University of Dundee

Dr Gopal Sapkota

Untangling the TGFβ and Wnt signalling networks in cells, development and human diseases
Position: 
Programme Leader in Protein Phosphorylation
Address: 
MRC Protein Phosphorylation Unit, The Sir James Black Centre, University of Dundee, Dundee DD1 5EH
Full Telephone: 
+44 (0) 1382 386330, int ext 86330
Email: 

Research

Synopsis of areas we work on:

  • Transforming Growth Factor β (TGFβ), Bone Morphogenetic Protein (BMP) and Wnt signalling pathways.
  • PAWS1 and the FAM83 family of uncharacterised proteins.
  • Regulation of proteins through post-translational modifications, including reversible phosphorylation and ubiquitylation.
  • Innovative biotechnology

Specific disease areas we focus on:

  • Cancer, fibrosis, and skin diseases

Technologies we employ:

  • CRISPR/Cas9 genome editing
  • Mass spectrometry
  • Transcriptomics
  • Advanced fluorescence microscopy
  • Cell and Molecular Biology
  • Biochemistry and Enzymology
  • X-ray crystallography
  • In vivo model systems

Key external collaborations:

  • Jim Smith (CRICK)
  • Alex Bullock (Oxford)

Specific projects and key aims:

1. Function and regulation of PAWS1: One of the key discoveries we have made in the lab is the identification of a previously uncharacterised SMAD1-interacting protein FAM83G, which we have termed PAWS1 (Protein Associated With SMAD1) (Vogt et al, 2014). In addition to its role in BMP signalling, our findings indicate that PAWS1 controls transcription, cell proliferation and migration and regulates embryogenesis. The precise molecular mechanisms through which PAWS1 exerts these outcomes remain to be defined. Delineating the molecular function and regulation of PAWS1 in cells, development and cancer is one of the key goals of the lab.

2. The DUF1669 domain and the FAM83 family of proteins: PAWS1 is a member of the poorly characterised FAM83 family of proteins that are linked through the conserved DUF1669 domain of unknown function, which possesses a pseudo-Phospholipase D catalytic motif. We aim to assign the biochemical role to this domain, through proteomic, biochemical, cellular and biophysical approaches. By employing proteomic and cell-based approaches, we have determined that each FAM83 member has unique interacting partners and subcellular localization profile, suggesting specific cellular roles. We aim to understand how the DUF1669 domain controls the function of the FAM83 family of proteins in their potentially diverse cellular roles. 

3. Phosphorylation and ubiquitylation in the TGFβ pathway: Building on the identification of OTUB1 as a player in the TGFβ pathway (Herhaus et al, 2013), we want to establish the molecular basis through which OTUB1 is recruited to the active phospho-SMAD2/3 complex. In doing so, we might reveal how DUBs like OTUB1 are directed to selective targets through recognition of specific post-translational modifications on targets. We are also interested in developing TGFβ/BMP pathway DUB (Herhaus et al, 2014; Herhaus et al 2015; Al-Salihi et al, 2012) inhibitors and assessing the efficacy of in TGFβ/BMP signalling.
Having established PAWS1 as the first non-SMAD substrate of type I BMP receptor (Vogt et al, 2014), we want to explore whether there are other non-SMAD targets of type I TGFβ and BMP receptors. For this, we will exploit a quantitative phospho-proteomics approach to identify new substrates of TGFBR1 (ALK5), using ALK5-null cells restored with WT or catalytically inactive ALK5. We will characterise the most promising targets further and establish their roles in cells and physiology.

