RhoBTB2 is a substrate of the mammalian Cul3 ubiquitin ligase complex - PubMed (original) (raw)

RhoBTB2 is a substrate of the mammalian Cul3 ubiquitin ligase complex

Andrew Wilkins et al. Genes Dev. 2004.

Abstract

Rhobtb2 is a candidate tumor suppressor located on human chromosome 8p21, a region commonly deleted in cancer. Rhobtb2 is homozygously deleted in 3.5% of primary breast cancers, and gene expression is ablated in approximately 50% of breast and lung cancer cell lines. RhoBTB2 is an 83-kD, atypical Rho GTPase of unknown function, comprising an N-terminal Rho GTPase domain and two tandem BTB domains. In this report, we demonstrate that RhoBTB2 binds to the ubiquitin ligase scaffold, Cul3, via its first BTB domain and show in vitro and in vivo that RhoBTB2 is a substrate for a Cul3-based ubiquitin ligase complex. Moreover, we show that a RhoBTB2 missense mutant identified in a lung cancer cell line is neither able to bind Cul3 nor is it regulated by the ubiquitin/proteasome system, resulting in increased RhoBTB2 protein levels in vivo. We suggest a model in which RhoBTB2 functions as a tumor suppressor by recruiting proteins to a Cul3 ubiquitin ligase complex for degradation.

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Figures

Figure 1.

RhoBTB2 interacts with Cul3 but not other cullin family members. (A) Interaction of RhoBTB2 and Cul3 in the yeast two-hybrid system. PJ69-4A yeast were cotransformed with the indicated plasmids and grown in the absence of leucine and uracil (Leu–, Ura–) to maintain both plasmids. Protein/protein interactions were determined by growth in the absence of adenine and histidine (Leu–, Ura–, Ade–, His–). For photography, 10 μL of appropriate clonal yeast suspensions were spotted on the indicated agar plates and grown for 3–5 d before being photographed against a black background. (B) Interaction of endogenous RhoBTB2 and Cul3. HeLa or 293T cell lysates were subject to immunoprecipitation by preimmune, RhoBTB2, or Cul3 antibodies and immune complexes probed for both Cul3 and RhoBTB2 by Western blotting. (C) RhoBTB2 binds specifically to Cul3. 293T cells were transfected with the indicated plasmids, then anti-HA immune complexes isolated and analyzed sequentially for the presence of RhoBTB2, cullin, and Nedd8 by Western blotting.

Figure 2.

RhoBTB2 interacts with the N terminus of Cul3 via its first BTB domain. (A) A schematic representation of the RhoBTB2 and Cul3 mutants used. The exact residue numbers where truncations are made are indicated. The approximate binding site of the ring finger protein Roc1 on Cul3 is also shown. (B) Mapping the binding site of Cul3 on RhoBTB2. 293T cells were transfected with the indicated plasmids. GST complexes were isolated and analyzed sequentially for the presence of Cul3 and GST–RhoBTB2 proteins by Western blotting. (C) Mapping the binding site of RhoBTB2 on Cul3. 293T cells were transfected with the indicated plasmids. GST complexes were isolated and analyzed sequentially for the presence of RhoBTB2 and GST–Cul3 proteins by Western blotting.

Figure 3.

Point mutations in RhoBTB2 and Cul3 disrupt their interaction. (A) A Y284D mutation in RhoBTB2 disrupts the interaction with Cul3. 293T cells were transfected with the indicated plasmids and anti-HA immune complexes isolated and analyzed sequentially for the presence of RhoBTB2 and Cul3 by Western blotting. The Y284D RhoBTB2 mutant was consistently expressed at levels three to fivefold higher than wild type; consequently, four times less RhoBTB2-Y284D immunoprecipitate than wild type was loaded to allow easier assessment of the relative amounts of Cul3. (B) A Y58K mutation in Cul3 disrupts the interaction with RhoBTB2. 293T cells were transfected with the indicated plasmids and anti-HA immune complexes isolated and analyzed sequentially for the presence of RhoBTB2 and Cul3 by Western blotting.

Figure 4.

RhoBTB2 protein levels are regulated by both Cul3 and the ubiquitin/proteasome system in vivo. (A) RhoBTB2 protein levels are negatively regulated by the proteasome in transfected cells. 293T cells were transfected with either wild-type or Y284D RhoBTB2 for 16 h. To control for variability in transfection, we then split cells into two separate dishes and, after cell reattachment, treated them with either 10 μM MG132 or DMSO alone for a further 12 h. Cell lysates were then analyzed for RhoBTB2 expression by Western blotting. As a loading control, blots were also probed for α-actin. (B) Endogenous RhoBTB2 protein levels are negatively regulated by the proteasome. Hela and SK-Mes-1 cells were treated with either 10 μM MG132 or DMSO alone for 12 h. Equal amounts of lysates were then subject to immunoprecipitation by anti-RhoBTB2 antibody and analyzed for the presence of RhoBTB2 by Western blotting. (C) RhoBTB2 has a short half-life that is stabilized by proteasomal inhibition. 293T cells were transfected with either wild-type or Y284D RhoBTB2 for 16 h. To control for variability in transfection, we then split cells into 3-cm dishes and, after cell reattachment, treated them with 100 μM cycloheximide plus either 10 μM MG132 or DMSO alone for the indicated number of hours. Cell lysates were then analyzed for RhoBTB2 expression by Western blotting. As a loading control, blots were also probed for α-actin. (D) shRNA-mediated ablation of Cul3 results in elevated levels of endogenous RhoBTB2 protein. HeLa and SK-Mes-1 cells were infected twice in 24 h with retroviruses expressing shRNA for either Cul3 or GFP. An empty vector virus was used as a negative control. After 48 h of infection, cell lysates were subject to immunoprecipitation and Western blotting by RhoBTB2 or Cul3 antibodies. As a control for the specificity of the Cul3 shRNA, cell lysates were Western blotted for Cul1 levels.

Figure 5.

RhoBTB2 is a target for a Cul3 ubiquitin ligase complex in vitro. 293T cells were transfected with GST–Cul3, Myc-RhoBTB, and Roc1 or with appropriate controls. GST–Cul3 complexes were subsequently isolated with GSH-agarose and subject to in vitro ubquitylation assay (see Materials and Methods). (A) After assay, the reactions were resolved by SDS-PAGE and probed for RhoBTB2. (B) The blot was then stripped and reprobed for Cul3. (C) The constituents of each Cul3 complex were confirmed by Western blotting for Cul3, Roc1, and RhoBTB2. In A and B, the only components of assay that were varied are indicated. In C, the transfected constructs are indicated.

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RhoBTB2 is a substrate of the mammalian Cul3 ubiquitin ligase complex - PubMed (original) (raw)