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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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ALK3 undergoes proteasomal degradation. (a) HEK293 cells transfected with untagged ALK3 were treated with or without 20 μM cycloheximide for 24 h prior to lysis. Cells were treated with DMSO control, 100 nM bafilomycin A1 (to inhibit vacuolar-type H+ ATPase) or 10 μM bortezomib (to inhibit the proteasome) for 3 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (b) HEK293 cells were transiently transfected with siFoxO4 or siUSP15-3. Cells were serum-starved overnight, treated with or without 10 μM bortezomib for 3 h and then stimulated with or without 6.25 ng ml−1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1 and total SMAD1. (c) HEK293 cells were transiently transfected with siFoxO4 (–), siUSP15-3 or siSMAD6 as indicated. Twenty-four hours post siRNA transfection, cells were serum-starved overnight and stimulated with or without 6.25 ng ml−1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against pSMAD1, total SMAD1, USP15 and GAPDH. The SMAD6 knockdown was confirmed by qRT-PCR (electronic supplementary material, figure S7).
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RSOB140065F6: ALK3 undergoes proteasomal degradation. (a) HEK293 cells transfected with untagged ALK3 were treated with or without 20 μM cycloheximide for 24 h prior to lysis. Cells were treated with DMSO control, 100 nM bafilomycin A1 (to inhibit vacuolar-type H+ ATPase) or 10 μM bortezomib (to inhibit the proteasome) for 3 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (b) HEK293 cells were transiently transfected with siFoxO4 or siUSP15-3. Cells were serum-starved overnight, treated with or without 10 μM bortezomib for 3 h and then stimulated with or without 6.25 ng ml−1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1 and total SMAD1. (c) HEK293 cells were transiently transfected with siFoxO4 (–), siUSP15-3 or siSMAD6 as indicated. Twenty-four hours post siRNA transfection, cells were serum-starved overnight and stimulated with or without 6.25 ng ml−1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against pSMAD1, total SMAD1, USP15 and GAPDH. The SMAD6 knockdown was confirmed by qRT-PCR (electronic supplementary material, figure S7).

Mentions: Polyubiquitylation-dependent destruction of ALK3 in cells could be mediated by the proteasomal or lysosomal degradation pathways [30] or even both [31]. To test these possibilities, we investigated the turnover of untagged human ALK3 expressed in HEK293 cells in the presence of the proteasomal inhibitor bortezomib and lysosomal fusion inhibitor bafilomycin [25,32–34]. As expected, Bortezomib resulted in the accumulation of polyubiquitin chains in extracts, whereas bafilomycin yielded increased levels of LC3-II [32] (figure 6a). Treatment of cells with bortezomib resulted in enhanced levels of ALK3 compared with control, whereas bafilomycin did not (figure 6a). Similarly, when cells were treated with cycloheximide for 24 h prior to lysis to prevent de novo ALK3 synthesis, bortezomib but not bafilomycin resulted in enhanced levels of ALK3 compared with control (figure 6a). Analogous results were obtained when Xenopus ALK3-HA was expressed in HEK293 cells (electronic supplementary material, figure S6a). Consistently, the pretreatment of cells with bortezomib but not bafilomycin resulted in enhanced levels of polyubiquitylation in FLAG-ALK3 IPs (electronic supplementary material, figure S6b). Together, these results suggest that ALK3 polyubiquitylation leads to its proteasomal degradation.Figure 6.


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)

ALK3 undergoes proteasomal degradation. (a) HEK293 cells transfected with untagged ALK3 were treated with or without 20 μM cycloheximide for 24 h prior to lysis. Cells were treated with DMSO control, 100 nM bafilomycin A1 (to inhibit vacuolar-type H+ ATPase) or 10 μM bortezomib (to inhibit the proteasome) for 3 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (b) HEK293 cells were transiently transfected with siFoxO4 or siUSP15-3. Cells were serum-starved overnight, treated with or without 10 μM bortezomib for 3 h and then stimulated with or without 6.25 ng ml−1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1 and total SMAD1. (c) HEK293 cells were transiently transfected with siFoxO4 (–), siUSP15-3 or siSMAD6 as indicated. Twenty-four hours post siRNA transfection, cells were serum-starved overnight and stimulated with or without 6.25 ng ml−1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against pSMAD1, total SMAD1, USP15 and GAPDH. The SMAD6 knockdown was confirmed by qRT-PCR (electronic supplementary material, figure S7).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4042855&req=5

RSOB140065F6: ALK3 undergoes proteasomal degradation. (a) HEK293 cells transfected with untagged ALK3 were treated with or without 20 μM cycloheximide for 24 h prior to lysis. Cells were treated with DMSO control, 100 nM bafilomycin A1 (to inhibit vacuolar-type H+ ATPase) or 10 μM bortezomib (to inhibit the proteasome) for 3 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (b) HEK293 cells were transiently transfected with siFoxO4 or siUSP15-3. Cells were serum-starved overnight, treated with or without 10 μM bortezomib for 3 h and then stimulated with or without 6.25 ng ml−1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1 and total SMAD1. (c) HEK293 cells were transiently transfected with siFoxO4 (–), siUSP15-3 or siSMAD6 as indicated. Twenty-four hours post siRNA transfection, cells were serum-starved overnight and stimulated with or without 6.25 ng ml−1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against pSMAD1, total SMAD1, USP15 and GAPDH. The SMAD6 knockdown was confirmed by qRT-PCR (electronic supplementary material, figure S7).
Mentions: Polyubiquitylation-dependent destruction of ALK3 in cells could be mediated by the proteasomal or lysosomal degradation pathways [30] or even both [31]. To test these possibilities, we investigated the turnover of untagged human ALK3 expressed in HEK293 cells in the presence of the proteasomal inhibitor bortezomib and lysosomal fusion inhibitor bafilomycin [25,32–34]. As expected, Bortezomib resulted in the accumulation of polyubiquitin chains in extracts, whereas bafilomycin yielded increased levels of LC3-II [32] (figure 6a). Treatment of cells with bortezomib resulted in enhanced levels of ALK3 compared with control, whereas bafilomycin did not (figure 6a). Similarly, when cells were treated with cycloheximide for 24 h prior to lysis to prevent de novo ALK3 synthesis, bortezomib but not bafilomycin resulted in enhanced levels of ALK3 compared with control (figure 6a). Analogous results were obtained when Xenopus ALK3-HA was expressed in HEK293 cells (electronic supplementary material, figure S6a). Consistently, the pretreatment of cells with bortezomib but not bafilomycin resulted in enhanced levels of polyubiquitylation in FLAG-ALK3 IPs (electronic supplementary material, figure S6b). Together, these results suggest that ALK3 polyubiquitylation leads to its proteasomal degradation.Figure 6.

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