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Ubiquitination by the membrane-associated RING-CH-8 (MARCH-8) ligase controls steady-state cell surface expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptor 1.

van de Kooij B, Verbrugge I, de Vries E, Gijsen M, Montserrat V, Maas C, Neefjes J, Borst J - J. Biol. Chem. (2013)

Bottom Line: The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored.Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression.These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.

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MARCH proteins preferentially down-regulate TRAIL-R1 cell surface levels in MCF-7Casp-3 cells.A, MCF-7Casp-3 breast cancer cells were transfected to express WT or K44A dynamin together with GFP. To detect endogenous TRAIL-R1 or TRAIL-R2 at the cell surface, the cells were stained with specific antibodies and a second step reagent, or with second step reagent only (Control) followed by flow cytometric analysis. Histograms of TRAIL-R fluorescence intensity (FI) in the GFP positive populations are shown. B and C, MCF-7Casp-3 cells were transfected to express GFP-tagged MARCH-1, -2, -4, -8, -9, or GFP only (Control) and cell surface levels of TRAIL-R1 (left) and TRAIL-R2 (right) were determined by flow cytometry. Representative histograms of TRAIL-R intensity in the GFP positive populations are shown in B. Panel C shows the quantification of TRAIL-R1 and -R2 expression in MCF-7Casp-3 cells expressing the indicated MARCH-GFP proteins or GFP only (−). The MFI, denoting TRAIL-R cell surface levels in GFP+ cells expressing MARCH-GFP or GFP only is expressed as percentage of the MFI in untransfected GFP− cells in the same cell population. Data represent mean ± S.D. of values from at least 3 independent experiments. Asterisks indicate statistically significant differences between MARCH-transfected and GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001). D, this experiment was performed and quantified as outlined in B and C, but in this case using the melanoma cell line Mel JuSo. Data represent mean ± S.D. of values from 2 independent experiments.
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Figure 1: MARCH proteins preferentially down-regulate TRAIL-R1 cell surface levels in MCF-7Casp-3 cells.A, MCF-7Casp-3 breast cancer cells were transfected to express WT or K44A dynamin together with GFP. To detect endogenous TRAIL-R1 or TRAIL-R2 at the cell surface, the cells were stained with specific antibodies and a second step reagent, or with second step reagent only (Control) followed by flow cytometric analysis. Histograms of TRAIL-R fluorescence intensity (FI) in the GFP positive populations are shown. B and C, MCF-7Casp-3 cells were transfected to express GFP-tagged MARCH-1, -2, -4, -8, -9, or GFP only (Control) and cell surface levels of TRAIL-R1 (left) and TRAIL-R2 (right) were determined by flow cytometry. Representative histograms of TRAIL-R intensity in the GFP positive populations are shown in B. Panel C shows the quantification of TRAIL-R1 and -R2 expression in MCF-7Casp-3 cells expressing the indicated MARCH-GFP proteins or GFP only (−). The MFI, denoting TRAIL-R cell surface levels in GFP+ cells expressing MARCH-GFP or GFP only is expressed as percentage of the MFI in untransfected GFP− cells in the same cell population. Data represent mean ± S.D. of values from at least 3 independent experiments. Asterisks indicate statistically significant differences between MARCH-transfected and GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001). D, this experiment was performed and quantified as outlined in B and C, but in this case using the melanoma cell line Mel JuSo. Data represent mean ± S.D. of values from 2 independent experiments.

Mentions: Interestingly, this experiment revealed a differential impact of K44A dynamin-1 on the steady-state cell surface expression of TRAIL-R1 versus TRAIL-R2. In cells that expressed high levels of dynamin, as revealed by high GFP expression, the K44A mutant specifically up-regulated cell surface expression of TRAIL-R1, whereas it did not affect TRAIL-R2 expression (Fig. 1A). This indicated that, at steady-state, TRAIL-R1 has a higher turnover by dynamin-dependent endocytosis than TRAIL-R2.


