The ubiquitination and degradation of Akt are mediated by MULAN. (A) Cellular Akt protein levels were reduced by MULAN through the proteasomal degradation pathway in a RING-dependent manner. After transfection with plasmids as indicated, HEK293 cells were treated with MG132 and then subjected to immunoblotting with the indicated antibodies. The level of ectopic expression of GST was normalized to the transfection control. (B) Akt was ubiquitinated by MULAN in vitro. In vitro ubiquitination assays were performed as described in Materials and Methods. (C) Akt was ubiquitinated by MULAN in vivo. After transfection with plasmids as indicated, HEK293 cells were treated with MG132 and then subjected to in vivo ubiquitination assays as described in Materials and Methods. (D) The ubiquitination of Akt was ablated by MULAN siRNA transfection. After transfection with siRNAs and the HA-Ub plasmid as indicated, HEK293 cells were treated with 1 μM MG132 for 12 h and then subjected to in vivo ubiquitination assays. (E) K48-linked polyubiquitination was responsible for MULAN-dependent Akt ubiquitination. After transfection with plasmids as indicated into HeLa cells, an in vivo ubiquitination assay was performed.

Activated Akt is a preferential target of the ubiquitin E3-ligase MULAN. (A) MULAN interacted with constitutively active Akt in vivo. After transfection with plasmids as indicated, HEK293 cells were treated with MG132 and then subjected to immunoprecipitation with an anti-myc antibody, followed by immunoblotting with the indicated antibodies. (B) Activated Akt was efficiently ubiquitinated by MULAN in vivo. After transfection with plasmids as indicated, HEK293 cells were treated with MG132 and then subjected to in vivo ubiquitination assays as described in Materials and Methods. (C) The MULAN-mediated proteolytic degradation of Akt was inhibited by LY294002. HEK293 cells were cotransfected with plasmids as indicated. After 24 h of incubation, cells were treated with 10 μM LY29402 for 4 h. Cells were lysed and immunoblotted with the indicated antibodies. The level of ectopic expression of GST was normalized to the transfection control. The graph represents the mean ± SD of the relative intensities of Akt from triplicate experiments.

The cell growth function of Akt is suppressed by MULAN. (A) Cell growth is attenuated by MULAN. After transfection with plasmids as indicated in HeLa cells, the number of viable cells were counted using a hemocytometer every 24 h. The results are representative of three independent experiments. (B) MULAN inhibits cell viability in a dose-dependent manner. HeLa cells were transfected with plasmids as indicated. After 24 h of incubation, cell viability was determined by the MTS assay. (C) MULAN suppresses clonogenic growth. HeLa cells were transfected with plasmids as indicated, subsequently incubated in media containing G418 (500 μg/ml) for 2 weeks, and stained with crystal violet. (D) MULAN inhibits anchorage-independent growth. The soft-agar assay was performed with HeLa cells transfected with indicated plasmids. Colonies were counted 2 weeks after seeding. The results are representative of three independent experiments (mean ± SD are shown). The student's t-test was performed for statistical significance (*P< 0.05). (E-G) Akt downstream signaling is regulated by MULAN. HeLa cells were transfected with the indicated MULAN plasmids (E) and MULAN siRNA (F). After 24 h of incubation, the cells were lysed and immunoblotted with the indicated antibodies. The HeLa cells were cotransfected with TCF/LEF1 reporter plasmid, pTOPFLASH, and other plasmids as indicated. After 24 h of incubation, cells were lysed and the reporter luciferase activities driven by the TCF response element were determined (G). (H, I) MULAN inhibited Akt-induced cell migration (H) but not K284R-Akt-induced cell migration (I). HeLa cells were cotransfected with plasmids as indicated. After 48 h of transfection, 100% confluent cells were scratched to form a wound. The migration of the cells was visualized on a phase-contrast microscope at 72 h after inducing a wound. (J) The cell growth of K284R-Akt-expressing cells is not inhibited by MULAN. After transfection of HeLa cells with plasmids as indicated, the number of viable cells was counted using a hemocytometer every 24 h.

Akt interacts with the MULAN E3 ubiquitin ligase in vitro and in vivo. (A) In vitro interaction between Akt and MULAN. ³⁵S-methionine-labeled Akt was tested for an interaction with GST-tagged MULAN (GST-MULAN) using pull-down assays. (B) In vivo association between Akt and MULAN. After transfection with plasmids as indicated, HEK293 cells were treated with MG132 and then subjected to immunoprecipitation with an anti-Akt antibody and immunoblotting with anti-EGFP. Immunoglobulin G was used as a negative control. (C) Endogenous interaction between MULAN and Akt isoforms. MG132-pretreated HeLa cells were subjected to immunoprecipitation with Akt isoform-specific antibodies, and immunoblotting was performed using an anti-MULAN antibody. (D) The functional domain structure and map of the plasmids of the Akt deletion mutant. (E) The KD of Akt interacts with MULAN. The ³⁵S-methionine-labeled Akt deletion mutants were examined for interaction with GST-MULAN using pull-down assays.

pAkt is a preferred substrate for ubiquitination by MULAN. (A) The interaction between MULAN and Akt was dependent on phosphorylated Akt. Serum-starved HeLa cells were treated with 10% serum or insulin along with MG132 for 6 h. An immunoprecipitation assay was then performed with an Akt antibody, followed by immunoblotting with an anti-MULAN antibody. (B) The cellular level of pAkt was decreased by MULAN. HeLa cells were transfected with plasmids as indicated. After 24 h of incubation, the medium was replaced with serum-free medium and the cells were subsequently stimulated with 10% serum for 2 h. Cells were lysed and immunoblotted with antibodies as indicated. (C) Cellular pAkt protein was ubiquitinated by MULAN. After transfection with plasmids as indicated, HeLa cells were incubated in a serum-free medium for 18 h. Cells were treated with 10 μM LY294002 and subsequently stimulated with 10% serum in the presence of 10 μM MG132. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (D) Dephosphorylation of pAkt decreased the MULAN-mediated Akt ubiquitination in vitro. In vitro ubiquitination assays were performed with purified human Akt protein derived from Sf9 cells and λ-PPase. Ubiquitinated Akt and pAkt were detected by immunoblotting with the indicated antibodies. (E) MULAN siRNA treatment increased Akt protein stability. HeLa cells were transfected with control and MULAN siRNA and treated with 40 μg/ml cycloheximide for the indicated times, after which Akt protein turnover was investigated by immunoblotting.

Lysine 284 of Akt is a site for MULAN-mediated polyubiquitination. (A) The KD of Akt is ubiquitinated by MULAN. A series of Akt deletion constructs were transcribed and translated in the presence of ³⁵S-methionine. The ³⁵S-labeled Akt mutants were subjected to ubiquitination by MULAN with in vitro ubiquitination assays. (B) Lysine 284 of Akt is a polyubiquitination site. HEK293 cells were cotransfected with plasmids as indicated. At 24 h after transfection, cells were treated with 10 μM MG132 and further subjected to in vivo ubiquitination assays as described in Materials and Methods. Whole-cell lysates were analyzed by immunoblotting with the indicated antibodies. (C) K284R-Akt is more stable than WT Akt. HeLa cells were cotransfected with the indicated plasmids and subsequently treated with 40 μg/ml cycloheximide. The cells were subjected to immunoblotting with the indicated antibodies.