DUSP3 phosphatase: structure, function and regulation.

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VHR/DUSP3 phosphatase: Structure, function and regulation

Karolina Pavic, Guangyou Duan, Maja Köhn European Molecular Biology Laboratory, Genome Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany

Corresponding author:

Maja Köhn Phone: +49 6221 3878544 Fax: +49 6221 387518 Email: [email protected]

Running title: Structure, function and regulation of VHR/DUSP3 phosphatase

Keywords: Vaccinia H1-Related phosphatase, dual specificity phosphatases, protein phosphatases, cell cycle progression, cancer, DUSP13B, DUSP26, DUSP27

Article type

: Review Article

Abbreviations: A-DUSP, atypical dual-specificity phosphatase; Ap1, activating protein 1; APC, antigen-presenting cell; ATP, adenosine triphosphate; BRCA1, breast cancer 1; Cdc14, cell division cycle 14; CLEC-2, C-type lectin type receptor 2; DDR, DNA damage response; DIFMUP, 6, 8-difluoro-4-methylumbelliferyl phosphate; EGF(R), epidermal growth factor (receptor); ErbB2, erythroblastic leukemia viral oncogene homolog 2; ERK, Extracellular signal regulated kinase; FCRγ, GPVI/Fc receptor γ-chain; FGF, fibroblast growth factor; FPR, formyl peptide receptor; GATPT, (Glucosamine-aminoethoxy) triphenyltin; GPVI, collagen This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/febs.13263 This article is protected by copyright. All rights reserved.

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receptor glycoprotein VI; HAD, haloacid dehalogenase; HME, human mammary epithelial; HnRNP, heterogeneous nuclear ribonucleoprotein; HUVEC, human umbilical vein endothelial cells; IFN, interferon; IR, insulin receptor; ITAM, immunoreceptor tyrosine-based activation motif; JNK, c-Jun N-terminal kinase; KGFR, keratinocyte growth factor receptor; LCC, Lung Lewis Carcinoma; MAPK, mitogen activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MKB, MAPK binding; MKP, MAPK phosphatase; NSCLC, non–small cell lung cancer; NPM, nucleophosmin; NSCLC, non-small cell lung cancer; NUCL, nucleolin; pBPA, para-benzoylphenylalanine; PDGFR, platelet-derived growth factor receptor; PK, protein

kinase;

PKC,

protein

kinase

C;

PLC,

phospholipase

C;

pNPP,

para-

nitrophenolphosphate; PP, protein phosphatase; PRL, phosphatase of regenerating liver; PTEN, phosphatase and tensin homologues; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; ROS, reactive oxygen species; SAR, structure-affinity relationship; SH2, Src homology 2; SRK, Src family kinases; STAT, signal transducers and activators of transcription; TCR, T-cell antigen receptor; Tyk2, tyrosine kinase 2; VH1, Vaccinia H1; VHR, Vaccinia H1-Related; VRK, Vaccinia-related kinase; wt, wild type.

Summary Vaccinia H1-Related (VHR) phosphatase, also known as dual-specificity phosphatase (DUSP) 3, is a small member of the DUSP (also called DSP) family of phosphatases. VHR has a preference for phospho-tyrosine substrates, and plays important roles in cellular signaling ranging from cell cycle regulation and DNA damage response to MAPK signaling, platelet activation and angiogenesis. VHR/DUSP3 has been implicated in several human cancers, where its tumor suppressing and promoting properties have been described. We give here a detailed overview of the VHR/DUSP3 phosphatase and compare it to its most closely related phosphatases DUSP13B, DUSP26 and DUSP27.

Introduction- general properties of the DUSPs The equilibrium of protein phosphorylation in cells is achieved through complementary activities of protein kinases (PKs), which catalyze the transfer of a phosphoryl moiety

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reported to both up- and down-regulate the activities of MAPK-pathways, but also have substrates outside of the MAPK families (13,35). Nevertheless, the involvement of A-DUSPs in MAPK signalling is still controversial (35). In general, substrates, roles and pathways are not well understood for A-DUSPs. Further studies are needed to shed light on the cellular targets of the A-DUSPs and mechanisms behind their involvement in various cellular processes, in normal or pathological states.

