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USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling.

Herhaus L, Al-Salihi MA, Dingwell KS, Cummins TD, Wasmus L, Vogt J, Ewan R, Bruce D, Macartney T, Weidlich S, Smith JC, Sapkota GP - Open Biol (2014)

Bottom Line: We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3.We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation.Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dow St., Dundee DD1 5EH, UK.

ABSTRACT
Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis.

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USP15 impacts osteoblastic differentiation in C2C12 myoblasts and modulates BMP signalling in Xenopus embryogenesis. (a) Mouse myoblast cell line C2C12 were transfected with siRNAs targeting mouse FoxO4 or USP15. Cells were serum-starved overnight and treated with or without BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and immunoblotted with antibodies against USP15, pSMAD1, total SMAD1 and GAPDH. (b) C2C12 cells transfected with mouse siFoxO4 or mouse siUSP15 were grown for up to 4 days in the presence of BMP. Cells were lysed and the alkaline phosphatase activity measured using a fluorescence plate reader. Data are represented as mean of three biological replicates and error bars indicate s.d. Representative extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against USP15 and GAPDH. (c) Xenopus embryos were injected with 80 ng of either xUSP15- (xUSP15-MO) or control-MO morpholinos at the one-cell stage and then collected at the indicated stages. Lysates were resolved by SDS-PAGE and immunoblotted with antibodies against pSMAD1 and α-tubulin. (d) qRT-PCR analysis of xVENT1 mRNA expression. Embryos were injected with 80 ng of either USP15-MO or control-MO at the one-cell stage and then animal caps were cut at stage 8.5. The animal caps were collected at the equivalent embryo stage of 10.5 and processed for qRT-PCR.
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RSOB140065F7: USP15 impacts osteoblastic differentiation in C2C12 myoblasts and modulates BMP signalling in Xenopus embryogenesis. (a) Mouse myoblast cell line C2C12 were transfected with siRNAs targeting mouse FoxO4 or USP15. Cells were serum-starved overnight and treated with or without BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and immunoblotted with antibodies against USP15, pSMAD1, total SMAD1 and GAPDH. (b) C2C12 cells transfected with mouse siFoxO4 or mouse siUSP15 were grown for up to 4 days in the presence of BMP. Cells were lysed and the alkaline phosphatase activity measured using a fluorescence plate reader. Data are represented as mean of three biological replicates and error bars indicate s.d. Representative extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against USP15 and GAPDH. (c) Xenopus embryos were injected with 80 ng of either xUSP15- (xUSP15-MO) or control-MO morpholinos at the one-cell stage and then collected at the indicated stages. Lysates were resolved by SDS-PAGE and immunoblotted with antibodies against pSMAD1 and α-tubulin. (d) qRT-PCR analysis of xVENT1 mRNA expression. Embryos were injected with 80 ng of either USP15-MO or control-MO at the one-cell stage and then animal caps were cut at stage 8.5. The animal caps were collected at the equivalent embryo stage of 10.5 and processed for qRT-PCR.

Mentions: BMP signalling plays a key role in inducing myoblast progenitor differentiation to the osteoblast lineage, as marked by the acquisition of alkaline phosphatase activity [35]. We investigated the effects of USP15 depletion on BMP-induced SMAD1 phosphorylation and the development of alkaline phosphatase activity in mouse myoblast C2C12 cells [26,35]. RNAi-mediated depletion of USP15 in C2C12 cells resulted in a more than 90% reduction in USP15 protein expression (figure 7a). Under these conditions, BMP-induced phosphorylation of SMAD1 was significantly reduced compared with controls (figure 7a), a reduction that was not due to a decrease in total levels of SMAD1 (figure 7a). Loss of USP15 significantly reduced BMP-induced alkaline phosphatase activity in C2C12 cells at both 48 h and 96 h post-BMP stimulation (figure 7b).Figure 7.


USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling.

