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

Dominic P Byrne,Pawin Ngamlert,David G Fernig,David H Drewry,William J Zuercher,Patrick A Eyers,Krithika Ramakrishnan,Carrow Wells,Neil G Berry,Yong Li,Claire E Eyers

doi:10.1042/bcj20180266

Dominic P Byrne, Pawin Ngamlert + Show 9 more

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https://doi.org/10.1042/bcj20180266

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Abstract

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.

Highlights

Like tyrosine phosphorylation [1], reversible tyrosine sulfation is a critical covalent modification that occurs post-translationally on proteins [2]
In conjunction with analytical differential scanning fluorimetry (DSF), we identified a variety of known ligands as new TPST1 and TPST2 inhibitors, including the promiscuous protein kinase inhibitor rottlerin and a family of oxindole-based rapidly accelerated fibrosarcoma (RAF) kinase inhibitors from the Published Kinase Inhibitor Set (PKIS)
To drive the development of new approaches to assay and inhibit protein tyrosine sulfation, we developed a DSF assay to examine the thermal stability of TPST1 or TPST2 in the presence or absence of biochemical ligands (Figure 1A)

Summary

Introduction

Like tyrosine phosphorylation [1], reversible tyrosine sulfation is a critical covalent modification that occurs post-translationally on proteins [2]. Sulfation can change the affinity of extracellular protein–protein interactions, such as those involved in chemotaxis [4], and host–pathogen interactions [5]. It controls the proteolytic processing of both bioactive peptides [6,7] and secreted antibodies [8], and multi-. Earlier studies had implicated tyrosine sulfation in the proteolytic control of the complement cascade component through decreased activity of C4 [13], the generation of gastrin from progastrin [14], and in regulating the binding of amino-terminal sulfated P-selectin glycoprotein ligand-1 (PSGL-1) to P-selectin [15]. The binding of L-selectin on lymphocytes to mucin-like glycoproteins on endothelial cells is regulated by sulfation, the sialyl LewisX surface antigen is modified by a distinct carbohydrate 6-O sulfotransferase [16]

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