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Tumour suppressor TRIM33 targets nuclear β-catenin degradation.

Xue J, Chen Y, Wu Y, Wang Z, Zhou A, Zhang S, Lin K, Aldape K, Majumder S, Lu Z, Huang S - Nat Commun (2015)

Bottom Line: Here we demonstrate that the tripartite motif-containing protein 33 (TRIM33), acting as an E3 ubiquitin ligase, reduces the abundance of nuclear β-catenin protein.In human glioblastoma specimens, endogenous TRIM33 levels are inversely correlated with β-catenin.In summary, our findings identify TRIM33 as a tumour suppressor that can abolish tumour cell proliferation and tumorigenesis by degrading nuclear β-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
Aberrant activation of β-catenin in the nucleus has been implicated in a variety of human cancers, but the fate of nuclear β-catenin is unknown. Here we demonstrate that the tripartite motif-containing protein 33 (TRIM33), acting as an E3 ubiquitin ligase, reduces the abundance of nuclear β-catenin protein. TRIM33-mediated β-catenin is destabilized and is GSK-3β or β-TrCP independent. TRIM33 interacts with and ubiquitylates nuclear β-catenin. Moreover, protein kinase Cδ, which directly phosphorylates β-catenin at Ser715, is required for the TRIM33-β-catenin interaction. The function of TRIM33 in suppressing tumour cell proliferation and brain tumour development depends on TRIM33-promoted β-catenin degradation. In human glioblastoma specimens, endogenous TRIM33 levels are inversely correlated with β-catenin. In summary, our findings identify TRIM33 as a tumour suppressor that can abolish tumour cell proliferation and tumorigenesis by degrading nuclear β-catenin. This work suggests a new therapeutic strategy against human cancers caused by aberrant activation of β-catenin.

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TRIM33 ubiquitylates nuclear β-catenina, HEK 293T cells with or without TRIM33 depletion by siRNA were transfected as indicated. Cells were treated with MG132 (10 μM for 4 h) prior to lysis and then subjected to anti-β-catenin IP followed by anti-HA with immunoblot analysis. b, HEK 293T cells with or without TRIM33 depletion by shRNA were transfected as indicated. Experiments were performed as described for a. c, Nuclear extracts of U87/EGFRvIII cells pretreated with MG132 (10 μM for 4 h) were immunoprecipitated. ΔRING, TRIM33 lacking the RING domain; CAmut, TRIM33 with point mutations (C125A/C128A) in the RING domain. d, In vivo ubiquitination assays performed in HEK 293T cells transiently transfected with HA-tagged K63-only ubiquitin or K48-only ubiquitin. e, TRIM33 ubiquitylation of β-catenin in vitro. Purified β-catenin was subjected to in vitro ubiquitylation by TRIM33 in the absence or presence of active PKCδ. β-Catenin was detected with anti-GST antibody. f & g, U87 cells with or without TRIM33 depletion were transfected with β-TrCP siRNA or control siRNA. Cells were treated for 8 h with 100 ng/ml Wnt-3a (Wnt-on) (f) or with 100 ng DKK1 (Wnt-off) (g). Experiments were performed as described for a.
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Figure 4: TRIM33 ubiquitylates nuclear β-catenina, HEK 293T cells with or without TRIM33 depletion by siRNA were transfected as indicated. Cells were treated with MG132 (10 μM for 4 h) prior to lysis and then subjected to anti-β-catenin IP followed by anti-HA with immunoblot analysis. b, HEK 293T cells with or without TRIM33 depletion by shRNA were transfected as indicated. Experiments were performed as described for a. c, Nuclear extracts of U87/EGFRvIII cells pretreated with MG132 (10 μM for 4 h) were immunoprecipitated. ΔRING, TRIM33 lacking the RING domain; CAmut, TRIM33 with point mutations (C125A/C128A) in the RING domain. d, In vivo ubiquitination assays performed in HEK 293T cells transiently transfected with HA-tagged K63-only ubiquitin or K48-only ubiquitin. e, TRIM33 ubiquitylation of β-catenin in vitro. Purified β-catenin was subjected to in vitro ubiquitylation by TRIM33 in the absence or presence of active PKCδ. β-Catenin was detected with anti-GST antibody. f & g, U87 cells with or without TRIM33 depletion were transfected with β-TrCP siRNA or control siRNA. Cells were treated for 8 h with 100 ng/ml Wnt-3a (Wnt-on) (f) or with 100 ng DKK1 (Wnt-off) (g). Experiments were performed as described for a.

Mentions: TRIM33 acts as an E3 ubiquitin ligase for several substrates18. Thus, we investigated whether TRIM33 could promote the ubiquitination of β-catenin. We found that TRIM33 depletion with siRNA or shRNA decreased the level of ubiquitination of nuclear β-catenin in HEK 293T cells or U87 cells (Fig. 4a, 4b and Supplementary Fig. 4d). Conversely, TRIM33 overexpression considerably increased the level of ubiquitination of nuclear β-catenin in U87/EGFRvIII cells (Fig. 4c). Furthermore, TRIM33 lacking the RING domain and TRIM33 with point mutations (C125A/C128A) in the RING domain each substantially reduced the ability of TRIM33 to promote the ubiquitination of nuclear β-catenin (Fig. 4c), which confirmed that the RING domain or its ubiquitin ligase activity is indispensable for TRIM33-induced β-catenin degradation.