General properties and roles of VHR VHR/DUSP3 is an A-DUSP comprised of 185 amino acids organized in a single catalytic domain (35,36). VHR was the first DUSP to have its crystal structure determined (37). It is a prototypic member of the A-DUSPs, smaller in size compared to MKPs and lacking not only the N-terminal MKB, but also any additional adapter or regulatory domains or sequences that may confer substrate specificity (8,37). VHR was identified by Ishibashi et al. by using an expression cloning strategy (36). The newly discovered enzyme bore restricted sequence resemblance to VH1, a key gene in Vaccinia virus and poxvirus (18), hence it was named VH1-Related phosphatase. Apart from the residues of the PTP signature motif, VHR shares no discernible sequence homology to the classical PTPs (37,38). There are only ten identical residues between VHR and the classical PTPs, most of them implicated in the phosphosubstrate hydrolysis (37). An evolutionary conservation analysis for VHR using ConSurf (39) based on 150 homologous sequences for VHR from UniRef90 (40) reveals highly conserved and non-conserved regions and amino acids in VHR (Figure 1A). As expected, residues His123, Cys124, Arg130 and Asp92 are found highly conserved as they are involved in the catalytic mechanism. Regions such as the “variable insert region” (see below) and the “substrate recognition region” (see below) on the other hand show very low conservation scores, making them unique features of VHR.

In the structure-sequence-based classification of the human phosphatome (3,12), the closest homologues of VHR are the A-DUPSs DUSP13A (MDSP) and DUSP13B (TMDP), DUSP26 (MKP-8) and DUSP27 (DUPD1). In Figure 1B we show an amino acid sequence


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of regenerating liver (PRLs, 3 members), slingshots (3 members), phosphatase and tensin homologues (PTENs, 6 members), myotubularins (16 members) and cell division cycle 14 homologues (Cdc14s, 5 members) (12,14). VHR is a member of the A-DUSPs, with the substrate specificity similar to members of the MKPs, and therefore these two families will be discussed below. DUSPs share the catalytic mechanism employed by the classical PTPs, which encompasses two distinct enzymatic steps (8). It involves residues of two distinct loops. The first is the active site, called the phosphate-binding loop (P-loop), which contains the conserved PTP signature motif with the catalytically active Cys. The second, called the general acid/base (WPD for tryptophan-proline-aspartate)-loop, is separated from the signature motif in the primary sequence, but moves close to the active site during catalysis. The Asp in the WPDloop functions both as a general acid to facilitate thiolysis of the scissile P-O bond in the phosphate ester of a substrate, and as a general base in the following hydrolysis of the thiophosphate intermediate (8,19).

MKPs versus atypical DUSPs MKPs represent a subgroup of 10 active phosphatases within the DUSP-superfamily that are involved in the downregulation of MAPK-mediated signaling events. These events are triggered in response to mitogens, growth factors, differentiation factors or stress via the dephosphorylation of Thr and/or Tyr residues in the Thr-X-Tyr motif of the kinase’s activation loop (where X represents any amino acid) (13,20). The MAPKs are a final constituent in conserved tripartite kinase signaling cascades, and are activated by the upstream dual-specificity kinases termed MAPK kinases (MAPKK, MKK or MEK), which, in turn, are activated by their upstream kinases (MAPKKK or MEKK) (20,21). In mammals, there are four major families of MAPKs (21). These comprise extracellular signal-regulated kinase (ERK) 1/2 (ERK1 is also known as p44 MAPK or as MAPK1 and ERK2 as p42 MAPK or as MAPK2), p38 (with α, β, γ and δ isoforms), c-jun N-terminal kinases (JNK), also known as stress-activated protein kinase (SAPK) (isoforms 1-3) and ERK5, also known as Big MAP kinase (BMK) 1 (20,22). Distinct MAPK pathways are activated in response to a particular stimulus: ERK1/2 are commonly activated by growth or differentiation factors, ERK5 by


