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The hereditary spastic paraplegia proteins NIPA1, spastin and spartin are inhibitors of mammalian BMP signalling.

Tsang HT, Edwards TL, Wang X, Connell JW, Davies RJ, Durrington HJ, O'Kane CJ, Luzio JP, Reid E - Hum. Mol. Genet. (2009)

Bottom Line: NIPA1 physically interacts with the type II BMP receptor (BMPRII) and we demonstrate that this interaction does not require the cytoplasmic tail of BMPRII.Disease-associated mutant versions of NIPA1 alter the trafficking of BMPRII and are less efficient at promoting BMPRII degradation than wild-type NIPA1.In addition, we demonstrate that two other members of the endosomal group of HSP proteins, spastin and spartin, are inhibitors of BMP signalling.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, Addenbrooke's Hospital, UK.

ABSTRACT
The hereditary spastic paraplegias (HSPs) are genetic conditions characterized by distal axonopathy of the longest corticospinal tract axons, and so their study provides an important opportunity to understand mechanisms involved in axonal maintenance and degeneration. A group of HSP genes encode proteins that localize to endosomes. One of these is NIPA1 (non-imprinted in Prader-Willi/Angelman syndrome 1) and we have shown recently that its Drosophila homologue spichthyin inhibits bone morphogenic protein (BMP) signalling, although the relevance of this finding to the mammalian protein was not known. We show here that mammalian NIPA1 is also an inhibitor of BMP signalling. NIPA1 physically interacts with the type II BMP receptor (BMPRII) and we demonstrate that this interaction does not require the cytoplasmic tail of BMPRII. We show that the mechanism by which NIPA1 inhibits BMP signalling involves downregulation of BMP receptors by promoting their endocytosis and lysosomal degradation. Disease-associated mutant versions of NIPA1 alter the trafficking of BMPRII and are less efficient at promoting BMPRII degradation than wild-type NIPA1. In addition, we demonstrate that two other members of the endosomal group of HSP proteins, spastin and spartin, are inhibitors of BMP signalling. Since BMP signalling is important for distal axonal function, we propose that dysregulation of BMP signalling could be a unifying pathological component in this endosomal group of HSPs, and perhaps of importance in other conditions in which distal axonal degeneration is found.

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NIPA1 co-immunoprecipitates with BMPRII. (A) NSC34 cells stably expressing NIPA1-GFP were first treated with chloroquine to increase the concentration of endogenous BMPRII and used in immunoprecipitation experiments with anti-GFP, or anti-actin as a control antibody. Total cell lysates (input lane) and the immunoprecipitated samples were then immunoblotted with anti-GFP or anti-BMPRII antibodies. The expected size of full-length endogenous BMPRII is indicated. (B and C) HeLa cells stably expressing NIPA1-GFP and transiently transfected with either myc-BMPRII (B) or myc-BMPRIIΔtail (C) were used in immunoprecipitation experiments with anti-GFP, or anti-actin as a control antibody. Total cell lysates (input lanes) and immunoprecipitated samples were then immunoblotted with anti-GFP or anti-myc. Immunoglobulin bands are marked ‘*’. Control experiments in which the immunoprecipitation was carried out in cells stably expressing GFP-Rab5 are shown on the right-hand side of (B) and (C).
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DDP324F6: NIPA1 co-immunoprecipitates with BMPRII. (A) NSC34 cells stably expressing NIPA1-GFP were first treated with chloroquine to increase the concentration of endogenous BMPRII and used in immunoprecipitation experiments with anti-GFP, or anti-actin as a control antibody. Total cell lysates (input lane) and the immunoprecipitated samples were then immunoblotted with anti-GFP or anti-BMPRII antibodies. The expected size of full-length endogenous BMPRII is indicated. (B and C) HeLa cells stably expressing NIPA1-GFP and transiently transfected with either myc-BMPRII (B) or myc-BMPRIIΔtail (C) were used in immunoprecipitation experiments with anti-GFP, or anti-actin as a control antibody. Total cell lysates (input lanes) and immunoprecipitated samples were then immunoblotted with anti-GFP or anti-myc. Immunoglobulin bands are marked ‘*’. Control experiments in which the immunoprecipitation was carried out in cells stably expressing GFP-Rab5 are shown on the right-hand side of (B) and (C).

Mentions: We next examined whether there is an interaction between NIPA1 and BMPRII. We first immunoprecipitated NIPA1-GFP from NSC34 cells stably expressing the protein, using anti-GFP, or anti-actin as a spurious control antibody, and immunoblotted with anti-BMPRII. In samples immunoprecipitated with anti-GFP, but not anti-actin, we observed faint immunoblot bands corresponding to the size of BMPRII (data not shown). In order to more clearly demonstrate this co-immunoprecipitation, we treated the NIPA1-GFP-expressing cells with chloroquine, to increase BMPRII concentration. In this situation, we found clear co-immunoprecipitation of endogenous BMPRII in cells immunoprecipitated with anti-GFP, but not with the anti-actin control antibody (Fig. 6A).