Ubiquitination by the membrane-associated RING-CH-8 (MARCH-8) ligase controls steady-state cell surface expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptor 1.

van de Kooij B, Verbrugge I, de Vries E, Gijsen M, Montserrat V, Maas C, Neefjes J, Borst J - J. Biol. Chem. (2013)

MARCH proteins preferentially down-regulate TRAIL-R1 cell surface levels in MCF-7Casp-3 cells.A, MCF-7Casp-3 breast cancer cells were transfected to express WT or K44A dynamin together with GFP. To detect endogenous TRAIL-R1 or TRAIL-R2 at the cell surface, the cells were stained with specific antibodies and a second step reagent, or with second step reagent only (Control) followed by flow cytometric analysis. Histograms of TRAIL-R fluorescence intensity (FI) in the GFP positive populations are shown. B and C, MCF-7Casp-3 cells were transfected to express GFP-tagged MARCH-1, -2, -4, -8, -9, or GFP only (Control) and cell surface levels of TRAIL-R1 (left) and TRAIL-R2 (right) were determined by flow cytometry. Representative histograms of TRAIL-R intensity in the GFP positive populations are shown in B. Panel C shows the quantification of TRAIL-R1 and -R2 expression in MCF-7Casp-3 cells expressing the indicated MARCH-GFP proteins or GFP only (−). The MFI, denoting TRAIL-R cell surface levels in GFP+ cells expressing MARCH-GFP or GFP only is expressed as percentage of the MFI in untransfected GFP− cells in the same cell population. Data represent mean ± S.D. of values from at least 3 independent experiments. Asterisks indicate statistically significant differences between MARCH-transfected and GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001). D, this experiment was performed and quantified as outlined in B and C, but in this case using the melanoma cell line Mel JuSo. Data represent mean ± S.D. of values from 2 independent experiments.
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Related In: Results  -  Collection

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Figure 1: MARCH proteins preferentially down-regulate TRAIL-R1 cell surface levels in MCF-7Casp-3 cells.A, MCF-7Casp-3 breast cancer cells were transfected to express WT or K44A dynamin together with GFP. To detect endogenous TRAIL-R1 or TRAIL-R2 at the cell surface, the cells were stained with specific antibodies and a second step reagent, or with second step reagent only (Control) followed by flow cytometric analysis. Histograms of TRAIL-R fluorescence intensity (FI) in the GFP positive populations are shown. B and C, MCF-7Casp-3 cells were transfected to express GFP-tagged MARCH-1, -2, -4, -8, -9, or GFP only (Control) and cell surface levels of TRAIL-R1 (left) and TRAIL-R2 (right) were determined by flow cytometry. Representative histograms of TRAIL-R intensity in the GFP positive populations are shown in B. Panel C shows the quantification of TRAIL-R1 and -R2 expression in MCF-7Casp-3 cells expressing the indicated MARCH-GFP proteins or GFP only (−). The MFI, denoting TRAIL-R cell surface levels in GFP+ cells expressing MARCH-GFP or GFP only is expressed as percentage of the MFI in untransfected GFP− cells in the same cell population. Data represent mean ± S.D. of values from at least 3 independent experiments. Asterisks indicate statistically significant differences between MARCH-transfected and GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001). D, this experiment was performed and quantified as outlined in B and C, but in this case using the melanoma cell line Mel JuSo. Data represent mean ± S.D. of values from 2 independent experiments.
Mentions: Interestingly, this experiment revealed a differential impact of K44A dynamin-1 on the steady-state cell surface expression of TRAIL-R1 versus TRAIL-R2. In cells that expressed high levels of dynamin, as revealed by high GFP expression, the K44A mutant specifically up-regulated cell surface expression of TRAIL-R1, whereas it did not affect TRAIL-R2 expression (Fig. 1A). This indicated that, at steady-state, TRAIL-R1 has a higher turnover by dynamin-dependent endocytosis than TRAIL-R2.

Bottom Line: The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored.Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression.These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.

Show MeSH
Related in: MedlinePlus