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Science 325 (5944): 1134-1138

Copyright © 2009 by the American Association for the Advancement of Science

The E3 Ligase TRAF6 Regulates Akt Ubiquitination and Activation

Wei-Lei Yang1,2, Jing Wang1, Chia-Hsin Chan1, Szu-Wei Lee1,2, Alejandro D. Campos3, Betty Lamothe3, Lana Hur3, Brian C. Grabiner1,2, Xin Lin1,2, Bryant G. Darnay3, and Hui-Kuan Lin1,2,*

1 Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
2 The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA.
3 Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.


Figure 1
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Fig. 1. TRAF6 is an E3 ubiquitin ligase for Akt. (A) Immunoblot (IB) of lysed 293T cells transfected with Akt along with His-ubiquitin (His-Ub), His–Ub K48R, or His–Ub K63R constructs. WT indicates wild type; Ni–nitrilotriacetic acid (NTA), nickel bead precipitate; and WCE, whole-cell extracts. (B) IB of lysed 293T cells transfected with hemagglutinin (HA)-Akt and His-Ub, along with various E3 ligases. (C) IB of lysed 293T cells transfected with HA-Akt and His-Ub, along with TRAF6 or TRAF6 C70A. (D) IB of lysed 293T cells transfected with Akt and TRAF6, along with HA–Ub K48 (K48-only ubiquitin) or HA–Ub K63 (K63-only ubiquitin). IP, immunoprecipitation. (E) GST-Akt-Flag proteins were incubated with adenosine triphosphate, E1, and E2 along with GST, GST-TRAF6, or GST–TRAF6 C70A proteins for in vitro ubiquitination of Akt. (F) WCE from 293T cells transfected with indicated plasmids was collected for IB analysis.

 

Figure 2
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Fig. 2. TRAF6 is required for ubiquitination and phosphorylation of Akt. (A) Traf6+/+ and Traf6–/– MEFs were serum-starved and treated with or without IGF-1 for 30 min; WCE were collected for immunoprecipitation with Akt, followed by IB analysis. (B) MEFs were serum-starved, treated with IGF-1 for various time points, and harvested for IB analysis. The quantification results are shown on the graphs to the right. (C) Primary Traf6–/– MEFs infected with mock, TRAF6, or TRAF6 C70A mutant were treated with IGF-1 at various time points and harvested for IB analysis. (D) MEFs were cultured in 10% FBS or serum-starved for 2 days, and apoptosis was determined by annexin V staining, followed by flow cytometry analysis. Results are presented as mean values ± SD. (E) Primary MEFs were infected with mock, constitutively active Akt (Mri-Akt), TRAF6, or TRAF6 C70A; and apoptosis and IB analysis were determined. Results are presented as mean values ± SD. (F) Heart and skeletal muscles isolated from WT and Traf6–/– mice (n = 4) injected with IGF-1 at various time points were subjected to an in vitro Akt kinase assay and IB analysis.

 

Figure 3
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Fig. 3. Identification of Akt ubiquitination sites. (A) IB of lysed 293T cells transfected with His-Ub along with HA-Akt or HA–Akt {Delta}PH. (B) IB of 293T cells transfected with His-Ub along with HA-Akt or various HA-Akt mutants and lysed for ubiquitination and phosphorylation of Akt. (C) WCE from 293T cells transfected with HA-Akt or various Akt mutants were incubated with control or PIP3 beads for overnight, washed, and subjected to IB analysis. The PIP3 binding was calculated as the ratio between amounts of Akt bound with PIP3 beads and total amounts of Akt. (D and E) IBs of lysed 293T cells transfected with indicated plasmids.

 

Figure 4
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Fig. 4. Ubiquitination of Akt is required for membrane recruitment of Akt. (A) The membrane (Mem) and cytosolic (Cyto) fractions from 293T cells transfected with mock or TRAF6 were subjected to IB analysis. (B) MEFs were serum-starved and treated with IGF-1, and the membrane and cytosolic fractions were isolated for IB analysis. (C) COS-1 cells were transfected with indicated plasmids, serum-starved, and treated with IGF-1 for 15 min; and the membrane fractions were isolated for IB analysis. (D) PC-3 cells silenced with control or TRAF6 shRNAs were harvested for IB analysis. (E) PC-3 cells silenced with control or TRAF6 shRNA were serum-starved, treated with IGF-1 for various times, and harvested for IB analysis. (F) PC-3 cells silenced with control or TRAF6 shRNAs were injected into nude mice (n = 6 for each group) and monitored for tumorigenesis. Results are presented as mean values ± SD. *P < 0.05, using Student’s t test.

 


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