Tumour suppressor TRIM33 targets nuclear β-catenin degradation.

Xue J, Chen Y, Wu Y, Wang Z, Zhou A, Zhang S, Lin K, Aldape K, Majumder S, Lu Z, Huang S - Nat Commun (2015)

TRIM33 ubiquitylates nuclear β-catenina, HEK 293T cells with or without TRIM33 depletion by siRNA were transfected as indicated. Cells were treated with MG132 (10 μM for 4 h) prior to lysis and then subjected to anti-β-catenin IP followed by anti-HA with immunoblot analysis. b, HEK 293T cells with or without TRIM33 depletion by shRNA were transfected as indicated. Experiments were performed as described for a. c, Nuclear extracts of U87/EGFRvIII cells pretreated with MG132 (10 μM for 4 h) were immunoprecipitated. ΔRING, TRIM33 lacking the RING domain; CAmut, TRIM33 with point mutations (C125A/C128A) in the RING domain. d, In vivo ubiquitination assays performed in HEK 293T cells transiently transfected with HA-tagged K63-only ubiquitin or K48-only ubiquitin. e, TRIM33 ubiquitylation of β-catenin in vitro. Purified β-catenin was subjected to in vitro ubiquitylation by TRIM33 in the absence or presence of active PKCδ. β-Catenin was detected with anti-GST antibody. f & g, U87 cells with or without TRIM33 depletion were transfected with β-TrCP siRNA or control siRNA. Cells were treated for 8 h with 100 ng/ml Wnt-3a (Wnt-on) (f) or with 100 ng DKK1 (Wnt-off) (g). Experiments were performed as described for a.
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Related In: Results  -  Collection

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Figure 4: TRIM33 ubiquitylates nuclear β-catenina, HEK 293T cells with or without TRIM33 depletion by siRNA were transfected as indicated. Cells were treated with MG132 (10 μM for 4 h) prior to lysis and then subjected to anti-β-catenin IP followed by anti-HA with immunoblot analysis. b, HEK 293T cells with or without TRIM33 depletion by shRNA were transfected as indicated. Experiments were performed as described for a. c, Nuclear extracts of U87/EGFRvIII cells pretreated with MG132 (10 μM for 4 h) were immunoprecipitated. ΔRING, TRIM33 lacking the RING domain; CAmut, TRIM33 with point mutations (C125A/C128A) in the RING domain. d, In vivo ubiquitination assays performed in HEK 293T cells transiently transfected with HA-tagged K63-only ubiquitin or K48-only ubiquitin. e, TRIM33 ubiquitylation of β-catenin in vitro. Purified β-catenin was subjected to in vitro ubiquitylation by TRIM33 in the absence or presence of active PKCδ. β-Catenin was detected with anti-GST antibody. f & g, U87 cells with or without TRIM33 depletion were transfected with β-TrCP siRNA or control siRNA. Cells were treated for 8 h with 100 ng/ml Wnt-3a (Wnt-on) (f) or with 100 ng DKK1 (Wnt-off) (g). Experiments were performed as described for a.
Mentions: TRIM33 acts as an E3 ubiquitin ligase for several substrates18. Thus, we investigated whether TRIM33 could promote the ubiquitination of β-catenin. We found that TRIM33 depletion with siRNA or shRNA decreased the level of ubiquitination of nuclear β-catenin in HEK 293T cells or U87 cells (Fig. 4a, 4b and Supplementary Fig. 4d). Conversely, TRIM33 overexpression considerably increased the level of ubiquitination of nuclear β-catenin in U87/EGFRvIII cells (Fig. 4c). Furthermore, TRIM33 lacking the RING domain and TRIM33 with point mutations (C125A/C128A) in the RING domain each substantially reduced the ability of TRIM33 to promote the ubiquitination of nuclear β-catenin (Fig. 4c), which confirmed that the RING domain or its ubiquitin ligase activity is indispensable for TRIM33-induced β-catenin degradation.

Bottom Line: Here we demonstrate that the tripartite motif-containing protein 33 (TRIM33), acting as an E3 ubiquitin ligase, reduces the abundance of nuclear β-catenin protein.In human glioblastoma specimens, endogenous TRIM33 levels are inversely correlated with β-catenin.In summary, our findings identify TRIM33 as a tumour suppressor that can abolish tumour cell proliferation and tumorigenesis by degrading nuclear β-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
Aberrant activation of β-catenin in the nucleus has been implicated in a variety of human cancers, but the fate of nuclear β-catenin is unknown. Here we demonstrate that the tripartite motif-containing protein 33 (TRIM33), acting as an E3 ubiquitin ligase, reduces the abundance of nuclear β-catenin protein. TRIM33-mediated β-catenin is destabilized and is GSK-3β or β-TrCP independent. TRIM33 interacts with and ubiquitylates nuclear β-catenin. Moreover, protein kinase Cδ, which directly phosphorylates β-catenin at Ser715, is required for the TRIM33-β-catenin interaction. The function of TRIM33 in suppressing tumour cell proliferation and brain tumour development depends on TRIM33-promoted β-catenin degradation. In human glioblastoma specimens, endogenous TRIM33 levels are inversely correlated with β-catenin. In summary, our findings identify TRIM33 as a tumour suppressor that can abolish tumour cell proliferation and tumorigenesis by degrading nuclear β-catenin. This work suggests a new therapeutic strategy against human cancers caused by aberrant activation of β-catenin.

Show MeSH
Related in: MedlinePlus