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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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Endogenous MARCH-8 regulates cell surface expression of endogenous TRAIL-R1.A, MCF-7Casp-3 cells were transfected to express GFP (−), together with either a control vector (−), with MARCH-8 targeting shRNA (8), or with two different MARCH-1-targeting shRNAs (1b and 1c). Endogenous TRAIL-R1 cell surface expression was determined by flow cytometric analysis and data were evaluated as described in the legend to Fig. 2. Data represent mean values ± S.D. from 3 independent experiments. Asterisks indicate statistically significant differences compared with GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; ***, p < 0.001). B, validation of the MARCH-8 rescue construct. MCF-7Casp-3 cells were transfected to express GFP-tagged MARCH-8 WT, or a MARCH-8 rescue (Rs) variant carrying silent mutations to allow escape from RNAi. Cells were cotransfected with empty vector (−) or MARCH-8 shRNA construct. MARCH-8.GFP expression was analyzed by immunoblotting for GFP in total cell lysates. C, MCF-7Casp-3 cells were transfected to express GFP (−), GFP-tagged MARCH-8 WT or Rs alone (−), or in conjunction MARCH-8 targeting shRNA (+). Endogenous TRAIL-R1 cell surface expression was determined by flow cytometric analysis and data were evaluated as described in the legend to Fig. 2. Data represent mean ± S.D. from at least 4 independent experiments. Asterisks indicate statistically significant differences compared with GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; ***, p < 0.001).
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Figure 8: Endogenous MARCH-8 regulates cell surface expression of endogenous TRAIL-R1.A, MCF-7Casp-3 cells were transfected to express GFP (−), together with either a control vector (−), with MARCH-8 targeting shRNA (8), or with two different MARCH-1-targeting shRNAs (1b and 1c). Endogenous TRAIL-R1 cell surface expression was determined by flow cytometric analysis and data were evaluated as described in the legend to Fig. 2. Data represent mean values ± S.D. from 3 independent experiments. Asterisks indicate statistically significant differences compared with GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; ***, p < 0.001). B, validation of the MARCH-8 rescue construct. MCF-7Casp-3 cells were transfected to express GFP-tagged MARCH-8 WT, or a MARCH-8 rescue (Rs) variant carrying silent mutations to allow escape from RNAi. Cells were cotransfected with empty vector (−) or MARCH-8 shRNA construct. MARCH-8.GFP expression was analyzed by immunoblotting for GFP in total cell lysates. C, MCF-7Casp-3 cells were transfected to express GFP (−), GFP-tagged MARCH-8 WT or Rs alone (−), or in conjunction MARCH-8 targeting shRNA (+). Endogenous TRAIL-R1 cell surface expression was determined by flow cytometric analysis and data were evaluated as described in the legend to Fig. 2. Data represent mean ± S.D. from at least 4 independent experiments. Asterisks indicate statistically significant differences compared with GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; ***, p < 0.001).

Mentions: We next used MARCH-8 shRNA to test whether endogenous MARCH-8 regulated endogenous TRAIL-R1 cell surface expression. MCF-7Casp-3 cells were transfected with either a control vector or a MARCH-8 targeting shRNA. Subsequently, cell surface expression of TRAIL-R1 and TRAIL-R2 was examined by flow cytometry. Expression of a MARCH-8 shRNA significantly up-regulated TRAIL-R1 cell surface expression, whereas TRAIL-R2 surface expression was unaffected (Fig. 8A). In contrast, expression of two different validated MARCH-1 targeting shRNAs had no effect on either TRAIL-R1 or TRAIL-R2 cell surface expression (supplemental Fig. S7 and Fig. 8A).


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)

Endogenous MARCH-8 regulates cell surface expression of endogenous TRAIL-R1.A, MCF-7Casp-3 cells were transfected to express GFP (−), together with either a control vector (−), with MARCH-8 targeting shRNA (8), or with two different MARCH-1-targeting shRNAs (1b and 1c). Endogenous TRAIL-R1 cell surface expression was determined by flow cytometric analysis and data were evaluated as described in the legend to Fig. 2. Data represent mean values ± S.D. from 3 independent experiments. Asterisks indicate statistically significant differences compared with GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; ***, p < 0.001). B, validation of the MARCH-8 rescue construct. MCF-7Casp-3 cells were transfected to express GFP-tagged MARCH-8 WT, or a MARCH-8 rescue (Rs) variant carrying silent mutations to allow escape from RNAi. Cells were cotransfected with empty vector (−) or MARCH-8 shRNA construct. MARCH-8.GFP expression was analyzed by immunoblotting for GFP in total cell lysates. C, MCF-7Casp-3 cells were transfected to express GFP (−), GFP-tagged MARCH-8 WT or Rs alone (−), or in conjunction MARCH-8 targeting shRNA (+). Endogenous TRAIL-R1 cell surface expression was determined by flow cytometric analysis and data were evaluated as described in the legend to Fig. 2. Data represent mean ± S.D. from at least 4 independent experiments. Asterisks indicate statistically significant differences compared with GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; ***, p < 0.001).
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Figure 8: Endogenous MARCH-8 regulates cell surface expression of endogenous TRAIL-R1.A, MCF-7Casp-3 cells were transfected to express GFP (−), together with either a control vector (−), with MARCH-8 targeting shRNA (8), or with two different MARCH-1-targeting shRNAs (1b and 1c). Endogenous TRAIL-R1 cell surface expression was determined by flow cytometric analysis and data were evaluated as described in the legend to Fig. 2. Data represent mean values ± S.D. from 3 independent experiments. Asterisks indicate statistically significant differences compared with GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; ***, p < 0.001). B, validation of the MARCH-8 rescue construct. MCF-7Casp-3 cells were transfected to express GFP-tagged MARCH-8 WT, or a MARCH-8 rescue (Rs) variant carrying silent mutations to allow escape from RNAi. Cells were cotransfected with empty vector (−) or MARCH-8 shRNA construct. MARCH-8.GFP expression was analyzed by immunoblotting for GFP in total cell lysates. C, MCF-7Casp-3 cells were transfected to express GFP (−), GFP-tagged MARCH-8 WT or Rs alone (−), or in conjunction MARCH-8 targeting shRNA (+). Endogenous TRAIL-R1 cell surface expression was determined by flow cytometric analysis and data were evaluated as described in the legend to Fig. 2. Data represent mean ± S.D. from at least 4 independent experiments. Asterisks indicate statistically significant differences compared with GFP-transfected control cells (one-way analysis of variance, Bonferroni correction; ***, p < 0.001).
Mentions: We next used MARCH-8 shRNA to test whether endogenous MARCH-8 regulated endogenous TRAIL-R1 cell surface expression. MCF-7Casp-3 cells were transfected with either a control vector or a MARCH-8 targeting shRNA. Subsequently, cell surface expression of TRAIL-R1 and TRAIL-R2 was examined by flow cytometry. Expression of a MARCH-8 shRNA significantly up-regulated TRAIL-R1 cell surface expression, whereas TRAIL-R2 surface expression was unaffected (Fig. 8A). In contrast, expression of two different validated MARCH-1 targeting shRNAs had no effect on either TRAIL-R1 or TRAIL-R2 cell surface expression (supplemental Fig. S7 and Fig. 8A).

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