiquitin-dependent coactivation of Forkhead proteins
Hui-Hua Li, … , David J. Glass, Cam Patterson
Hui-Hua Li, … , David J. Glass, Cam Patterson
Published October 25, 2007
Citation Information: J Clin Invest. 2007;117(11):3211-3223. https://doi.org/10.1172/JCI31757.
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Research Article Cardiology

Atrogin-1 inhibits Akt-dependent cardiac hypertrophy in mice via ubiquitin-dependent coactivation of Forkhead proteins

Abstract

Cardiac hypertrophy is a major cause of human morbidity and mortality. Although much is known about the pathways that promote hypertrophic responses, mechanisms that antagonize these pathways have not been as clearly defined. Atrogin-1, also known as muscle atrophy F-box, is an F-box protein that inhibits pathologic cardiac hypertrophy by participating in a ubiquitin ligase complex that triggers degradation of calcineurin, a factor involved in promotion of pathologic hypertrophy. Here we demonstrated that atrogin-1 also disrupted Akt-dependent pathways responsible for physiologic cardiac hypertrophy. Our results indicate that atrogin-1 does not affect the activity of Akt itself, but serves as a coactivator for members of the Forkhead family of transcription factors that function downstream of Akt. This coactivator function of atrogin-1 was dependent on its ubiquitin ligase activity and the deposition of polyubiquitin chains on lysine 63 of Foxo1 and Foxo3a. Transgenic mice expressing atrogin-1 in the heart displayed increased Foxo1 ubiquitylation and upregulation of known Forkhead target genes concomitant with suppression of cardiac hypertrophy, while mice lacking atrogin-1 displayed the opposite physiologic phenotype. These experiments define a role for lysine 63–linked ubiquitin chains in transcriptional coactivation and demonstrate that atrogin-1 uses this mechanism to disrupt physiologic cardiac hypertrophic signaling through its effects on Forkhead transcription factors.

Authors

Hui-Hua Li, Monte S. Willis, Pamela Lockyer, Nathaniel Miller, Holly McDonough, David J. Glass, Cam Patterson

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Figure 2

Atrogin-1 interacts with Foxo1 and Foxo3a in vivo and in vitro.

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Atrogin-1 interacts with Foxo1 and Foxo3a in vivo and in vitro. (A) We t...
(A) We transfected 293 cells with the indicated plasmids. Equal amounts of protein lysates were immunoprecipitated with anti-HA or anti-Myc antibody and analyzed by IB with the indicated antibodies. WCE, whole cell extract. (B) The ability of Foxo1 and Foxo3a expressed in 293 cells to be retained by GST or a GST–atrogin-1 fusion protein was analyzed by IB after GST pulldown. (C) Endogenous protein interactions were examined in cardiomyocyte cell lysates immunoprecipitated with rabbit IgG or anti–atrogin-1 antibody and analyzed by IB with antibodies to detect Foxo1, Foxo3a, and atrogin-1. (D) Domains of atrogin-1 involved in binding to Foxo1 and Foxo3a expressed in 293 cells were mapped with GST pulldown assays. GST–atrogin-1 proteins were affinity purified and analyzed by SDS-PAGE and Coomassie blue staining. The ability of the truncated atrogin-1 fusion proteins to bind to Foxo1 and Foxo3a from whole-cell lysates was determined by blotting with antibodies against Flag and HA, respectively. (E) Deletion constructs of atrogin-1 in D that bind to Foxo1 and Foxo3a. NLS, nuclear localization sequence; PDZ, PDZ-binding domain. (F) Interaction domains of Foxo1 required for binding to atrogin-1 were mapped by co-IP. We transfected 293 cells with the indicated Flag-Foxo1 and HA–atrogin-1 plasmids. Equal amounts of lysates were prepared for IP with a Flag antibody and immunoblotted with antibodies for HA or Flag. (G) Deletion constructs of Foxo1 in F that bind to atrogin-1. FK, Forkhead domain; NES, nuclear export sequence; LxxLL, nuclear receptor–interacting domain or LxxLL motif; TAD, transactivation domain.