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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 interacts and co-localizes with SMAD6 and ALK3. (a) HEK293 cells were transfected with GFP-USP15 with control vector or mammalian expression vectors encoding N-terminal FLAG-tagged ALK5, ALK3, ALK2 or ALK6. Cells were lysed and extracts (1 mg) subjected to GFP-IPs. GFP-IPs (40%) or extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (b) HEK293 cells expressing GFP control or GFP-USP15 were transfected with FLAG-ALK3, HA-SMAD6 or both as indicated. Cells were lysed and extracts (1 mg) subjected to GFP-IPs. GFP-IPs (40%) or extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (c) Fixed cell immunofluorescence was performed on U2OS cells transfected with FLAG-ALK3, HA-SMAD6 and GFP-USP15. Individual and merged pictures are shown, indicating localization of FLAG-ALK3 mainly in the cytosol, HA-SMAD6 in the nucleus and GFP-USP15 in both compartments. GFP-USP15 mainly co-localizes with FLAG-ALK3. Pictures were taken using a 60× lens, scale bar represents 30 μm.
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RSOB140065F4: USP15 interacts and co-localizes with SMAD6 and ALK3. (a) HEK293 cells were transfected with GFP-USP15 with control vector or mammalian expression vectors encoding N-terminal FLAG-tagged ALK5, ALK3, ALK2 or ALK6. Cells were lysed and extracts (1 mg) subjected to GFP-IPs. GFP-IPs (40%) or extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (b) HEK293 cells expressing GFP control or GFP-USP15 were transfected with FLAG-ALK3, HA-SMAD6 or both as indicated. Cells were lysed and extracts (1 mg) subjected to GFP-IPs. GFP-IPs (40%) or extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (c) Fixed cell immunofluorescence was performed on U2OS cells transfected with FLAG-ALK3, HA-SMAD6 and GFP-USP15. Individual and merged pictures are shown, indicating localization of FLAG-ALK3 mainly in the cytosol, HA-SMAD6 in the nucleus and GFP-USP15 in both compartments. GFP-USP15 mainly co-localizes with FLAG-ALK3. Pictures were taken using a 60× lens, scale bar represents 30 μm.

Mentions: To explore this idea, we first tested the ability of USP15 to interact with various ALKs, including ALK3, upon co-expression in HEK293 cells. GFP-USP15 IPs from HEK293 extracts interacted with FLAG-ALK5, FLAG-ALK3, FLAG-ALK2 and FLAG-ALK6 (figure 4a). Under these conditions, the association between USP15 and ALK2 appeared to be the strongest (figure 4a). Next, we tested how SMAD6 affected the interaction between GFP-USP15 and FLAG-ALK3 (figure 4b). Expression of SMAD6 caused a reduction in the ability of GFP-USP15 to interact with FLAG-ALK3, suggesting that SMAD6 disrupts USP15 : ALK3 association. Interestingly, the interaction between GFP-USP15 and HA-SMAD6 was completely abolished by FLAG-ALK3 overexpression (figure 4b), suggesting that USP15 : SMAD6 and USP15 : ALK3 interactions could be mutually exclusive.Figure 4.


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 interacts and co-localizes with SMAD6 and ALK3. (a) HEK293 cells were transfected with GFP-USP15 with control vector or mammalian expression vectors encoding N-terminal FLAG-tagged ALK5, ALK3, ALK2 or ALK6. Cells were lysed and extracts (1 mg) subjected to GFP-IPs. GFP-IPs (40%) or extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (b) HEK293 cells expressing GFP control or GFP-USP15 were transfected with FLAG-ALK3, HA-SMAD6 or both as indicated. Cells were lysed and extracts (1 mg) subjected to GFP-IPs. GFP-IPs (40%) or extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (c) Fixed cell immunofluorescence was performed on U2OS cells transfected with FLAG-ALK3, HA-SMAD6 and GFP-USP15. Individual and merged pictures are shown, indicating localization of FLAG-ALK3 mainly in the cytosol, HA-SMAD6 in the nucleus and GFP-USP15 in both compartments. GFP-USP15 mainly co-localizes with FLAG-ALK3. Pictures were taken using a 60× lens, scale bar represents 30 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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RSOB140065F4: USP15 interacts and co-localizes with SMAD6 and ALK3. (a) HEK293 cells were transfected with GFP-USP15 with control vector or mammalian expression vectors encoding N-terminal FLAG-tagged ALK5, ALK3, ALK2 or ALK6. Cells were lysed and extracts (1 mg) subjected to GFP-IPs. GFP-IPs (40%) or extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (b) HEK293 cells expressing GFP control or GFP-USP15 were transfected with FLAG-ALK3, HA-SMAD6 or both as indicated. Cells were lysed and extracts (1 mg) subjected to GFP-IPs. GFP-IPs (40%) or extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (c) Fixed cell immunofluorescence was performed on U2OS cells transfected with FLAG-ALK3, HA-SMAD6 and GFP-USP15. Individual and merged pictures are shown, indicating localization of FLAG-ALK3 mainly in the cytosol, HA-SMAD6 in the nucleus and GFP-USP15 in both compartments. GFP-USP15 mainly co-localizes with FLAG-ALK3. Pictures were taken using a 60× lens, scale bar represents 30 μm.
Mentions: To explore this idea, we first tested the ability of USP15 to interact with various ALKs, including ALK3, upon co-expression in HEK293 cells. GFP-USP15 IPs from HEK293 extracts interacted with FLAG-ALK5, FLAG-ALK3, FLAG-ALK2 and FLAG-ALK6 (figure 4a). Under these conditions, the association between USP15 and ALK2 appeared to be the strongest (figure 4a). Next, we tested how SMAD6 affected the interaction between GFP-USP15 and FLAG-ALK3 (figure 4b). Expression of SMAD6 caused a reduction in the ability of GFP-USP15 to interact with FLAG-ALK3, suggesting that SMAD6 disrupts USP15 : ALK3 association. Interestingly, the interaction between GFP-USP15 and HA-SMAD6 was completely abolished by FLAG-ALK3 overexpression (figure 4b), suggesting that USP15 : SMAD6 and USP15 : ALK3 interactions could be mutually exclusive.Figure 4.

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