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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 augments BMP signalling. (a) HEK293 cells transiently expressing control HA-vector or HA-USP15 were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP-2 for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against HA, endogenous pSMAD1, total SMAD1 and GAPDH. (b) HEK293 cells stably expressing GFP or GFP-USP15 were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP for 1 h prior to separation into cytoplasmic and nuclear fractions. The fractions were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against GFP, Lamin A/C, GAPDH, endogenous pSMAD1 and total SMAD1.
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RSOB140065F3: USP15 augments BMP signalling. (a) HEK293 cells transiently expressing control HA-vector or HA-USP15 were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP-2 for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against HA, endogenous pSMAD1, total SMAD1 and GAPDH. (b) HEK293 cells stably expressing GFP or GFP-USP15 were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP for 1 h prior to separation into cytoplasmic and nuclear fractions. The fractions were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against GFP, Lamin A/C, GAPDH, endogenous pSMAD1 and total SMAD1.

Mentions: As depletion of USP15 inhibits BMP signalling, we asked whether elevation of USP15 has the opposite effect. Indeed, overexpression of HA-USP15 in HEK293 cells increased the levels of pSMAD1 in response to BMP signalling (figure 3a), and this was true in both nuclear [27–29] and cytoplasmic fractions (figure 3b). Cytoplasmic fractions also contained the majority of GFP-USP15 (figure 3b).Figure 3.


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 augments BMP signalling. (a) HEK293 cells transiently expressing control HA-vector or HA-USP15 were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP-2 for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against HA, endogenous pSMAD1, total SMAD1 and GAPDH. (b) HEK293 cells stably expressing GFP or GFP-USP15 were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP for 1 h prior to separation into cytoplasmic and nuclear fractions. The fractions were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against GFP, Lamin A/C, GAPDH, endogenous pSMAD1 and total SMAD1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140065F3: USP15 augments BMP signalling. (a) HEK293 cells transiently expressing control HA-vector or HA-USP15 were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP-2 for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against HA, endogenous pSMAD1, total SMAD1 and GAPDH. (b) HEK293 cells stably expressing GFP or GFP-USP15 were serum-starved overnight and stimulated with 6.25 ng ml−1 BMP for 1 h prior to separation into cytoplasmic and nuclear fractions. The fractions were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against GFP, Lamin A/C, GAPDH, endogenous pSMAD1 and total SMAD1.
Mentions: As depletion of USP15 inhibits BMP signalling, we asked whether elevation of USP15 has the opposite effect. Indeed, overexpression of HA-USP15 in HEK293 cells increased the levels of pSMAD1 in response to BMP signalling (figure 3a), and this was true in both nuclear [27–29] and cytoplasmic fractions (figure 3b). Cytoplasmic fractions also contained the majority of GFP-USP15 (figure 3b).Figure 3.

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