Herhaus L et al. (2014), USP15 targets ALK3/BMPR1A for deubiquitylation ...

XB-ART-49057

Open Biol 2014 May 01;45:140065. doi: 10.1098/rsob.140065.

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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 .

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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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Species referenced: Xenopus laevis
Genes referenced: acvr1 bmp2 bmpr1a bmpr1b foxo4 isyna1 lmna prss1 smad1 smad6 smad6.2 tgfbr1 usp15
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	FigureÂ 1. 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.
	FigureÂ 2. 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 mlâ1 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 mlâ1 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.
	FigureÂ 3. 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 mlâ1 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 mlâ1 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.
	FigureÂ 4. 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 60Ã lens, scale bar represents 30 Î¼m.
	FigureÂ 5. 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 Î¼M) 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 Î¼M 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 Î¼M bortezomib for 3 h then stimulated with 6.25 ng mlâ1 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 Î¼M bortezomib for 3 h then stimulated with 6.25 ng mlâ1 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.
	FigureÂ 6. ALK3 undergoes proteasomal degradation. (a) HEK293 cells transfected with untagged ALK3 were treated with or without 20 Î¼M cycloheximide for 24 h prior to lysis. Cells were treated with DMSO control, 100 nM bafilomycin A1 (to inhibit vacuolar-type H+ ATPase) or 10 Î¼M bortezomib (to inhibit the proteasome) for 3 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. (b) HEK293 cells were transiently transfected with siFoxO4 or siUSP15-3. Cells were serum-starved overnight, treated with or without 10 Î¼M bortezomib for 3 h and then stimulated with or without 6.25 ng mlâ1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against endogenous USP15, pSMAD1 and total SMAD1. (c) HEK293 cells were transiently transfected with siFoxO4 (â), siUSP15-3 or siSMAD6 as indicated. Twenty-four hours post siRNA transfection, cells were serum-starved overnight and stimulated with or without 6.25 ng mlâ1 BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against pSMAD1, total SMAD1, USP15 and GAPDH. The SMAD6 knockdown was confirmed by qRT-PCR (electronic supplementary material, figure S7).
	FigureÂ 7. USP15 impacts osteoblastic differentiation in C2C12 myoblasts and modulates BMP signalling in Xenopus embryogenesis. (a) Mouse myoblast cell line C2C12 were transfected with siRNAs targeting mouse FoxO4 or USP15. Cells were serum-starved overnight and treated with or without BMP for 1 h prior to lysis. Extracts were resolved by SDS-PAGE and immunoblotted with antibodies against USP15, pSMAD1, total SMAD1 and GAPDH. (b) C2C12 cells transfected with mouse siFoxO4 or mouse siUSP15 were grown for up to 4 days in the presence of BMP. Cells were lysed and the alkaline phosphatase activity measured using a fluorescence plate reader. Data are represented as mean of three biological replicates and error bars indicate s.d. Representative extracts were resolved by SDS-PAGE and subjected to immunoblotting with antibodies against USP15 and GAPDH. (c) Xenopus embryos were injected with 80 ng of either xUSP15- (xUSP15-MO) or control-MO morpholinos at the one-cell stage and then collected at the indicated stages. Lysates were resolved by SDS-PAGE and immunoblotted with antibodies against pSMAD1 and Î±-tubulin. (d) qRT-PCR analysis of xVENT1 mRNA expression. Embryos were injected with 80 ng of either USP15-MO or control-MO at the one-cell stage and then animal caps were cut at stage 8.5. The animal caps were collected at the equivalent embryo stage of 10.5 and processed for qRT-PCR.

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