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. 2012 Jan 20;287(4):2485-99.
doi: 10.1074/jbc.M111.302216. Epub 2011 Nov 30.

Clusterin and COMMD1 independently regulate degradation of the mammalian copper ATPases ATP7A and ATP7B

Affiliations

Clusterin and COMMD1 independently regulate degradation of the mammalian copper ATPases ATP7A and ATP7B

Stephanie Materia et al. J Biol Chem. .

Abstract

ATP7A and ATP7B are copper-transporting P(1B)-type ATPases (Cu-ATPases) that are critical for regulating intracellular copper homeostasis. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and copper toxicity disorders, Menkes and Wilson diseases, respectively. Clusterin and COMMD1 were previously identified as interacting partners of these Cu-ATPases. In this study, we confirmed that clusterin and COMMD1 interact to down-regulate both ATP7A and ATP7B. Overexpression and knockdown of clusterin/COMMD1 decreased and increased, respectively, endogenous levels of ATP7A and ATP7B, consistent with a role in facilitating Cu-ATPase degradation. We demonstrate that whereas the clusterin/ATP7B interaction was enhanced by oxidative stress or mutation of ATP7B, the COMMD1/ATP7B interaction did not change under oxidative stress conditions, and only increased with ATP7B mutations that led to its misfolding. Clusterin and COMMD1 facilitated the degradation of ATP7B containing the same Wilson disease-causing C-terminal mutations via different degradation pathways, clusterin via the lysosomal pathway and COMMD1 via the proteasomal pathway. Furthermore, endogenous ATP7B existed in a complex with clusterin and COMMD1, but these interactions were neither competitive nor cooperative and occurred independently of each other. Together these data indicate that clusterin and COMMD1 represent alternative and independent systems regulating Cu-ATPase quality control, and consequently contributing to the maintenance of copper homeostasis.