The hereditary spastic paraplegia proteins NIPA1, spastin and spartin are inhibitors of mammalian BMP signalling.

Tsang HT, Edwards TL, Wang X, Connell JW, Davies RJ, Durrington HJ, O'Kane CJ, Luzio JP, Reid E - Hum. Mol. Genet. (2009)

NIPA1 co-immunoprecipitates with BMPRII. (A) NSC34 cells stably expressing NIPA1-GFP were first treated with chloroquine to increase the concentration of endogenous BMPRII and used in immunoprecipitation experiments with anti-GFP, or anti-actin as a control antibody. Total cell lysates (input lane) and the immunoprecipitated samples were then immunoblotted with anti-GFP or anti-BMPRII antibodies. The expected size of full-length endogenous BMPRII is indicated. (B and C) HeLa cells stably expressing NIPA1-GFP and transiently transfected with either myc-BMPRII (B) or myc-BMPRIIΔtail (C) were used in immunoprecipitation experiments with anti-GFP, or anti-actin as a control antibody. Total cell lysates (input lanes) and immunoprecipitated samples were then immunoblotted with anti-GFP or anti-myc. Immunoglobulin bands are marked ‘*’. Control experiments in which the immunoprecipitation was carried out in cells stably expressing GFP-Rab5 are shown on the right-hand side of (B) and (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2748891&req=5

DDP324F6: NIPA1 co-immunoprecipitates with BMPRII. (A) NSC34 cells stably expressing NIPA1-GFP were first treated with chloroquine to increase the concentration of endogenous BMPRII and used in immunoprecipitation experiments with anti-GFP, or anti-actin as a control antibody. Total cell lysates (input lane) and the immunoprecipitated samples were then immunoblotted with anti-GFP or anti-BMPRII antibodies. The expected size of full-length endogenous BMPRII is indicated. (B and C) HeLa cells stably expressing NIPA1-GFP and transiently transfected with either myc-BMPRII (B) or myc-BMPRIIΔtail (C) were used in immunoprecipitation experiments with anti-GFP, or anti-actin as a control antibody. Total cell lysates (input lanes) and immunoprecipitated samples were then immunoblotted with anti-GFP or anti-myc. Immunoglobulin bands are marked ‘*’. Control experiments in which the immunoprecipitation was carried out in cells stably expressing GFP-Rab5 are shown on the right-hand side of (B) and (C).
Mentions: We next examined whether there is an interaction between NIPA1 and BMPRII. We first immunoprecipitated NIPA1-GFP from NSC34 cells stably expressing the protein, using anti-GFP, or anti-actin as a spurious control antibody, and immunoblotted with anti-BMPRII. In samples immunoprecipitated with anti-GFP, but not anti-actin, we observed faint immunoblot bands corresponding to the size of BMPRII (data not shown). In order to more clearly demonstrate this co-immunoprecipitation, we treated the NIPA1-GFP-expressing cells with chloroquine, to increase BMPRII concentration. In this situation, we found clear co-immunoprecipitation of endogenous BMPRII in cells immunoprecipitated with anti-GFP, but not with the anti-actin control antibody (Fig. 6A).

Bottom Line: NIPA1 physically interacts with the type II BMP receptor (BMPRII) and we demonstrate that this interaction does not require the cytoplasmic tail of BMPRII.Disease-associated mutant versions of NIPA1 alter the trafficking of BMPRII and are less efficient at promoting BMPRII degradation than wild-type NIPA1.In addition, we demonstrate that two other members of the endosomal group of HSP proteins, spastin and spartin, are inhibitors of BMP signalling.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, Addenbrooke's Hospital, UK.

ABSTRACT
The hereditary spastic paraplegias (HSPs) are genetic conditions characterized by distal axonopathy of the longest corticospinal tract axons, and so their study provides an important opportunity to understand mechanisms involved in axonal maintenance and degeneration. A group of HSP genes encode proteins that localize to endosomes. One of these is NIPA1 (non-imprinted in Prader-Willi/Angelman syndrome 1) and we have shown recently that its Drosophila homologue spichthyin inhibits bone morphogenic protein (BMP) signalling, although the relevance of this finding to the mammalian protein was not known. We show here that mammalian NIPA1 is also an inhibitor of BMP signalling. NIPA1 physically interacts with the type II BMP receptor (BMPRII) and we demonstrate that this interaction does not require the cytoplasmic tail of BMPRII. We show that the mechanism by which NIPA1 inhibits BMP signalling involves downregulation of BMP receptors by promoting their endocytosis and lysosomal degradation. Disease-associated mutant versions of NIPA1 alter the trafficking of BMPRII and are less efficient at promoting BMPRII degradation than wild-type NIPA1. In addition, we demonstrate that two other members of the endosomal group of HSP proteins, spastin and spartin, are inhibitors of BMP signalling. Since BMP signalling is important for distal axonal function, we propose that dysregulation of BMP signalling could be a unifying pathological component in this endosomal group of HSPs, and perhaps of importance in other conditions in which distal axonal degeneration is found.

Show MeSH
Related in: MedlinePlus