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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 targets endogenous TRAIL-R1 for lysosomal degradation.A and B, H358 cells were treated with cycloheximide (CHX; 50 μg/ml) alone, or with CHX in combination with bafilomycin A1 (Baf A1; 200 nm) or MG132 (10 μm) for 16 h. Total cell lysates were analyzed by immunoblotting with α-TRAIL-R1 and α-actin antibodies. A, data of a representative experiment. B, mean ± S.D. of quantified values from 3 independent experiments. TRAIL-R1 intensity was corrected for actin expression, the control value was set to 100%. Asterisk indicates statistically significant difference (Student's t test; *, p < 0.05). C and D, MCF-7Casp-3 cells were transfected to express GFP only (−), or GFP-tagged MARCH-8. Cells were treated with BafA1 (100 nm) or left untreated for 16 h. Total cell lysates of GFP+ cells, obtained by flow cytometric sorting, were analyzed by immunoblotting with α-TRAIL-R1 and α-actin antibodies, and α-GFP antibody to detect MARCH-8. Solid and open arrowheads indicate, respectively, MARCH-8.GFP and GFP only. C, data of a representative experiment. D, mean ± S.D. of quantified values from 3 independent experiments. TRAIL-R1 intensity was corrected for actin expression, control value was set to 100%. Asterisk indicates a statistically significant difference (Student's t test; *, p < 0.05).
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Figure 6: MARCH-8 targets endogenous TRAIL-R1 for lysosomal degradation.A and B, H358 cells were treated with cycloheximide (CHX; 50 μg/ml) alone, or with CHX in combination with bafilomycin A1 (Baf A1; 200 nm) or MG132 (10 μm) for 16 h. Total cell lysates were analyzed by immunoblotting with α-TRAIL-R1 and α-actin antibodies. A, data of a representative experiment. B, mean ± S.D. of quantified values from 3 independent experiments. TRAIL-R1 intensity was corrected for actin expression, the control value was set to 100%. Asterisk indicates statistically significant difference (Student's t test; *, p < 0.05). C and D, MCF-7Casp-3 cells were transfected to express GFP only (−), or GFP-tagged MARCH-8. Cells were treated with BafA1 (100 nm) or left untreated for 16 h. Total cell lysates of GFP+ cells, obtained by flow cytometric sorting, were analyzed by immunoblotting with α-TRAIL-R1 and α-actin antibodies, and α-GFP antibody to detect MARCH-8. Solid and open arrowheads indicate, respectively, MARCH-8.GFP and GFP only. C, data of a representative experiment. D, mean ± S.D. of quantified values from 3 independent experiments. TRAIL-R1 intensity was corrected for actin expression, control value was set to 100%. Asterisk indicates a statistically significant difference (Student's t test; *, p < 0.05).

Mentions: Because MARCH-1 and -8 down-regulated endogenous TRAIL-R1 from the cell surface (Fig. 1) and reduced total expression levels of exogenous TRAIL-R1 (Fig. 5), we hypothesized that MARCH-1 and -8 targeted TRAIL-R1 for degradation. To test this, we first studied the mechanism by which endogenous TRAIL-R1 was turned over at steady state. For this purpose, we used H358 lung cancer cells, as they express larger amounts of TRAIL-R1 than MCF-7Casp-3 cells (supplemental Fig. S6). After a 16-h treatment with the translation inhibitor cycloheximide (CHX), endogenous TRAIL-R1 levels were reduced to about 60% of untreated control levels (Fig. 6, A and B). Recoveries of TRAIL-R1 from Nonidet P-40 lysates and lysates prepared with more stringent RIPA buffer were similar, indicating that the receptor did not reside in compartments that were insoluble after Nonidet P-40 lysis (results not shown). Thus, CHX treatment visualized steady-state degradation of endogenous TRAIL-R1. This degradation could be blocked by bafilomycin A1 (BafA1), a lysosomal inhibitor (Fig. 6, A and B). Inhibiting the proteasome with MG132 also partially inhibited TRAIL-R1 degradation. Statistical evaluation of data from three independent experiments indicated that rescue of TRAIL-R1 by BafA1, but not rescue by MG132, was significant (Fig. 6B). This indicates that at steady-state, a large pool of endogenous TRAIL-R1 is targeted for degradation in lysosomes.


