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New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors

Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein–protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (...

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Autores principales: Byrne, Dominic P., Li, Yong, Ngamlert, Pawin, Ramakrishnan, Krithika, Eyers, Claire E., Wells, Carrow, Drewry, David H., Zuercher, William J., Berry, Neil G., Fernig, David G., Eyers, Patrick A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094398/
https://www.ncbi.nlm.nih.gov/pubmed/29934490
http://dx.doi.org/10.1042/BCJ20180266
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author Byrne, Dominic P.
Li, Yong
Ngamlert, Pawin
Ramakrishnan, Krithika
Eyers, Claire E.
Wells, Carrow
Drewry, David H.
Zuercher, William J.
Berry, Neil G.
Fernig, David G.
Eyers, Patrick A.
author_facet Byrne, Dominic P.
Li, Yong
Ngamlert, Pawin
Ramakrishnan, Krithika
Eyers, Claire E.
Wells, Carrow
Drewry, David H.
Zuercher, William J.
Berry, Neil G.
Fernig, David G.
Eyers, Patrick A.
author_sort Byrne, Dominic P.
collection PubMed
description Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein–protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based inhibitors of the Ser/Thr kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro. We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.
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spelling pubmed-60943982018-09-05 New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors Byrne, Dominic P. Li, Yong Ngamlert, Pawin Ramakrishnan, Krithika Eyers, Claire E. Wells, Carrow Drewry, David H. Zuercher, William J. Berry, Neil G. Fernig, David G. Eyers, Patrick A. Biochem J Research Articles Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein–protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based inhibitors of the Ser/Thr kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro. We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors. Portland Press Ltd. 2018-08-16 2018-08-14 /pmc/articles/PMC6094398/ /pubmed/29934490 http://dx.doi.org/10.1042/BCJ20180266 Text en © 2018 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Articles
Byrne, Dominic P.
Li, Yong
Ngamlert, Pawin
Ramakrishnan, Krithika
Eyers, Claire E.
Wells, Carrow
Drewry, David H.
Zuercher, William J.
Berry, Neil G.
Fernig, David G.
Eyers, Patrick A.
New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors
title New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors
title_full New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors
title_fullStr New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors
title_full_unstemmed New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors
title_short New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors
title_sort new tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (tpsts) are novel targets for raf protein kinase inhibitors
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094398/
https://www.ncbi.nlm.nih.gov/pubmed/29934490
http://dx.doi.org/10.1042/BCJ20180266
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