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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 deubiquitylates ALK3. (a) Human recombinant GST-USP15 expressed in Escherichia coli was employed in an in vitro deubiquitylation assay using K48-, K63- and K11-linked and linear di-ubiquitin (Ub) molecules as substrates. The reactions were quenched by adding SDS sample buffer and boiling for 5 min. The samples were resolved by SDS-PAGE, Coomassie stained and then imaged. (b) HEK293 cell transfected with FLAG control or FLAG-ALK3 vectors were treated with bortezomib (10 μM) for 3 h prior to lysis. FLAG-IPs from extracts (1 mg protein) were used as substrates for GST-USP15 in an in vitro deubiquitylation assay. The reactions were stopped by adding SDS sample buffer and boiling for 5 min. The samples were resolved by SDS-PAGE and subjected to immunoblotting analysis using the indicated antibodies. (c) HEK293 cells were transiently transfected with FLAG control or FLAG-ALK3 vectors with or without HA-USP15. Prior to lysis, cells were treated with 10 μM bortezomib for 3 h. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting analysis using the indicated antibodies. (d) HEK293 cells transiently expressing FLAG-ALK3, HA-USP15 and USP15 C269S DUB dead mutant (DD) were serum-starved overnight, pretreated with 10 μM bortezomib for 3 h then stimulated with 6.25 ng ml–1 BMP for 1 h prior to lysis. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (e) HEK293 cells transiently expressing siUSP15-3, FLAG-ALK3 and siUSP15-3 resistant silent mutant of HA-USP15 (HA-USP15R) were serum-starved overnight, pretreated with 10 μM bortezomib for 3 h then stimulated with 6.25 ng ml−1 BMP for 1 h prior to lysis. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies.
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RSOB140065F5: USP15 deubiquitylates ALK3. (a) Human recombinant GST-USP15 expressed in Escherichia coli was employed in an in vitro deubiquitylation assay using K48-, K63- and K11-linked and linear di-ubiquitin (Ub) molecules as substrates. The reactions were quenched by adding SDS sample buffer and boiling for 5 min. The samples were resolved by SDS-PAGE, Coomassie stained and then imaged. (b) HEK293 cell transfected with FLAG control or FLAG-ALK3 vectors were treated with bortezomib (10 μM) for 3 h prior to lysis. FLAG-IPs from extracts (1 mg protein) were used as substrates for GST-USP15 in an in vitro deubiquitylation assay. The reactions were stopped by adding SDS sample buffer and boiling for 5 min. The samples were resolved by SDS-PAGE and subjected to immunoblotting analysis using the indicated antibodies. (c) HEK293 cells were transiently transfected with FLAG control or FLAG-ALK3 vectors with or without HA-USP15. Prior to lysis, cells were treated with 10 μM bortezomib for 3 h. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting analysis using the indicated antibodies. (d) HEK293 cells transiently expressing FLAG-ALK3, HA-USP15 and USP15 C269S DUB dead mutant (DD) were serum-starved overnight, pretreated with 10 μM bortezomib for 3 h then stimulated with 6.25 ng ml–1 BMP for 1 h prior to lysis. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (e) HEK293 cells transiently expressing siUSP15-3, FLAG-ALK3 and siUSP15-3 resistant silent mutant of HA-USP15 (HA-USP15R) were serum-starved overnight, pretreated with 10 μM bortezomib for 3 h then stimulated with 6.25 ng ml−1 BMP for 1 h prior to lysis. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies.

Mentions: The strong interaction between USP15 and ALK3 and their co-localization at the plasma membrane suggested that USP15 could act as a DUB for ALK3. Human recombinant USP15 expressed in bacteria displays DUB activity in vitro and is capable of cleaving not only K48-linked but also K63- and K11-linked diubiquitin chains (figure 5a). It did not cleave linear diubiquitin chain (figure 5a). To test whether USP15 can deubiquitylate polyubiquitylated ALK3 in vitro, we immunoprecipitated FLAG-ALK3 from HEK293 cells treated with the proteasome inhibitor bortezomib (to enrich the pool of polyubiquitylated FLAG-ALK3) and subjected the FLAG-IPs to in vitro deubiquitylation by GST-USP15 (figure 5b). In the absence of USP15, FLAG-ALK3 IPs displayed robust polyubiquitylation, while the introduction of GST-USP15 caused efficient deubiquitylation, leading to the accumulation of mono-ubiquitin (figure 5b).Figure 5.


