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. 2009 Jan 15;417(2):601-9.
doi: 10.1042/BJ20080854.

COMMD1 expression is controlled by critical residues that determine XIAP binding

Gabriel N Maine  1 , Xicheng Mao, Patricia A Muller, Christine M Komarck, Leo W J Klomp, Ezra Burstein
Affiliations

COMMD1 expression is controlled by critical residues that determine XIAP binding

Gabriel N Maine et al. Biochem J. .

Abstract

COMMD {COMM [copper metabolism Murr1 (mouse U2af1-rs1 region 1)] domain-containing} proteins participate in several cellular processes, ranging from NF-kappaB (nuclear factor kappaB) regulation, copper homoeostasis, sodium transport and adaptation to hypoxia VSports手机版. The best-studied member of this family is COMMD1, but relatively little is known about its regulation, except that XIAP [X-linked IAP (inhibitor of apoptosis)] functions as its ubiquitin ligase. In the present study, we identified that the COMM domain of COMMD1 is required for its interaction with XIAP, and other COMMD proteins can similarly interact with IAPs. Two conserved leucine repeats within the COMM domain were found to be critically required for XIAP binding. A COMMD1 mutant which was unable to bind to XIAP demonstrated a complete loss of basal ubiquitination and great stabilization of the protein. Underscoring the importance of IAP-mediated ubiquitination, we found that long-term expression of wild-type COMMD1 results in nearly physiological protein levels as a result of increased ubiquitination, but this regulatory event is circumvented when a mutant form that cannot bind XIAP is expressed. In summary, our findings indicate that COMMD1 expression is controlled primarily by protein ubiquitination, and its interaction with IAP proteins plays an essential role. .