4. Drug discovery and signalling crosstalk in the TGFβ pathway: Using robust endogenously driven BMP and TGFβ reporter cells (Rojas-Fernandez et al, 2015) that we have generated using CRISPR/Cas9, we have undertaken a chemical screen using highly potent and selective kinase inhibitors. From this, we have identified a handful of novel kinases that appear to either inhibit or activate TGFβ and BMP signalling, downstream of SMAD phosphorylation. We want to pursue functional tests on these kinases to establish how they regulate TGFβ and BMP signalling and whether these can target pathologies associated with TGFβ and BMP. Our endogenous reporter cells provide a robust platform for further high throughput chemical and genetic screening. The technology we have described to rapidly produce endogenous reporter systems using CRISPR/Cas9 can be readily applied to study the regulation of transcription of any gene.

5. Advancing innovative technologies to aid fundamental research and drug discovery:Advancing and exploiting new technologies to address fundamental research questions and aid drug discovery remains a top priority of the lab. We are combining the rapid genome editing capability afforded by CRISPR/Cas9 with advanced knowledge of protein chemistry to engineer robust molecular tools capable of selectively targeting individual proteins for desired functional modulation in cells. Affinity-directed PROtein Missile (AdPROM) system (Fulcher et al, 2016) for targeted proteolysis of proteins of interest is an example of the type of technologies we want to develop and exploit.

Outreach:
We are committed to communicating our research ideas and outcomes to the public. The School of Life Sciences at the University of Dundee has Open Days, during which laboratory tours can be arranged for members of the public interested in or curious about our work.
Our laboratory actively supports initiatives aimed at encouraging young students from taking up interest in science and research. Every year, we are committed to hosting one or two S4-S6 pupils to spend a week in the laboratory to gain a first-hand experience in biomedical research. Anyone interested should make enquiries to Gopal Sapkota (g.sapkota@dundee.ac.uk).

Key References

  1. Cammareri P, Rose AM, Vincent DF, Wang J, Nagano A, Libertini S, Ridgway RA, Athineos D, Coates PJ, McHugh A, Pourreyron C, Dayal JH, Larsson J, Weidlich S, Spender LC, Sapkota GP, Purdie KJ, Proby CM, Harwood CA, Leigh IM, Clevers H, Barker N, Karlsson S, Pritchard C, Marais R, Chelala C, South AP, Sansom OJ, Inman GJ. (2016) Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma. Nat Commun. 2016 Aug 25;7:12493. doi: 10.1038/ncomms12493.
  2. Fulcher, A. J., Macartney, T., Bozatzi, P., Hornberger, A., Rojas-Fernandez, A., and Sapkota, G. P. (2016) An Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis. Open Biol. 
  3. Rojas-Fernandez, A., Herhaus, L., Macartney, T., Lachaud, C., Hay, R. T., and Sapkota, G. P. (2015) Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9. Sci Rep 5:9811 doi: 10.1038/srep09811
  4. Herhaus, L., Perez-Oliva, A.B., Cozza, G., Gourlay, R., Weidlich, S., Campbell, D. G., Pinna, L. A. and Sapkota, G. P. (2015) Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser16 to trigger its nuclear localization. Sci Signal. 2015 Apr 14;8(372):ra35. doi: 10.1126/scisignal.aaa0441.
  5. Herhaus, L., and Sapkota, G. P. (2014) The emerging roles of deubiquitylating enzymes (DUBs) in the TGFβ and BMP pathways. Cell Signal. 2014 Oct;26(10):2186-92. doi: 10.1016/j.cellsig.2014.06.012.
  6. Herhaus, L., Al-Salihi, M. A., Dingwell, K. S., Cummins, T. D., Wasmus, L., Vogt, J., Bruce, D. L., Macartney, T., Weidlich, S., Smith, J. C. and Sapkota, G. P. (2014). USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling. Open Biol. 4: 140065.
  7. Vogt, J., Dingwell, K. S., Herhaus, L., Gourlay, R., Macartney, T., Campbell, D., Smith, J. C. and Sapkota, G. P. (2014). Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling. Open Biol 4, pp. 130210
  8. Herhaus, L., Al-Salihi, M.A., Macartney, T., Weidlich, S. and Sapkota, G.P. (2013) OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3. Nature Communications, 4:2519 | DOI: 10.1038/ncomms3519
  9. Al-Salihi, M. A., Herhaus, L., Macartney, T. and Sapkota, G. P. (2012). USP11 augments TGFbeta signalling by deubiquitylating ALK5. Open Biology 2, pp. 120063
  10. Al-Salihi, M. A., Herhaus, L. and Sapkota, G. P. (2012). Regulation of the transforming growth factor beta pathway by reversible ubiquitylation. Open Biology 2, pp. 120082

