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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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Identification and characterization of USP15 as an interactor of SMAD6. (a) Coomassie stained gel image showing anti-GFP-IPs from HEK293 extracts expressing GFP-SMAD6. The interacting proteins were excised as 2 mm gel pieces, digested with trypsin and identified by mass spectrometry. The location where USP11 and USP15 were identified is indicated. All protein interactors of GFP-SMAD6 identified by mass spectrometry are indicated in the right panel. Protein interactors of GFP control were removed from the list of GFP-SMAD6 interactors. (b) HEK293 cells were transfected transiently with FLAG-SMADs. Extract inputs were resolved by SDS-PAGE and subjected to immunoblotting (IB) with anti-USP11, anti-USP15 and anti-FLAG antibodies as indicated. (c) FLAG-IPs were subjected to immunoblotting with anti-USP11 and anti-FLAG antibodies. (d) Endogenous pre-immune IgG or anti-USP15 IPs were subjected to immunoblotting with anti-FLAG and anti-USP15 antibodies as indicated.
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RSOB140065F1: Identification and characterization of USP15 as an interactor of SMAD6. (a) Coomassie stained gel image showing anti-GFP-IPs from HEK293 extracts expressing GFP-SMAD6. The interacting proteins were excised as 2 mm gel pieces, digested with trypsin and identified by mass spectrometry. The location where USP11 and USP15 were identified is indicated. All protein interactors of GFP-SMAD6 identified by mass spectrometry are indicated in the right panel. Protein interactors of GFP control were removed from the list of GFP-SMAD6 interactors. (b) HEK293 cells were transfected transiently with FLAG-SMADs. Extract inputs were resolved by SDS-PAGE and subjected to immunoblotting (IB) with anti-USP11, anti-USP15 and anti-FLAG antibodies as indicated. (c) FLAG-IPs were subjected to immunoblotting with anti-USP11 and anti-FLAG antibodies. (d) Endogenous pre-immune IgG or anti-USP15 IPs were subjected to immunoblotting with anti-FLAG and anti-USP15 antibodies as indicated.

Mentions: In an effort to uncover new regulators of the BMP pathway, we employed a proteomic approach to identify interactors of SMAD6. We stably integrated a single copy of GFP-tagged SMAD6 into human embryonic kidney (HEK293) cells under a tetracycline-inducible promoter. Treatment of these cells with tetracycline resulted in a robust expression of GFP-SMAD6. Immunoprecipitates (IPs) of GFP-SMAD6 from cell extracts were resolved by SDS-PAGE and the interacting proteins were excised, digested with trypsin and identified by mass spectrometry. Consistent with the reported roles of SMAD6 in recruiting E3 ubiquitin ligases to ALK3 [14,17,19,24], we identified several members of the HECT E3 ubiquitin ligase family, including SMURF2, WWP1/2, NEDD4L and ITCH, as interactors of GFP-SMAD6 (figure 1a). Interestingly, two DUBs (USP11 and USP15) were identified as novel interactors of GFP-SMAD6 (figure 1a). USP11 and USP15 did not feature as interactors of either GFP alone or of GFP-tagged SMADs 1–5 and 8 in similar proteomic assays [20,25]. Several other proteins, including NASP, CTBP1/2, KPNA2, PIGR, PLUNC and LYZ were also identified as interactors of only GFP-SMAD6 (figure 1a).Figure 1.


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)

Identification and characterization of USP15 as an interactor of SMAD6. (a) Coomassie stained gel image showing anti-GFP-IPs from HEK293 extracts expressing GFP-SMAD6. The interacting proteins were excised as 2 mm gel pieces, digested with trypsin and identified by mass spectrometry. The location where USP11 and USP15 were identified is indicated. All protein interactors of GFP-SMAD6 identified by mass spectrometry are indicated in the right panel. Protein interactors of GFP control were removed from the list of GFP-SMAD6 interactors. (b) HEK293 cells were transfected transiently with FLAG-SMADs. Extract inputs were resolved by SDS-PAGE and subjected to immunoblotting (IB) with anti-USP11, anti-USP15 and anti-FLAG antibodies as indicated. (c) FLAG-IPs were subjected to immunoblotting with anti-USP11 and anti-FLAG antibodies. (d) Endogenous pre-immune IgG or anti-USP15 IPs were subjected to immunoblotting with anti-FLAG and anti-USP15 antibodies as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4042855&req=5

RSOB140065F1: Identification and characterization of USP15 as an interactor of SMAD6. (a) Coomassie stained gel image showing anti-GFP-IPs from HEK293 extracts expressing GFP-SMAD6. The interacting proteins were excised as 2 mm gel pieces, digested with trypsin and identified by mass spectrometry. The location where USP11 and USP15 were identified is indicated. All protein interactors of GFP-SMAD6 identified by mass spectrometry are indicated in the right panel. Protein interactors of GFP control were removed from the list of GFP-SMAD6 interactors. (b) HEK293 cells were transfected transiently with FLAG-SMADs. Extract inputs were resolved by SDS-PAGE and subjected to immunoblotting (IB) with anti-USP11, anti-USP15 and anti-FLAG antibodies as indicated. (c) FLAG-IPs were subjected to immunoblotting with anti-USP11 and anti-FLAG antibodies. (d) Endogenous pre-immune IgG or anti-USP15 IPs were subjected to immunoblotting with anti-FLAG and anti-USP15 antibodies as indicated.
Mentions: In an effort to uncover new regulators of the BMP pathway, we employed a proteomic approach to identify interactors of SMAD6. We stably integrated a single copy of GFP-tagged SMAD6 into human embryonic kidney (HEK293) cells under a tetracycline-inducible promoter. Treatment of these cells with tetracycline resulted in a robust expression of GFP-SMAD6. Immunoprecipitates (IPs) of GFP-SMAD6 from cell extracts were resolved by SDS-PAGE and the interacting proteins were excised, digested with trypsin and identified by mass spectrometry. Consistent with the reported roles of SMAD6 in recruiting E3 ubiquitin ligases to ALK3 [14,17,19,24], we identified several members of the HECT E3 ubiquitin ligase family, including SMURF2, WWP1/2, NEDD4L and ITCH, as interactors of GFP-SMAD6 (figure 1a). Interestingly, two DUBs (USP11 and USP15) were identified as novel interactors of GFP-SMAD6 (figure 1a). USP11 and USP15 did not feature as interactors of either GFP alone or of GFP-tagged SMADs 1–5 and 8 in similar proteomic assays [20,25]. Several other proteins, including NASP, CTBP1/2, KPNA2, PIGR, PLUNC and LYZ were also identified as interactors of only GFP-SMAD6 (figure 1a).Figure 1.

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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Related in: MedlinePlus