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References 1. Hunter T (2000) Signaling-2000 and beyond. Cell 100, 113-127. 2. Barford D (1995) Protein phosphatases. Curr Opin Struct Biol 5, 728-734. 3. Li X, Wilmanns M, Thornton J & Köhn M (2013) Elucidating human phosphatase-substrate networks. Sci Signal 6, rs10. 4. Julien SG, Dubé N, Hardy S & Tremblay ML (2011) Inside the human cancer tyrosine phosphatome. Nat Rev Cancer 11, 35-49. 5. Rios P, Nunes-Xavier CE, Tabernero L, Köhn M & Pulido R (2014) Dual-specificity phosphatases as molecular targets for inhibition in human diseases. Antioxid Redox Signal 20, 2251-2273. 6. De Munter S, Köhn M & Bollen M (2013) Challenges and opportunities in the development of protein phosphatase-directed therapeutics. ACS Chem Biol. 8, 36-45. 7. Tonks NK (2006) Protein tyrosine phosphatases: from genes, to function, to disease. Nat. Rev. Mol Cell Biol 7, 833-846. 8. Tonks NK (2013) Protein tyrosine phosphatases-from housekeeping enzymes to master regulators of signal transduction. FEBS J 280, 346-378. 9. Alonso A, Sasin J, Bottini N, Friedberg I, Friedberg I, Osterman A, Godzik A, Hunter T, Dixon J & Mustelin T (2004) Protein tyrosine phosphatases in the human genome. Cell 117, 699-711. 10. Barr AJ, Ugochukwu E, Lee WH, King ONF, Flippakopoulos P, Alfano I, Savitsky P, BurgessBrown NA, Müller S & Knapp S (2009) Large-scale structural analysis of the classical human protein tyrosine phosphatome. Cell 136, 352-363. 11. Tiganis T & Bennett AM (2007) Protein tyrosine phosphatase function: the substrate perspective. Biochem J 402, 1-15. 12. Duan G, Li X & Köhn M (2015) The human DEPhOsphorylation database DEPOD: a 2015 update. Nucleic Acid Res 43, D531-535. 13. Patterson KI, Brummer T, Brien PMO & Daly RJ (2009) Dual-specificity phosphatases: critical regulators with diverse cellular targets. Biochem J 489, 475-489. 14. www.depod.org 15. Maehama T, Taylor GS & Dixon JE (2001) PTEN and myotubularin: Novel phosphoinositide phosphatases. Annu Rev Biochem 70, 247−279. 16. Deshpande T, Takagi T, Hao L, Buratowski S & Charbonneau H (1999) Human PIR1 of the protein-tyrosine phosphatase superfamily has RNA 5′-triphosphatase and diphosphatase activities. J Biol Chem 274, 16590−16594. 17. Tagliabracci VS, Turnbull J, Wang W, Girard JM, Zhao X, Skurat AV, Delgado-Escueta A V, Minassian BA, Depaoli-Roach AA & Roach PJ (2007) Laforin is a glycogen phosphatase, This article is protected by copyright. All rights reserved.

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Figure 4 Reported physiological substrates of VHR (STAT5, ERK1/2, JNK1/2, p38, EGFR, ErbB2) and their involvement in different pathways (simplified, schematic). Information about the pathways was gathered from the following sources and references: 23,70,71, KEGG PATHWAY: ErbB signaling pathway, MAPK signaling pathway, JAK-STAT signaling pathway. The question marks signify that it is unclear if VHR acts on the MAPKs ERK1/2, JNK1/2 and p38 also in the cytoplasm in addition to in the nucleus. L = hormones, growth factors, cytokines, interleukins, stress stimuli, lypopolysaccharide, mitogens, GPCR activation (corresponding to the receptors); ILR = interleukin receptor; TNFR = tumor necrosis factor receptor; GPCR = G-protein coupled receptor; CR = cytokine receptor; JAK = janus kinase; Raf = rapidly accelerated fibrosarcoma.


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Figure 5 Examples of small-molecule inhibitors of VHR. Details are discussed in the main text.