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 deubiquitylates ALK3. (a) Human recombinant GST-USP15 expressed in Escherichia coli was employed in an in vitro deubiquitylation assay using K48-, K63- and K11-linked and linear di-ubiquitin (Ub) molecules as substrates. The reactions were quenched by adding SDS sample buffer and boiling for 5 min. The samples were resolved by SDS-PAGE, Coomassie stained and then imaged. (b) HEK293 cell transfected with FLAG control or FLAG-ALK3 vectors were treated with bortezomib (10 μM) for 3 h prior to lysis. FLAG-IPs from extracts (1 mg protein) were used as substrates for GST-USP15 in an in vitro deubiquitylation assay. The reactions were stopped by adding SDS sample buffer and boiling for 5 min. The samples were resolved by SDS-PAGE and subjected to immunoblotting analysis using the indicated antibodies. (c) HEK293 cells were transiently transfected with FLAG control or FLAG-ALK3 vectors with or without HA-USP15. Prior to lysis, cells were treated with 10 μM bortezomib for 3 h. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting analysis using the indicated antibodies. (d) HEK293 cells transiently expressing FLAG-ALK3, HA-USP15 and USP15 C269S DUB dead mutant (DD) were serum-starved overnight, pretreated with 10 μM bortezomib for 3 h then stimulated with 6.25 ng ml–1 BMP for 1 h prior to lysis. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (e) HEK293 cells transiently expressing siUSP15-3, FLAG-ALK3 and siUSP15-3 resistant silent mutant of HA-USP15 (HA-USP15R) were serum-starved overnight, pretreated with 10 μM bortezomib for 3 h then stimulated with 6.25 ng ml−1 BMP for 1 h prior to lysis. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140065F5: USP15 deubiquitylates ALK3. (a) Human recombinant GST-USP15 expressed in Escherichia coli was employed in an in vitro deubiquitylation assay using K48-, K63- and K11-linked and linear di-ubiquitin (Ub) molecules as substrates. The reactions were quenched by adding SDS sample buffer and boiling for 5 min. The samples were resolved by SDS-PAGE, Coomassie stained and then imaged. (b) HEK293 cell transfected with FLAG control or FLAG-ALK3 vectors were treated with bortezomib (10 μM) for 3 h prior to lysis. FLAG-IPs from extracts (1 mg protein) were used as substrates for GST-USP15 in an in vitro deubiquitylation assay. The reactions were stopped by adding SDS sample buffer and boiling for 5 min. The samples were resolved by SDS-PAGE and subjected to immunoblotting analysis using the indicated antibodies. (c) HEK293 cells were transiently transfected with FLAG control or FLAG-ALK3 vectors with or without HA-USP15. Prior to lysis, cells were treated with 10 μM bortezomib for 3 h. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting analysis using the indicated antibodies. (d) HEK293 cells transiently expressing FLAG-ALK3, HA-USP15 and USP15 C269S DUB dead mutant (DD) were serum-starved overnight, pretreated with 10 μM bortezomib for 3 h then stimulated with 6.25 ng ml–1 BMP for 1 h prior to lysis. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (e) HEK293 cells transiently expressing siUSP15-3, FLAG-ALK3 and siUSP15-3 resistant silent mutant of HA-USP15 (HA-USP15R) were serum-starved overnight, pretreated with 10 μM bortezomib for 3 h then stimulated with 6.25 ng ml−1 BMP for 1 h prior to lysis. FLAG-IPs and extract inputs were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies.
Mentions: The strong interaction between USP15 and ALK3 and their co-localization at the plasma membrane suggested that USP15 could act as a DUB for ALK3. Human recombinant USP15 expressed in bacteria displays DUB activity in vitro and is capable of cleaving not only K48-linked but also K63- and K11-linked diubiquitin chains (figure 5a). It did not cleave linear diubiquitin chain (figure 5a). To test whether USP15 can deubiquitylate polyubiquitylated ALK3 in vitro, we immunoprecipitated FLAG-ALK3 from HEK293 cells treated with the proteasome inhibitor bortezomib (to enrich the pool of polyubiquitylated FLAG-ALK3) and subjected the FLAG-IPs to in vitro deubiquitylation by GST-USP15 (figure 5b). In the absence of USP15, FLAG-ALK3 IPs displayed robust polyubiquitylation, while the introduction of GST-USP15 caused efficient deubiquitylation, leading to the accumulation of mono-ubiquitin (figure 5b).Figure 5.

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