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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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Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery.A, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 (WT) or its K273A lysine mutant (K/A). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates (IP) were analyzed by immunoblotting (IB) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments. B, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 (WT) or the K273A TRAIL-R1 mutant (K/A), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments. C, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 (WT) or its K273A lysine mutant (K/A). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for panel A. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments. D, alignment of primary amino acid sequence of part of the transmembrane segment (italic) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in E. Relevant potential ubiquitination sites are shown in bold. E, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in D. TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments. Asterisk denotes the heavy chain of the antibody used for IP.
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Figure 4: Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery.A, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 (WT) or its K273A lysine mutant (K/A). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates (IP) were analyzed by immunoblotting (IB) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments. B, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 (WT) or the K273A TRAIL-R1 mutant (K/A), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments. C, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 (WT) or its K273A lysine mutant (K/A). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for panel A. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments. D, alignment of primary amino acid sequence of part of the transmembrane segment (italic) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in E. Relevant potential ubiquitination sites are shown in bold. E, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in D. TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments. Asterisk denotes the heavy chain of the antibody used for IP.

Mentions: Cells were harvested and lysed in Nonidet P-40 buffer consisting of 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mm NaCl, 1 mm PMSF and Complete Protease Inhibitors (Roche). For the experiment depicted in Fig. 4C, cells were lysed in 50 mm Tris-HCl, pH 8.0, 1% SDS, 10 mm DTT, 0.5 mm EDTA, for 10 min at 95 °C. The SDS was quenched by addition of 9 volumes of Nonidet P-40 buffer. Cell lysates were clarified by centrifugation for 10 min at 13,000 × g and protein content was measured by Bio-Rad protein assay. Immunoprecipitation was performed with antibody to mRFP, followed by Protein G-Sepharose beads (GE Healthcare). Immunoprecipitates were washed, resuspended in reducing NuPAGE sample buffer (with 0.1 m DTT), and heated for 10 min at 95 °C. SDS-PAGE was done on pre-cast 4–12% NuPAGE minigels, according to the manufacturer's protocol (Invitrogen). Total cell lysate (taken prior to immunoprecipitation) was run at 30 μg of protein per lane, as determined by Bio-Rad protein assay. Proteins were transferred to nitrocellulose membranes by wet blotting for 90 min at 70 V. Membranes were blocked for 1 h at room temperature with 5% (w/v) skim milk (Oxoid) in Tris-buffered saline (TBS). Antibody probing was performed in TBS with 1% (w/v) skim milk and 0.05% (v/v) Tween 20. For detection by ECL (Pierce Biotechnology), blots were incubated with HRP-conjugated anti-HA or anti-FLAG mAb, or with rabbit anti-mRFP followed by HRP-conjugated swine anti-rabbit Ig. Alternatively, blots were incubated with unconjugated primary antibody, followed by IRDye-conjugated second step antibody and proteins were detected on the Odyssey infrared imager (LI-COR). Quantification of signals was done using ImageLab software (Bio-Rad) or Odyssey software (LI-COR), respectively.


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)

Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery.A, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 (WT) or its K273A lysine mutant (K/A). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates (IP) were analyzed by immunoblotting (IB) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments. B, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 (WT) or the K273A TRAIL-R1 mutant (K/A), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments. C, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 (WT) or its K273A lysine mutant (K/A). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for panel A. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments. D, alignment of primary amino acid sequence of part of the transmembrane segment (italic) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in E. Relevant potential ubiquitination sites are shown in bold. E, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in D. TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments. Asterisk denotes the heavy chain of the antibody used for IP.
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Figure 4: Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery.A, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 (WT) or its K273A lysine mutant (K/A). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates (IP) were analyzed by immunoblotting (IB) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments. B, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 (WT) or the K273A TRAIL-R1 mutant (K/A), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments. C, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 (WT) or its K273A lysine mutant (K/A). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for panel A. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments. D, alignment of primary amino acid sequence of part of the transmembrane segment (italic) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in E. Relevant potential ubiquitination sites are shown in bold. E, MCF-7Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in D. TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments. Asterisk denotes the heavy chain of the antibody used for IP.
Mentions: Cells were harvested and lysed in Nonidet P-40 buffer consisting of 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mm NaCl, 1 mm PMSF and Complete Protease Inhibitors (Roche). For the experiment depicted in Fig. 4C, cells were lysed in 50 mm Tris-HCl, pH 8.0, 1% SDS, 10 mm DTT, 0.5 mm EDTA, for 10 min at 95 °C. The SDS was quenched by addition of 9 volumes of Nonidet P-40 buffer. Cell lysates were clarified by centrifugation for 10 min at 13,000 × g and protein content was measured by Bio-Rad protein assay. Immunoprecipitation was performed with antibody to mRFP, followed by Protein G-Sepharose beads (GE Healthcare). Immunoprecipitates were washed, resuspended in reducing NuPAGE sample buffer (with 0.1 m DTT), and heated for 10 min at 95 °C. SDS-PAGE was done on pre-cast 4–12% NuPAGE minigels, according to the manufacturer's protocol (Invitrogen). Total cell lysate (taken prior to immunoprecipitation) was run at 30 μg of protein per lane, as determined by Bio-Rad protein assay. Proteins were transferred to nitrocellulose membranes by wet blotting for 90 min at 70 V. Membranes were blocked for 1 h at room temperature with 5% (w/v) skim milk (Oxoid) in Tris-buffered saline (TBS). Antibody probing was performed in TBS with 1% (w/v) skim milk and 0.05% (v/v) Tween 20. For detection by ECL (Pierce Biotechnology), blots were incubated with HRP-conjugated anti-HA or anti-FLAG mAb, or with rabbit anti-mRFP followed by HRP-conjugated swine anti-rabbit Ig. Alternatively, blots were incubated with unconjugated primary antibody, followed by IRDye-conjugated second step antibody and proteins were detected on the Odyssey infrared imager (LI-COR). Quantification of signals was done using ImageLab software (Bio-Rad) or Odyssey software (LI-COR), respectively.

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