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Figures

FIGURE 1.
FIGURE 1.
ATP7B, clusterin, and COMMD1 exist in a complex together. Co-immunoprecipitation of endogenous (A) ATP7B and clusterin, (B) ATP7B and COMMD1, (C) ATP7A and COMMD1, and (D) clusterin and COMMD1 from HEK293T cells. Dynabeads (Protein G) were bound with the antibody as indicated (IP Ab). Proteins were co-immunoprecipitated (IP) from whole cell lysates and then fractionated and immunoblotted with anti-ATP7A, anti-ATP7B, anti-clusterin, and anti-COMMD1 antibodies (WB Ab). Preimmune serum was used as a negative control. E, sequential co-immunoprecipitation of ATP7B, clusterin, and COMMD1. Dynabeads bound with anti-ATP7B antibody (IP Ab) were used to co-immunoprecipitate ATP7B and any interacting proteins from HEK293T cell lysates. Once eluted, the protein samples were re-precipitated with the anti-COMMD1 antibody (IP Ab). Proteins were again eluted, then fractionated and immunoblotted with anti-ATP7B, anti-clusterin, and anti-COMMD1 antibodies (WB Ab). Preimmune serum was used as a negative control. WB, Western blot.
FIGURE 2.
FIGURE 2.
Oxidative stress has no affect on the interaction between the Cu-ATPases and COMMD1. Co-immunoprecipitation of endogenous (A, i) ATP7B and COMMD1 and (B, i) ATP7A and COMMD1 from HEK293T cells that were untreated or treated with CuCl2 (200 μm, 24 h), bathocuproinedisulfonic acid (BCS)/d-penicillamine (D-PEN) (200 μm 72 h), diamide (10 mm, 15 min), or H2O2 (2 mm, 20 min). Cell lysates and co-immunoprecipitated (IP) proteins were fractionated and immunoblotted with anti-ATP7B, anti-ATP7A, anti-COMMD1, or anti-β-actin antibodies (WB Ab). A (ii) and B (ii) densitometric analysis of co-precipitated COMMD1 relative to ATP7B or ATP7A, respectively, expressed as relative optical density and representing the mean ± S.E. (n = 3). Asterisks indicate values that are significantly different, *, p < 0.05. WB, Western blot.
FIGURE 3.
FIGURE 3.
Increased binding of COMMD1 to unstable ATP7B mutants MBD1–6del and MBD3–5del. A, schematic representation of the N terminus of WT-ATP7B and truncated mutants MBD1–6del and MBD3–5del. B, co-immunoprecipitation (IP) of endogenous COMMD1 and stably expressed WT-ATP7B, MBD1–6del, or MBD3–5del from CHO-K1 cells cultured at either 37 or 30 °C. Cell lysates and co-immunoprecipitated proteins were fractionated and immunoblotted with anti-ATP7B, anti-COMMD1, and anti-β-actin antibodies (WB Ab). Densitometric analysis of co-immunoprecipitated COMMD1 relative to immunoprecipitated ATP7B is shown, expressed as relative optical density and representing the mean ± S.E. (n = 3). Asterisks indicate values that are significantly different, *, p < 0.05. WB, Western blot.
FIGURE 4.
FIGURE 4.
COMMD1 and clusterin interact independently with ATP7B. Co-immunoprecipitation (IP) of endogenous ATP7B and COMMD1 from HEK293T cells transiently transfected with a clusterin cDNA construct (A, i) and a clusterin shRNA plasmid construct (B, i). Cell lysates and co-immunoprecipitated proteins were fractionated and immunoblotted with anti-ATP7B, anti-clusterin, anti-COMMD1, and anti-β-actin antibodies (WB Ab). Preimmune serum was used as a negative control. C, (i) co-immunoprecipitation of endogenous ATP7B and clusterin from HEK293T cells transiently transfected with either a COMMD1 shRNA plasmid construct or a COMMD1-FLAG cDNA construct. Cell lysates and co-immunoprecipitated proteins were fractionated and immunoblotted with anti-ATP7B, anti-clusterin, anti-COMMD1, and anti-β-tubulin antibodies (WB Ab). Preimmune serum was used as a negative control. Densitometric analysis of co-immunoprecipitated COMMD1 ((A, ii) and (B, ii)) and clusterin (C, ii) relative to immunoprecipitated ATP7B is shown, expressed as relative optical density and representing the mean ± S.E. (n = 3). Asterisks indicate values that are significantly different, *, p < 0.05. WB, Western blot.
FIGURE 5.
FIGURE 5.
Clusterin and COMMD1 affect the levels of endogenous ATP7A and ATP7B. A, (i) HEK293T cells were transiently transfected with an empty vector or plasmids encoding clusterin or COMMD1 shRNAs. Proteins were fractionated and immunoblotted with anti-ATP7A, anti-ATP7B, anti-clusterin, anti-COMMD1, and anti-β-actin antibodies. B, (i) HEK293T cells were transiently transfected with an empty vector or either clusterin or COMMD1 cDNA constructs. Proteins were fractionated and immunoblotted with anti-ATP7A, anti-ATP7B, anti-clusterin, anti-COMMD1, and anti-β-actin antibodies. A (ii) and B (ii), densitometric analysis of ATP7A and ATP7B levels, expressed as relative optical density and representing the mean ± S.E. (n = 3). Asterisks indicate values that are significantly different, *, p < 0.05. WB, Western blot.
FIGURE 6.
FIGURE 6.
COMMD1 plays a role in determining the stability of ATP7A. A, schematic representation of the N terminus of WT-ATP7A, ATP7Am4–6, and ATP7Am1–6. B (i), HEK293T cells were transiently transfected with the empty vector or cDNA constructs encoding WT-ATP7A, WT-ATP7A, and COMMD1-FLAG, or ATP7Am4–6 and COMMD1-FLAG for 48 h. Proteins were fractionated and immunoblotted with anti-ATP7A, anti-COMMD1, and anti-β-actin antibodies. C, CHO-K1 cells stably expressing WT-ATP7A, ATP7Am4–6, and ATP7Am1–6 were transiently transfected with the empty vector or cDNA constructs encoding COMMD1-FLAG for 48 h. Proteins were fractionated and immunoblotted with anti-ATP7A, anti-COMMD1, and anti-β-actin antibodies. D (i), co-immunoprecipitation of endogenous COMMD1 and either WT-ATP7A, ATP7Am4–6, or ATP7Am1–6 stably expressed in CHO-K1 cells. Cell lysates and co-immunoprecipitated proteins were fractionated and immunoblotted with anti-ATP7A, anti-COMMD1, and anti-β-actin antibodies. Preimmune serum was used as a negative control. B (ii) and D (ii), densitometric analysis of ATP7A levels or co-immunoprecipitated COMMD1 levels relative to ATP7A, respectively, expressed as relative optical density and representing the mean ± S.E. (n = 3). Asterisks indicate values that are significantly different, *, p < 0.05. WB, Western blot.
FIGURE 7.
FIGURE 7.
Clusterin and COMMD1 target ATP7B for degradation via different pathways. CHO-K1 cells stably expressing (A and B) Atp7b-tx or (C and D) ATP7B-4193del were co-transfected with cDNA constructs encoding either clusterin or COMMD1-FLAG for transient expression. Cells were incubated with CXH (53 μm, 8 h) in the absence and presence of (A and C) MG132 (5 μm, 8 h) (B and D) NH4Cl (5 μm, 20 min) or leupeptin (100 μm, 8 h). Cell lysates were fractionated and immunoblotted with anti-ATP7B, anti-clusterin, anti-FLAG, and anti-β-actin antibodies. A (ii), B (ii), C (ii), and D (ii), densitometric analysis of ATP7B levels, expressed as relative optical density and representing the mean ± S.E. (n = 3). Asterisks indicate values that are significantly different, *, p < 0.05.

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