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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 overexpression inhibits apoptosis induction by TRAIL.A, MCF-7Casp-3 cells were transfected to express either GFP only, or GFP-tagged MARCH-1, or -8. At 24 h after transfection, cells were stimulated with IZ-TRAIL for 5 h and caspase-3 cleavage was determined by flow cytometry in the GFP positive cell populations. B, as in A, with the following adaptations. Cells were transfected with WT MARCH-1 or -8, or with the MARCH-8 RING mutant described in the legend to Fig. 2. Cells were stimulated with TRAIL for 14 h, and cell death was read out by propidium iodide (PI) uptake. Data in A and B represent mean ± S.D. of values from 3 to 4 independent experiments. The percentage of cells with cleaved caspase-3 or PI uptake in the untreated control samples was subtracted. Asterisks indicate statistically significant differences between MARCH.GFP-transfected and GFP only-transfected control cells at the indicated concentration of TRAIL (Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001).
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Figure 3: MARCH overexpression inhibits apoptosis induction by TRAIL.A, MCF-7Casp-3 cells were transfected to express either GFP only, or GFP-tagged MARCH-1, or -8. At 24 h after transfection, cells were stimulated with IZ-TRAIL for 5 h and caspase-3 cleavage was determined by flow cytometry in the GFP positive cell populations. B, as in A, with the following adaptations. Cells were transfected with WT MARCH-1 or -8, or with the MARCH-8 RING mutant described in the legend to Fig. 2. Cells were stimulated with TRAIL for 14 h, and cell death was read out by propidium iodide (PI) uptake. Data in A and B represent mean ± S.D. of values from 3 to 4 independent experiments. The percentage of cells with cleaved caspase-3 or PI uptake in the untreated control samples was subtracted. Asterisks indicate statistically significant differences between MARCH.GFP-transfected and GFP only-transfected control cells at the indicated concentration of TRAIL (Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Mentions: To evaluate the possible implications of our findings for tumor therapy with TRAIL receptor agonists, we tested whether MARCH ligases altered the sensitivity of MCF-7Casp-3 cells to TRAIL-induced apoptosis. We focused on MARCH-1 and -8, because they are closely related (20), they are most clearly implicated in the endocytic trafficking of membrane proteins and consistently affected TRAIL-R1 cell surface expression in MCF-7Casp-3 and Mel JuSo cells. The cells were transiently transfected to express GFP-tagged MARCH-1 or MARCH-8, or GFP alone (control) and treated with soluble recombinant TRAIL, at different doses. Apoptosis signaling was read out at 5 h after TRAIL treatment, by flow cytometric detection of cleaved caspase-3 in the GFP-positive (MARCH- or control GFP expressing) cell population. In addition, cell death was read out by staining with propidium iodide at 14 h after TRAIL treatment, followed by flow cytometry. Both MARCH-1 and MARCH-8 expressing cells were significantly less sensitive to TRAIL-induced apoptosis than the control GFP expressing cells (Fig. 3, A and B). Data depicted here were obtained with isoleucine-zippered (IZ) TRAIL (26, 32), but similar results were obtained with FLAG-tagged TRAIL (supplemental Fig. S3). Expression of the MARCH-8 RING mutant did not confer resistance to TRAIL-induced cell death (Fig. 3B). These data indicate that MARCH ligase activity can determine the sensitivity of tumor cells to TRAIL-induced apoptosis.


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 overexpression inhibits apoptosis induction by TRAIL.A, MCF-7Casp-3 cells were transfected to express either GFP only, or GFP-tagged MARCH-1, or -8. At 24 h after transfection, cells were stimulated with IZ-TRAIL for 5 h and caspase-3 cleavage was determined by flow cytometry in the GFP positive cell populations. B, as in A, with the following adaptations. Cells were transfected with WT MARCH-1 or -8, or with the MARCH-8 RING mutant described in the legend to Fig. 2. Cells were stimulated with TRAIL for 14 h, and cell death was read out by propidium iodide (PI) uptake. Data in A and B represent mean ± S.D. of values from 3 to 4 independent experiments. The percentage of cells with cleaved caspase-3 or PI uptake in the untreated control samples was subtracted. Asterisks indicate statistically significant differences between MARCH.GFP-transfected and GFP only-transfected control cells at the indicated concentration of TRAIL (Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001).
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Figure 3: MARCH overexpression inhibits apoptosis induction by TRAIL.A, MCF-7Casp-3 cells were transfected to express either GFP only, or GFP-tagged MARCH-1, or -8. At 24 h after transfection, cells were stimulated with IZ-TRAIL for 5 h and caspase-3 cleavage was determined by flow cytometry in the GFP positive cell populations. B, as in A, with the following adaptations. Cells were transfected with WT MARCH-1 or -8, or with the MARCH-8 RING mutant described in the legend to Fig. 2. Cells were stimulated with TRAIL for 14 h, and cell death was read out by propidium iodide (PI) uptake. Data in A and B represent mean ± S.D. of values from 3 to 4 independent experiments. The percentage of cells with cleaved caspase-3 or PI uptake in the untreated control samples was subtracted. Asterisks indicate statistically significant differences between MARCH.GFP-transfected and GFP only-transfected control cells at the indicated concentration of TRAIL (Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001).
Mentions: To evaluate the possible implications of our findings for tumor therapy with TRAIL receptor agonists, we tested whether MARCH ligases altered the sensitivity of MCF-7Casp-3 cells to TRAIL-induced apoptosis. We focused on MARCH-1 and -8, because they are closely related (20), they are most clearly implicated in the endocytic trafficking of membrane proteins and consistently affected TRAIL-R1 cell surface expression in MCF-7Casp-3 and Mel JuSo cells. The cells were transiently transfected to express GFP-tagged MARCH-1 or MARCH-8, or GFP alone (control) and treated with soluble recombinant TRAIL, at different doses. Apoptosis signaling was read out at 5 h after TRAIL treatment, by flow cytometric detection of cleaved caspase-3 in the GFP-positive (MARCH- or control GFP expressing) cell population. In addition, cell death was read out by staining with propidium iodide at 14 h after TRAIL treatment, followed by flow cytometry. Both MARCH-1 and MARCH-8 expressing cells were significantly less sensitive to TRAIL-induced apoptosis than the control GFP expressing cells (Fig. 3, A and B). Data depicted here were obtained with isoleucine-zippered (IZ) TRAIL (26, 32), but similar results were obtained with FLAG-tagged TRAIL (supplemental Fig. S3). Expression of the MARCH-8 RING mutant did not confer resistance to TRAIL-induced cell death (Fig. 3B). These data indicate that MARCH ligase activity can determine the sensitivity of tumor cells to TRAIL-induced apoptosis.

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