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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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TRAIL-R1 is a substrate of endogenous MARCH-8.A, endogenous MARCH-8 expression in MCF-7Casp-3 (MCF-7) and Mel Juso (MJ) cells, as determined by RT-PCR on cDNA. Non-reverse transcribed RNA (RNA) was used as a control template to exclude amplification of genomic DNA. B, down-regulation of endogenous MARCH-8 by RNAi. MCF-7Casp-3 cells were transfected with MARCH-8 shRNA, together with GFP. MARCH-8 shRNA expressing cells (GFP+) and nonexpressing cells (GFP−) cells were separated by flow cytometric sorting and analyzed for endogenous MARCH-8 transcript levels by quantitative RT-PCR. Signals that were corrected for GAPDH and MARCH-8 transcript levels in the GFP+ population were normalized to the levels in the GFP− population. C, MCF-7Casp-3 cells were transfected to express mRFP alone, WT TRAIL-R1.mRFP, or K273A TRAIL-R1.mRFP, together with FLAG-ubiquitin and either an empty vector, or the MARCH-8 targeting shRNA. TRAIL-R1 was isolated by immunoprecipitation with α-mRFP antibody and immunoprecipitates (IP) were analyzed by immunoblotting for TRAIL-R1 (α-mRFP) or ubiquitin (α-FLAG). Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Data shown are representative of 3 independent experiments.
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Figure 7: TRAIL-R1 is a substrate of endogenous MARCH-8.A, endogenous MARCH-8 expression in MCF-7Casp-3 (MCF-7) and Mel Juso (MJ) cells, as determined by RT-PCR on cDNA. Non-reverse transcribed RNA (RNA) was used as a control template to exclude amplification of genomic DNA. B, down-regulation of endogenous MARCH-8 by RNAi. MCF-7Casp-3 cells were transfected with MARCH-8 shRNA, together with GFP. MARCH-8 shRNA expressing cells (GFP+) and nonexpressing cells (GFP−) cells were separated by flow cytometric sorting and analyzed for endogenous MARCH-8 transcript levels by quantitative RT-PCR. Signals that were corrected for GAPDH and MARCH-8 transcript levels in the GFP+ population were normalized to the levels in the GFP− population. C, MCF-7Casp-3 cells were transfected to express mRFP alone, WT TRAIL-R1.mRFP, or K273A TRAIL-R1.mRFP, together with FLAG-ubiquitin and either an empty vector, or the MARCH-8 targeting shRNA. TRAIL-R1 was isolated by immunoprecipitation with α-mRFP antibody and immunoprecipitates (IP) were analyzed by immunoblotting for TRAIL-R1 (α-mRFP) or ubiquitin (α-FLAG). Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Data shown are representative of 3 independent experiments.

Mentions: The findings outlined above strongly suggested that endogenous MARCH proteins were responsible for TRAIL-R1 ubiquitination and its down-regulation. To test the involvement of endogenous MARCH proteins, we used RNA interference. RT-PCR revealed that both MCF-7Casp-3 and Mel JuSo cells express MARCH-8 (Fig. 7A). A short hairpin (sh)RNA construct was made that efficiently silenced MARCH-8 expression, as tested on endogenous MARCH-8 mRNA (Fig. 7B).


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)

TRAIL-R1 is a substrate of endogenous MARCH-8.A, endogenous MARCH-8 expression in MCF-7Casp-3 (MCF-7) and Mel Juso (MJ) cells, as determined by RT-PCR on cDNA. Non-reverse transcribed RNA (RNA) was used as a control template to exclude amplification of genomic DNA. B, down-regulation of endogenous MARCH-8 by RNAi. MCF-7Casp-3 cells were transfected with MARCH-8 shRNA, together with GFP. MARCH-8 shRNA expressing cells (GFP+) and nonexpressing cells (GFP−) cells were separated by flow cytometric sorting and analyzed for endogenous MARCH-8 transcript levels by quantitative RT-PCR. Signals that were corrected for GAPDH and MARCH-8 transcript levels in the GFP+ population were normalized to the levels in the GFP− population. C, MCF-7Casp-3 cells were transfected to express mRFP alone, WT TRAIL-R1.mRFP, or K273A TRAIL-R1.mRFP, together with FLAG-ubiquitin and either an empty vector, or the MARCH-8 targeting shRNA. TRAIL-R1 was isolated by immunoprecipitation with α-mRFP antibody and immunoprecipitates (IP) were analyzed by immunoblotting for TRAIL-R1 (α-mRFP) or ubiquitin (α-FLAG). Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Data shown are representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: TRAIL-R1 is a substrate of endogenous MARCH-8.A, endogenous MARCH-8 expression in MCF-7Casp-3 (MCF-7) and Mel Juso (MJ) cells, as determined by RT-PCR on cDNA. Non-reverse transcribed RNA (RNA) was used as a control template to exclude amplification of genomic DNA. B, down-regulation of endogenous MARCH-8 by RNAi. MCF-7Casp-3 cells were transfected with MARCH-8 shRNA, together with GFP. MARCH-8 shRNA expressing cells (GFP+) and nonexpressing cells (GFP−) cells were separated by flow cytometric sorting and analyzed for endogenous MARCH-8 transcript levels by quantitative RT-PCR. Signals that were corrected for GAPDH and MARCH-8 transcript levels in the GFP+ population were normalized to the levels in the GFP− population. C, MCF-7Casp-3 cells were transfected to express mRFP alone, WT TRAIL-R1.mRFP, or K273A TRAIL-R1.mRFP, together with FLAG-ubiquitin and either an empty vector, or the MARCH-8 targeting shRNA. TRAIL-R1 was isolated by immunoprecipitation with α-mRFP antibody and immunoprecipitates (IP) were analyzed by immunoblotting for TRAIL-R1 (α-mRFP) or ubiquitin (α-FLAG). Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Data shown are representative of 3 independent experiments.
Mentions: The findings outlined above strongly suggested that endogenous MARCH proteins were responsible for TRAIL-R1 ubiquitination and its down-regulation. To test the involvement of endogenous MARCH proteins, we used RNA interference. RT-PCR revealed that both MCF-7Casp-3 and Mel JuSo cells express MARCH-8 (Fig. 7A). A short hairpin (sh)RNA construct was made that efficiently silenced MARCH-8 expression, as tested on endogenous MARCH-8 mRNA (Fig. 7B).

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