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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-8 requires a functional RING domain to down-regulate TRAIL-R1 cell surface expression. MCF-7Casp-3 cells were transfected to express GFP only (−), or GFP-tagged WT MARCH-8, or a MARCH-8 variant carrying ligase-inactivating mutations in its RING domain (MARCH-8 RING). Cell surface levels of TRAIL-R1 were determined by antibody staining, followed by flow cytometric analysis. A, schematic depiction of MARCH-8, on a relative scale, with indication of the RING-CH domain and transmembrane (TM) segments, as well as the three point mutations. B, primary data from a representative experiment, showing histograms of TRAIL-R1 cell surface expression (fluorescence intensity, FI) in GFP+ (MARCH-transfected) cells. C, TRAIL-R1 cell surface expression was quantified and statistically analyzed as described in the legend to Fig. 1C. Data represent mean ± S.D. of values from 3 independent experiments. Asterisk indicates statistically significant differences between cells with WT MARCH-8 versus control or the RING mutant (one-way analysis of variance, Bonferroni correction; *, p < 0.05).
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Figure 2: MARCH-8 requires a functional RING domain to down-regulate TRAIL-R1 cell surface expression. MCF-7Casp-3 cells were transfected to express GFP only (−), or GFP-tagged WT MARCH-8, or a MARCH-8 variant carrying ligase-inactivating mutations in its RING domain (MARCH-8 RING). Cell surface levels of TRAIL-R1 were determined by antibody staining, followed by flow cytometric analysis. A, schematic depiction of MARCH-8, on a relative scale, with indication of the RING-CH domain and transmembrane (TM) segments, as well as the three point mutations. B, primary data from a representative experiment, showing histograms of TRAIL-R1 cell surface expression (fluorescence intensity, FI) in GFP+ (MARCH-transfected) cells. C, TRAIL-R1 cell surface expression was quantified and statistically analyzed as described in the legend to Fig. 1C. Data represent mean ± S.D. of values from 3 independent experiments. Asterisk indicates statistically significant differences between cells with WT MARCH-8 versus control or the RING mutant (one-way analysis of variance, Bonferroni correction; *, p < 0.05).

Mentions: As MARCH proteins are ubiquitin ligases, we hypothesized that they down-regulate TRAIL-R1 cell surface expression by virtue of their ligase activity. To test this, we created a ligase-dead MARCH-8 variant (MARCH-8 RING) by mutating three conserved residues in its RING-CH domain (H107N, C110S, W114S; Fig. 2A). The impact of WT MARCH-8 versus the MARCH-8 RING mutant on TRAIL-R1 cell surface levels in MCF-7Casp-3 cells was analyzed by flow cytometry, as outlined above for Fig. 1. A representative histogram is shown in Fig. 2B. Quantification of multiple experiments demonstrated that in contrast to WT MARCH-8, the MARCH-8 RING mutant did not down-regulate TRAIL-R1 cell surface levels (Fig. 2C). Thus, MARCH-8 requires an intact RING domain to reduce TRAIL-R1 cell surface expression. This indicates that the ligase activity of MARCH-8 is essential for TRAIL-R1 down-regulation.


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-8 requires a functional RING domain to down-regulate TRAIL-R1 cell surface expression. MCF-7Casp-3 cells were transfected to express GFP only (−), or GFP-tagged WT MARCH-8, or a MARCH-8 variant carrying ligase-inactivating mutations in its RING domain (MARCH-8 RING). Cell surface levels of TRAIL-R1 were determined by antibody staining, followed by flow cytometric analysis. A, schematic depiction of MARCH-8, on a relative scale, with indication of the RING-CH domain and transmembrane (TM) segments, as well as the three point mutations. B, primary data from a representative experiment, showing histograms of TRAIL-R1 cell surface expression (fluorescence intensity, FI) in GFP+ (MARCH-transfected) cells. C, TRAIL-R1 cell surface expression was quantified and statistically analyzed as described in the legend to Fig. 1C. Data represent mean ± S.D. of values from 3 independent experiments. Asterisk indicates statistically significant differences between cells with WT MARCH-8 versus control or the RING mutant (one-way analysis of variance, Bonferroni correction; *, p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: MARCH-8 requires a functional RING domain to down-regulate TRAIL-R1 cell surface expression. MCF-7Casp-3 cells were transfected to express GFP only (−), or GFP-tagged WT MARCH-8, or a MARCH-8 variant carrying ligase-inactivating mutations in its RING domain (MARCH-8 RING). Cell surface levels of TRAIL-R1 were determined by antibody staining, followed by flow cytometric analysis. A, schematic depiction of MARCH-8, on a relative scale, with indication of the RING-CH domain and transmembrane (TM) segments, as well as the three point mutations. B, primary data from a representative experiment, showing histograms of TRAIL-R1 cell surface expression (fluorescence intensity, FI) in GFP+ (MARCH-transfected) cells. C, TRAIL-R1 cell surface expression was quantified and statistically analyzed as described in the legend to Fig. 1C. Data represent mean ± S.D. of values from 3 independent experiments. Asterisk indicates statistically significant differences between cells with WT MARCH-8 versus control or the RING mutant (one-way analysis of variance, Bonferroni correction; *, p < 0.05).
Mentions: As MARCH proteins are ubiquitin ligases, we hypothesized that they down-regulate TRAIL-R1 cell surface expression by virtue of their ligase activity. To test this, we created a ligase-dead MARCH-8 variant (MARCH-8 RING) by mutating three conserved residues in its RING-CH domain (H107N, C110S, W114S; Fig. 2A). The impact of WT MARCH-8 versus the MARCH-8 RING mutant on TRAIL-R1 cell surface levels in MCF-7Casp-3 cells was analyzed by flow cytometry, as outlined above for Fig. 1. A representative histogram is shown in Fig. 2B. Quantification of multiple experiments demonstrated that in contrast to WT MARCH-8, the MARCH-8 RING mutant did not down-regulate TRAIL-R1 cell surface levels (Fig. 2C). Thus, MARCH-8 requires an intact RING domain to reduce TRAIL-R1 cell surface expression. This indicates that the ligase activity of MARCH-8 is essential for TRAIL-R1 down-regulation.

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