Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex
Hui-Hua Li, … , Da-Zhi Wang, Cam Patterson
Hui-Hua Li, … , Da-Zhi Wang, Cam Patterson
Published October 15, 2004
Citation Information: J Clin Invest. 2004;114(8):1058-1071. https://doi.org/10.1172/JCI22220.
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Article Cardiology

Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex

Abstract

Calcineurin, which binds to the Z-disc in cardiomyocytes via α-actinin, promotes cardiac hypertrophy in response to numerous pathologic stimuli. However, the endogenous mechanisms regulating calcineurin activity in cardiac muscle are not well understood. We demonstrate that a muscle-specific F-box protein called atrogin-1, or muscle atrophy F-box, directly interacts with calcineurin A and α-actinin-2 at the Z-disc of cardiomyocytes. Atrogin-1 associates with Skp1, Cul1, and Roc1 to assemble an SCFatrogin-1 complex with ubiquitin ligase activity. Expression of atrogin-1 decreases levels of calcineurin A and promotes its ubiquitination. Moreover, atrogin-1 attenuates agonist-induced calcineurin activity and represses calcineurin-dependent transactivation and NFATc4 translocation. Conversely, downregulation of atrogin-1 using adenoviral small interfering RNA (siRNA) expression enhances agonist-induced calcineurin activity and cardiomyocyte hypertrophy. Consistent with these cellular observations, overexpression of atrogin-1 in hearts of transgenic mice reduces calcineurin protein levels and blunts cardiac hypertrophy after banding of the thoracic aorta. These studies indicate that the SCFatrogin-1 ubiquitin ligase complex interacts with and represses calcineurin by targeting calcineurin for ubiquitin-mediated proteolysis, leading to inhibition of cardiac hypertrophy in response to pathologic stimuli.

Authors

Hui-Hua Li, Vishram Kedar, Chunlian Zhang, Holly McDonough, Ranjana Arya, Da-Zhi Wang, Cam Patterson

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Atrogin-1 participates in an SCFatrogin-1 complex that ubiquitinates cal...
Atrogin-1 participates in an SCFatrogin-1 complex that ubiquitinates calcineurin A in vitro. (A) GST–atrogin-1 (WT), atrogin-1 ΔF-box, and atrogin-1 1–284 were purified from bacteria. The purity of each purified protein was verified by SDS-PAGE and Coomassie staining. (B) Association of Skp1 and Roc1 with Cul1 was confirmed by cotransfection with T7-Skp1, Myc-Cul1, and HA-Roc1 in COS-7 cells. Equal amounts of cell extract were immunoprecipitated with Myc antibody and analyzed by immuoblotting with antibodies against T7 (Skp1, top), Myc (Cul1, middle), and HA (Roc1, bottom), respectively. (C) Interaction of atrogin-1 with Skp1, Cul1, and Roc1 was analyzed in in vitro GST pull-down assays. The ability of Skp1 (top), Cul1 (middle), and Roc1 (bottom) to bind GST, GST–atrogin-1, or GST–atrogin-1 ΔF-box was analyzed by immunoblotting. (D) The SCFatrogin-1 complex was evaluated for ubiquitin ligase activity in the presence of recombinant E1, UbcH3/CDC34 (E2 UbcH3), and ubiquitin (Ub) as indicated. (E) The SCFatrogin-1 complex was evaluated for its capacity to ubiquitinate purified GFP-tagged calcineurin A. After the in vitro ubiquitylation reaction, the samples were analyzed by immunoblotting with an anti-ubiquitin antibody to identify ubiquitinated products or with an anti-GFP antibody directed to calcineurin A to reveal ubiquitinated calcineurin A species. (F) Purified GFP-tagged calcineurin A was incubated with SCFatrogin-1 complex for the indicated times, and calcineurin A ubiquitination was examined by anti-GFP (top) or anti-ubiquitin (bottom) antibodies. (G) Purified GFP-tagged calcineurin A was incubated with SCFatrogin-1 complex containing atrogin-1 WT, the ΔF-box mutant, or the 1–284 mutant, and calcineurin A ubiquitination was examined by immunoblotting.