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 targets endogenous TRAIL-R1 for lysosomal degradation.A and B, H358 cells were treated with cycloheximide (CHX; 50 μg/ml) alone, or with CHX in combination with bafilomycin A1 (Baf A1; 200 nm) or MG132 (10 μm) for 16 h. Total cell lysates were analyzed by immunoblotting with α-TRAIL-R1 and α-actin antibodies. A, data of a representative experiment. B, mean ± S.D. of quantified values from 3 independent experiments. TRAIL-R1 intensity was corrected for actin expression, the control value was set to 100%. Asterisk indicates statistically significant difference (Student's t test; *, p < 0.05). C and D, MCF-7Casp-3 cells were transfected to express GFP only (−), or GFP-tagged MARCH-8. Cells were treated with BafA1 (100 nm) or left untreated for 16 h. Total cell lysates of GFP+ cells, obtained by flow cytometric sorting, were analyzed by immunoblotting with α-TRAIL-R1 and α-actin antibodies, and α-GFP antibody to detect MARCH-8. Solid and open arrowheads indicate, respectively, MARCH-8.GFP and GFP only. C, data of a representative experiment. D, mean ± S.D. of quantified values from 3 independent experiments. TRAIL-R1 intensity was corrected for actin expression, control value was set to 100%. Asterisk indicates a statistically significant difference (Student's t test; *, p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: MARCH-8 targets endogenous TRAIL-R1 for lysosomal degradation.A and B, H358 cells were treated with cycloheximide (CHX; 50 μg/ml) alone, or with CHX in combination with bafilomycin A1 (Baf A1; 200 nm) or MG132 (10 μm) for 16 h. Total cell lysates were analyzed by immunoblotting with α-TRAIL-R1 and α-actin antibodies. A, data of a representative experiment. B, mean ± S.D. of quantified values from 3 independent experiments. TRAIL-R1 intensity was corrected for actin expression, the control value was set to 100%. Asterisk indicates statistically significant difference (Student's t test; *, p < 0.05). C and D, MCF-7Casp-3 cells were transfected to express GFP only (−), or GFP-tagged MARCH-8. Cells were treated with BafA1 (100 nm) or left untreated for 16 h. Total cell lysates of GFP+ cells, obtained by flow cytometric sorting, were analyzed by immunoblotting with α-TRAIL-R1 and α-actin antibodies, and α-GFP antibody to detect MARCH-8. Solid and open arrowheads indicate, respectively, MARCH-8.GFP and GFP only. C, data of a representative experiment. D, mean ± S.D. of quantified values from 3 independent experiments. TRAIL-R1 intensity was corrected for actin expression, control value was set to 100%. Asterisk indicates a statistically significant difference (Student's t test; *, p < 0.05).
Mentions: Because MARCH-1 and -8 down-regulated endogenous TRAIL-R1 from the cell surface (Fig. 1) and reduced total expression levels of exogenous TRAIL-R1 (Fig. 5), we hypothesized that MARCH-1 and -8 targeted TRAIL-R1 for degradation. To test this, we first studied the mechanism by which endogenous TRAIL-R1 was turned over at steady state. For this purpose, we used H358 lung cancer cells, as they express larger amounts of TRAIL-R1 than MCF-7Casp-3 cells (supplemental Fig. S6). After a 16-h treatment with the translation inhibitor cycloheximide (CHX), endogenous TRAIL-R1 levels were reduced to about 60% of untreated control levels (Fig. 6, A and B). Recoveries of TRAIL-R1 from Nonidet P-40 lysates and lysates prepared with more stringent RIPA buffer were similar, indicating that the receptor did not reside in compartments that were insoluble after Nonidet P-40 lysis (results not shown). Thus, CHX treatment visualized steady-state degradation of endogenous TRAIL-R1. This degradation could be blocked by bafilomycin A1 (BafA1), a lysosomal inhibitor (Fig. 6, A and B). Inhibiting the proteasome with MG132 also partially inhibited TRAIL-R1 degradation. Statistical evaluation of data from three independent experiments indicated that rescue of TRAIL-R1 by BafA1, but not rescue by MG132, was significant (Fig. 6B). This indicates that at steady-state, a large pool of endogenous TRAIL-R1 is targeted for degradation in lysosomes.

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