Teaching

MRes Cancer Biology

Publications

Top 10 Publications

  1. Cammareri P, Rose AM, Vincent DF, Wang J, Nagano A, Libertini S, Ridgway RA, Athineos D, Coates PJ, McHugh A, Pourreyron C, Dayal JH, Larsson J, Weidlich S, Spender LC, Sapkota GP, Purdie KJ, Proby CM, Harwood CA, Leigh IM, Clevers H, Barker N, Karlsson S, Pritchard C, Marais R, Chelala C, South AP, Sansom OJ, Inman GJ. (2016) Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma. Nat Commun. 2016 Aug 25;7:12493. doi: 10.1038/ncomms12493.
  2. Fulcher, A. J., Macartney, T., Bozatzi, P., Hornberger, A., Rojas-Fernandez, A., and Sapkota, G. P. (2016) An Affinity-directed PROtein Missile (AdPROM) system for targeted proteolysis. Open Biol. 
  3. Rojas-Fernandez, A., Herhaus, L., Macartney, T., Lachaud, C., Hay, R. T., and Sapkota, G. P. (2015) Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9. Sci Rep 5:9811 doi: 10.1038/srep09811
  4. Herhaus, L., Perez-Oliva, A.B., Cozza, G., Gourlay, R., Weidlich, S., Campbell, D. G., Pinna, L. A. and Sapkota, G. P. (2015) Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser16 to trigger its nuclear localization. Sci Signal. 2015 Apr 14;8(372):ra35. doi: 10.1126/scisignal.aaa0441.
  5. Herhaus, L., and Sapkota, G. P. (2014) The emerging roles of deubiquitylating enzymes (DUBs) in the TGFβ and BMP pathways. Cell Signal. 2014 Oct;26(10):2186-92. doi: 10.1016/j.cellsig.2014.06.012.
  6. Herhaus, L., Al-Salihi, M. A., Dingwell, K. S., Cummins, T. D., Wasmus, L., Vogt, J., Bruce, D. L., Macartney, T., Weidlich, S., Smith, J. C. and Sapkota, G. P. (2014). USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling. Open Biol. 4: 140065.
  7. Vogt, J., Dingwell, K. S., Herhaus, L., Gourlay, R., Macartney, T., Campbell, D., Smith, J. C. and Sapkota, G. P. (2014). Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling. Open Biol 4, pp. 130210
  8. Herhaus, L., Al-Salihi, M.A., Macartney, T., Weidlich, S. and Sapkota, G.P. (2013) OTUB1 enhances TGFβ signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3. Nature Communications, 4:2519 | DOI: 10.1038/ncomms3519
  9. Al-Salihi, M. A., Herhaus, L., Macartney, T. and Sapkota, G. P. (2012). USP11 augments TGFbeta signalling by deubiquitylating ALK5. Open Biology 2, pp. 120063
  10. Al-Salihi, M. A., Herhaus, L. and Sapkota, G. P. (2012). Regulation of the transforming growth factor beta pathway by reversible ubiquitylation. Open Biology 2, pp. 120082

Impact

Commercial Impact:

In collaboration with the pharmaceutical industry via the Division of Signal Transduction Therapy collaboration with AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Janssen Pharmaceutica, Merck Serono and Pfizer the research ouptuts from my group contribute to accelerating the development of company drug development programmes through access to research data and reagents. Reagents are also commercialised to provide access to the wider scientific community via license arrangements with companies such as Millipore, AbCam and Ubiquigent.