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Figure 1
Figure 1. IAPs regulate COMM domain containing proteins
(A) XIAP and c-IAP1 promote COMMD1 degradation. Cells were transfected with COMMD1-Flag along with the wild-type or RING inactive mutants of the indicated Flag-tagged IAP proteins (XIAP, c-IAP1, or c-IAP2). One day after transfection, the cells were lysed and the levels of the indicated proteins were analyzed by western blot (Flag or β-Actin immunoblotting). (B) The COMM domain mediates the COMMD1-XIAP interaction. Wild-type XIAP was co-expressed with full-length COMMD1 (F.L.), the amino-terminal 118 amino acids (N-term), or the COMM domain (amino acids 119-190) fused with GST. Cells were treated with MG-132 or the corresponding vehicle, and cell lysates were subsequently prepared for COMMD1 precipitation using glutathione (GSH) beads. (C) Other COMM domain containing proteins can interact with IAPs. As in (B), COMMD-GST fusion proteins and the indicated IAP proteins were co-expressed in 293 cells. Point mutations in the RING domains of XIAP, c-IAP1 and c-IAP2 were utilized (see text for details). Cell lysates were subsequently prepared and COMMD-GST proteins were precipitated. The presence of co-precipitated IAP proteins was detected by immunoblotting. Expression of COMMD-GST fusion proteins was confirmed by immunoblotting. (D) XIAP mediates the degradation of interacting COMMDs. COMMD1, 2, 4, 5, or 10 were expressed in 293 cells along with XIAP. Cell lysates were subsequently subjected to SDS-PAGE and immunoblotting for COMMD levels (Flag antibody), β-Actin (loading control) or XIAP.
Figure 2
Figure 2. Leucine repeats in the COMM domain determine XIAP binding
(A) Sequence alignment. The sequences for COMMD1, 2, 4, 5, and 10 were aligned across their COMM domains as previously described [1]. Highly conserved motifs are noted in the alignment. (B) Mutations introduced in the COMM domain. The alanine substitutions introduced are depicted. The W/P mutant targets W124 and P141, Mut1 targets the first leucine repeat (I145, L147, and L149A), Mut2 targets the second leucine repeat (L172, L175, and V178), and Mut1/2 targets both leucine repeats. (C) and (D) Effects of COMM domain mutations on XIAP binding. COMMD1 wild-type (WT) and the indicated mutations were expressed along with XIAP H467A in untreated cells (C) or along with either XIAP wild-type or XIAP H467A in cells treated with MG-132 for 3 hours (D). COMMD1 was subsequently precipitated from cell lysates and the presence of XIAP was determined by immunoblotting. (E) In vitro binding between XIAP and COMMD1. Recombinant COMMD1-GST fusion proteins were prepared in E. coli (wild-type or Mut1/2 versions) and were subsequently mixed with a lysate from cells expressing XIAP. After precipitation of COMMD1 and extensive washing of the GSH-sepharose beads, the material was resolved by SDS-PAGE and immunoblotted for XIAP (top panel) or for GST (bottom panel).
Figure 3
Figure 3. Mutations in the leucine repeats of COMMD1 stabilize the protein by impairing its ubiquitination
(A) and (B) Basal ubiquitination is greatly affected by mutations in the leucine repeats. COMMD1 was expressed in 293 cells and ubiquitinated proteins were subsequently precipitated from cell lysates with nickel-agarose beads (to precipitate expressed His6-tagged ubiquitin). The precipitated material was immunoblotted for COMMD1, and for ubiquitin (to ensure adequate precipitation). All mutants generated were tested in (A) and the mutations involving the leucine repeats were tested again in (B) in cells expressing increasing amounts of COMMD1. (C) Lack of ubiquitination of Mut1/2 in vitro. Radiolabeled wild-type of Mut1/2 COMMD1 were generated by in vitro translation and were subsequently offered as substrate for an in vitro ubiquitination reaction. At the end of the reaction, the material was resolved by SDS-PAGE and the proteins were detected by auto-radiography. Unmodified COMMD1 is indicated by arrows, while the ubiquitinated products are noted by markers to the left of the gel. (D) Mutations of the leucine repeats stabilize COMMD1. The wild-type protein or its mutants were transfected in a dose titrated manner and the resulting expression of COMMD1 was determined by immunoblotting. β-Actin was utilized as a loading control. (E) Mutations in the leucine repeats prevent XIAP-mediated degradation. COMMD1 wild-type or the indicated mutants were expressed along with XIAP and the levels of expression were determined by immunoblotting (upper panel). Expression of XIAP was confirmed (middle panel) and β-Actin was utilized as a loading control (bottom panel).
Figure 4
Figure 4. COMMD1 expression is controlled by post-transcriptional mechanisms
(A) and (B) Stable expression of COMMD1 Mut1/2 results in greater protein levels that COMMD1 wild-type. 293 cells were stably transfected with COMMD1 wild-type or Mut1/2 for over a month. Cell lysates for mRNA and protein extraction were prepared. COMMD1 mRNA levels were determined by qRT-PCR and normalized to the levels of GAPDH (top graph). COMMD1 protein levels were determined by immunoblotting, using β-Actin as a loading control (bottom panels). (C) RT-PCR detection strategy. Relative positions of oligonucleotides that recognize both endogenous and exogenous mRNA for COMMD1 are indicated. (D) Decreased expression of endogenous COMMD1 is not due to decreased endogenous mRNA expression. Utilizing the strategy depicted in (C), COMMD1 mRNA expression was determined by quantitative real-time RT-PCR (left and middle panels, total and endogenous expression). COMMD1 protein levels are also shown (right panel, SCHAD is a loading control).
Figure 5
Figure 5. COMMD1 ubiquitination is critical for controlling basal levels of COMMD1 expression
(A) Expression of wild-type COMMD1 results in increased ubiquitination. Cell lysates from the indicated stable cell lines were subjected to denaturing immunoprecipitation of COMMD1 using a rabbit polyclonal antibody. The recovered material was immunoblotted for COMMD1 using a mouse monoclonal antibody. Non-modified COMMD1 was readily detectable at the bottom of the gel (bottom panel) and modified high molecular weight (HMW) material was noticeable at the top of the gel on the WT lane only (upper panel). (B) Recovery of COMMD1 from a Triton-insoluble compartment. Lysates from the indicated stable cell lines were prepared with a Triton containing buffer (soluble fraction, middle panels), and the remaining pellet was lysed with an 8M urea buffer (insoluble fraction, bottom panels). In addition, a whole cell lysate was prepared by lysing cells directly in an 8M urea buffer (WCL, top panels). COMMD1 levels were determined by immunoblotting; β-Actin and histone 4 were utilized as loading controls. (C) IAP expression levels were unaffected in COMMD1 stable lines. Cell lysates were subjected to SDS-PAGE and were immunoblotted with an XIAP antibody (top panel) and then re-probed with an antibody that reacts with both c-IAP1 and c-IAP2 (middle panel). β-Actin serves as a loading control.
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References

    1. Burstein E, Hoberg JE, Wilkinson AS, Rumble JM, Csomos RA, Komarck CM, Maine GN, Wilkinson JC, Mayo MW, Duckett CS. COMMD proteins: A novel family of structural and functional homologs of MURR1. J Biol Chem. 2005;280:22222–22232. - PubMed
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