Abstract
The serine/threonine kinase Akt functions in multiple cellular processes, including cell survival and tumor development. Studies of the mechanisms that negatively regulate Akt have focused on dephosphorylation-mediated inactivation. In this study, we identified a negative regulator of Akt, MULAN, which possesses both a RING finger domain and E3 ubiquitin ligase activity. Akt was found to directly interact with MULAN and to be ubiquitinated by MULAN in vitro and in vivo. Other molecular assays demonstrated that phosphorylated Akt is a substantive target for both interaction with MULAN and ubiquitination by MULAN. The results of the functional studies suggest that the degradation of Akt by MULAN suppresses cell proliferation and viability. These data provide insight into the Akt ubiquitination signaling network.
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Acknowledgements
We would like to thank Dr Dirk Bohmann (University of Rochester, USA) for kindly providing the HA-Ub plasmid and Dr Zhijian Chen (University of Texas Southwestern Medical Center, USA) for the HA-Ub-K48R and HA-Ub-K63R mutant constructs. This work was supported by grants from the Ministry of Education, Science and Technology (grants 20110028646 to S An and 20100018768 to J H Lee), the National R&D Program for Cancer Control, the Ministry of Health & Welfare (0720070 to S An), and the Korea Foundation for Cancer Research (KFCR-2009-002 to S Bae) of the Republic of Korea.
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Supplementary information
Supplementary information, Data S1
Materials and Methods (PDF 111 kb)
Supplementary information, Figure S1
The siRNA-mediated ablation of MULAN expression stabilizes endogenous Akt1 and Akt2 proteins. (PDF 138 kb)
Supplementary information, Figure S2
MULAN induced Akt1 degradation and colocalized with Akt1 in mitochondria. (PDF 3514 kb)
Supplementary information, Figure S3
Serum decreased Akt protein stability. (PDF 202 kb)
Supplementary information, Figure S4
Geldanamycin induced Akt degradation in both control cells and MULAN-depleted cells. (PDF 162 kb)
Supplementary information, Figure S5
The MULAN-mediated proteolytic degradation of Akt was rescued by wortmannin. (PDF 132 kb)
Supplementary information, Figure S6
The mutation of lysine 284 prevents the proteolytic degradation of ectopically expressed Akt by MULAN. (PDF 274 kb)
Supplementary information, Figure S7
The depletion of MULAN in HeLa cells by shRNA increased cell growth. (PDF 60 kb)
Supplementary information, Figure S8
Akt-mediated increases in cell viability are reduced by MULAN in a RING domain-dependent manner. (PDF 70 kb)
Supplementary information, Figure S9
Depletion of endogenous MULAN inhibits cell migration. (PDF 95 kb)
Supplementary information, Figure S10
MULAN-induced NF-κB activation was independent of its E3 ligase activity, and Akt-induced NF-κB activation was decreased by MULAN in an E3 ligase activity-dependent manner. (PDF 71 kb)
Supplementary information, Figure S11
Ectopically expressed Akt is degraded by MULAN in HeLa cells in the presence of the caspase inhibitor Z-VAD. (PDF 132 kb)
Supplementary information, Table S1
Primer sequences used to generate constructs for Akt and MULAN. (PDF 54 kb)
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Bae, S., Kim, SY., Jung, J. et al. Akt is negatively regulated by the MULAN E3 ligase. Cell Res 22, 873–885 (2012). https://doi.org/10.1038/cr.2012.38
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DOI: https://doi.org/10.1038/cr.2012.38
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