Herhaus L, Al-Salihi MA, Dingwell KS, Cummins TD, Wasmus L, Vogt J, Ewan R, Bruce D, Macartney T, Weidlich S, Smith JC, Sapkota GP - Open Biol (2014)

USP15 impacts osteoblastic differentiation in C2C12 myoblasts and modulates BMP signalling in Xenopus embryogenesis. (a) Mouse myoblast cell line C2C12 were transfected with siRNAs targeting mouse FoxO4 or USP15. Cells were serum-starved overnight and treated with or without BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and immunoblotted with antibodies against USP15, pSMAD1, total SMAD1 and GAPDH. (b) C2C12 cells transfected with mouse siFoxO4 or mouse siUSP15 were grown for up to 4 days in the presence of BMP. Cells were lysed and the alkaline phosphatase activity measured using a fluorescence plate reader. Data are represented as mean of three biological replicates and error bars indicate s.d. Representative extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against USP15 and GAPDH. (c) Xenopus embryos were injected with 80 ng of either xUSP15- (xUSP15-MO) or control-MO morpholinos at the one-cell stage and then collected at the indicated stages. Lysates were resolved by SDS-PAGE and immunoblotted with antibodies against pSMAD1 and α-tubulin. (d) qRT-PCR analysis of xVENT1 mRNA expression. Embryos were injected with 80 ng of either USP15-MO or control-MO at the one-cell stage and then animal caps were cut at stage 8.5. The animal caps were collected at the equivalent embryo stage of 10.5 and processed for qRT-PCR.
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Related In: Results  -  Collection

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RSOB140065F7: USP15 impacts osteoblastic differentiation in C2C12 myoblasts and modulates BMP signalling in Xenopus embryogenesis. (a) Mouse myoblast cell line C2C12 were transfected with siRNAs targeting mouse FoxO4 or USP15. Cells were serum-starved overnight and treated with or without BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and immunoblotted with antibodies against USP15, pSMAD1, total SMAD1 and GAPDH. (b) C2C12 cells transfected with mouse siFoxO4 or mouse siUSP15 were grown for up to 4 days in the presence of BMP. Cells were lysed and the alkaline phosphatase activity measured using a fluorescence plate reader. Data are represented as mean of three biological replicates and error bars indicate s.d. Representative extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against USP15 and GAPDH. (c) Xenopus embryos were injected with 80 ng of either xUSP15- (xUSP15-MO) or control-MO morpholinos at the one-cell stage and then collected at the indicated stages. Lysates were resolved by SDS-PAGE and immunoblotted with antibodies against pSMAD1 and α-tubulin. (d) qRT-PCR analysis of xVENT1 mRNA expression. Embryos were injected with 80 ng of either USP15-MO or control-MO at the one-cell stage and then animal caps were cut at stage 8.5. The animal caps were collected at the equivalent embryo stage of 10.5 and processed for qRT-PCR.
Mentions: BMP signalling plays a key role in inducing myoblast progenitor differentiation to the osteoblast lineage, as marked by the acquisition of alkaline phosphatase activity [35]. We investigated the effects of USP15 depletion on BMP-induced SMAD1 phosphorylation and the development of alkaline phosphatase activity in mouse myoblast C2C12 cells [26,35]. RNAi-mediated depletion of USP15 in C2C12 cells resulted in a more than 90% reduction in USP15 protein expression (figure 7a). Under these conditions, BMP-induced phosphorylation of SMAD1 was significantly reduced compared with controls (figure 7a), a reduction that was not due to a decrease in total levels of SMAD1 (figure 7a). Loss of USP15 significantly reduced BMP-induced alkaline phosphatase activity in C2C12 cells at both 48 h and 96 h post-BMP stimulation (figure 7b).Figure 7.

Bottom Line: We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3.We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation.Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dow St., Dundee DD1 5EH, UK.

ABSTRACT
Protein kinase ALK3/BMPR1A mediates bone morphogenetic protein (BMP) signalling through phosphorylation and activation of SMADs 1/5/8. SMAD6, a transcriptional target of BMP, negatively regulates the BMP pathway by recruiting E3 ubiquitin ligases and targeting ALK3 for ubiquitin-mediated degradation. Here, we identify a deubiquitylating enzyme USP15 as an interactor of SMAD6 and ALK3. We show that USP15 enhances BMP-induced phosphorylation of SMAD1 by interacting with and deubiquitylating ALK3. RNAi-mediated depletion of USP15 increases ALK3 K48-linked polyubiquitylation, and reduces both BMP-induced SMAD1 phosphorylation and transcription of BMP target genes. We also show that loss of USP15 expression from mouse myoblast cells inhibits BMP-induced osteoblast differentiation. Furthermore, USP15 modulates BMP-induced phosphorylation of SMAD1 and transcription during Xenopus embryogenesis.

Show MeSH
Related in: MedlinePlus