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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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Depletion of USP15 inhibits BMP signalling. (a) HEK293 cells were transiently transfected with three individual siRNAs targeting USP15, serum-starved overnight and stimulated with 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, SMAD1 and GAPDH. (b) As in (a), except that siUSP15-3 was used to knockdown endogenous USP15 expression in HeLa cells. (c) As in (b) except that U2OS cells were used. (d) HEK293 cells were transiently transfected with siUSP15-3. Cells were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP for 1 h. Cells were then washed and harvested 2 h later. The expression of USP15 and the BMP-target gene ID1 were assessed by qRT-PCR. Results are average of six biological replicates. The error bars indicate s.d. (e) As in (d), except that HEK293 cells were transfected with siUSP11. The expression of USP11 and ID1 were assessed by qRT-PCR. Results are average of three biological replicates. The error bars indicate s.d.
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RSOB140065F2: Depletion of USP15 inhibits BMP signalling. (a) HEK293 cells were transiently transfected with three individual siRNAs targeting USP15, serum-starved overnight and stimulated with 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, SMAD1 and GAPDH. (b) As in (a), except that siUSP15-3 was used to knockdown endogenous USP15 expression in HeLa cells. (c) As in (b) except that U2OS cells were used. (d) HEK293 cells were transiently transfected with siUSP15-3. Cells were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP for 1 h. Cells were then washed and harvested 2 h later. The expression of USP15 and the BMP-target gene ID1 were assessed by qRT-PCR. Results are average of six biological replicates. The error bars indicate s.d. (e) As in (d), except that HEK293 cells were transfected with siUSP11. The expression of USP11 and ID1 were assessed by qRT-PCR. Results are average of three biological replicates. The error bars indicate s.d.

Mentions: We therefore investigated the effect of RNAi-mediated depletion of USP15 in BMP signalling in three different human cell lines. Three distinct siRNAs targeting USP15 and a control siRNA targeting FoxO4 were transfected in HEK293 cells (figure 2a), in which all three USP15 siRNAs caused an approximately 80–90% reduction in USP15 protein levels compared with the FoxO4 control (figure 2a). Depletion of USP15 caused a substantial reduction in the levels of BMP-induced pSMAD1 (tail-phosphorylated SMAD1) without significantly affecting total SMAD1 levels (figure 2a). The inhibition of BMP-induced pSMAD1 levels by siUSP15-3 was partially rescued by the restoration of FLAG-USP15 overexpression in cells (electronic supplementary material, figure S4).Figure 2.


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)

Depletion of USP15 inhibits BMP signalling. (a) HEK293 cells were transiently transfected with three individual siRNAs targeting USP15, serum-starved overnight and stimulated with 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, SMAD1 and GAPDH. (b) As in (a), except that siUSP15-3 was used to knockdown endogenous USP15 expression in HeLa cells. (c) As in (b) except that U2OS cells were used. (d) HEK293 cells were transiently transfected with siUSP15-3. Cells were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP for 1 h. Cells were then washed and harvested 2 h later. The expression of USP15 and the BMP-target gene ID1 were assessed by qRT-PCR. Results are average of six biological replicates. The error bars indicate s.d. (e) As in (d), except that HEK293 cells were transfected with siUSP11. The expression of USP11 and ID1 were assessed by qRT-PCR. Results are average of three biological replicates. The error bars indicate s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4042855&req=5

RSOB140065F2: Depletion of USP15 inhibits BMP signalling. (a) HEK293 cells were transiently transfected with three individual siRNAs targeting USP15, serum-starved overnight and stimulated with 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, SMAD1 and GAPDH. (b) As in (a), except that siUSP15-3 was used to knockdown endogenous USP15 expression in HeLa cells. (c) As in (b) except that U2OS cells were used. (d) HEK293 cells were transiently transfected with siUSP15-3. Cells were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP for 1 h. Cells were then washed and harvested 2 h later. The expression of USP15 and the BMP-target gene ID1 were assessed by qRT-PCR. Results are average of six biological replicates. The error bars indicate s.d. (e) As in (d), except that HEK293 cells were transfected with siUSP11. The expression of USP11 and ID1 were assessed by qRT-PCR. Results are average of three biological replicates. The error bars indicate s.d.
Mentions: We therefore investigated the effect of RNAi-mediated depletion of USP15 in BMP signalling in three different human cell lines. Three distinct siRNAs targeting USP15 and a control siRNA targeting FoxO4 were transfected in HEK293 cells (figure 2a), in which all three USP15 siRNAs caused an approximately 80–90% reduction in USP15 protein levels compared with the FoxO4 control (figure 2a). Depletion of USP15 caused a substantial reduction in the levels of BMP-induced pSMAD1 (tail-phosphorylated SMAD1) without significantly affecting total SMAD1 levels (figure 2a). The inhibition of BMP-induced pSMAD1 levels by siUSP15-3 was partially rescued by the restoration of FLAG-USP15 overexpression in cells (electronic supplementary material, figure S4).Figure 2.

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