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VHR was most often found in the nucleus, while in telophase it was concentrated between the daughter chromatids. When levels of VHR were reduced by small interfering (si) RNA, the cells arrested in G1/S and G2/M phases. These cells had increased levels of p21Cip/Waf1 (a cyclin dependent kinase (CDK) inhibitor that is involved in blocking cell cycle progression) whereas genes implicated in cell cycle regulation, DNA replication, repair or transcription were downregulated. Reduced levels of VHR in G1 phase were linked to the growthpromoting role of MAPKs. It was previously recognized that in the presence of growth arresting stimuli, such as DNA damaging agents or phorbol esters, prolonged activation of ERK1/2 led to cell cycle arrest, whereas activation of JNK was reported to activate p53, a regulator of growth arrest or apoptosis, and, subsequently, p21Cip/Waf1 (49,50). Thus, cell cycle dependent levels of VHR would enable activation of MAPKs for only as long as necessary to regulate normal cell cycle progression (49). A recent study described VHR as being highly expressed in endothelial cells and provided evidence that endogenous VHR is required for in vitro tubulogenesis. It was observed to also contribute to growth factor-induced angiogenic sprouting, but has no effect on the proliferation of endothelial cells (51). By creating a VHR knock-out mouse, the authors showed that VHR deficiency affects angiogenesis in vivo and ex vivo, together suggesting that in vivo, a physiological function of VHR is the mediation of neovascularization by affecting the b-FGF-induced endothelial cell sprouting via protein kinase C (PKC). The VHRdeficient mice were viable and without any obvious phenotypic differences (51). The health of the VHR knock-out mice seems to contradict the earlier finding in HeLa cells, where VHR deficiency by siRNA led to cell cycle arrest and senescence (49). However, different effects through acute and chronic loss of the protein and compensatory effects through, for example, functional redundance with other DUSPs could explain the different findings. A subsequent study involving these VHR knock out mice implicated VHR in platelet signalling. In human and mouse platelets VHR was found highly expressed, and the authors showed that VHR plays a selective and essential role in collagen receptor glycoprotein VI (GPVI) and C-type lectin type receptor 2 (CLEC-2) mediated platelet activation and aggregation in vivo (52) (for a description of the involved pathways see below).


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Structural characteristics of VHR The crystal structure of VHR was determined at 2.1 Å resolution (37, PDB entry: 1VHR) (Figure 2A). The overall protein fold of VHR was found to be similar to the fold of the classical PTPs (37). Compared to the classical PTPs, the active site pocket of VHR is shallower (no more than 6Å in depth). The depth of the active site pocket is the result of differences in the architecture of the N-terminal α1-β1 loop. The position of the conserved general acid/base residue Asp92 differed from its location with respect to the active site in PTP1B or Yersinia PTP, thus providing the possibility for proton transfer to the phosphorylated substrate’s leaving group from a different angle, and rendering the active site pocket-bound moiety less diffusion-restricted. The α1-β1 loop at the N-terminus in VHR is much shorter than the corresponding segment in classical PTPs (37). This region is referred to as “recognition region” (residues Gly19-Pro29) because it is implicated in the formation of the substrate-binding groove, together with the α1 helix. In addition, the architecture of the α1β1 loop solvent-exposes a positively charged patch containing the Arg158 of a secondary phosphate-binding site, where pThr moiety of a substrate is loosely tethered (38). Another region of the most prominent differences resides between chains β3-β7 and is termed “variable insert” (residues Asn61-Ile83, 37). The amino acid composition of this region is diverse among homologs (see also Figure 1). The residues localized at the edges of the β3β7 region contribute to the integrity of the active site. Met69 of this region in VHR forms a hydrogen bridge with the conserved Arg130, thus aiding to the proper orientation of the active site pocket. Phe68 and His70 in VHR form hydrogen bonds with the non-conserved Arg125 of the P-loop (Figure 1 and 2B). The latter is a truly unique feature of VHR, because Arg125 is replaced by small unpolar amino acids in the other closest related DUSPs (Figure 1B), and Phe68 is not present in them and is generally not conserved among DSPs (Figure 1). Phe68 was shown to be involved in VHR dimerization by covalent chemical trapping of the interaction in solution (53).

The preference of VHR to dephosphorylate pTyr was explained by the crystal structure of its catalytically inactive mutant C124S in complex with a peptide derived from the activation loop of p38 MAPK (PDB entry: 1J4X, 38). Substrate preference is seemingly dictated by the


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narrow entrance to the active site cleft determined by the side chains of Glu126 and Tyr128. Most of the dual-specific MKPs have smaller hydrophobic residues at these positions, Ala and Ile, respectively (8). When point mutations to small amino acids were simultaneously generated in these positions in VHR and the activity of the double mutant was assessed against a peptide derived from the activation loop of JNK, but with both phosphorylated residues in the peptide being pThr, the double mutant was nine-fold more active than the native enzyme (38). Interestingly, in the crystal structure of DUSP27 (54) the pTyr preference was also accounted for by the bulky Met149 and Arg151 residing in the analogous positions. The role of the conserved active site His123 was investigated in an extensive site-directed mutagenesis study coupled to kinetic profiling. In VHR, His123 is amongst only ten residues conserved between VHR and the classical PTPs (37,55). His123 helps to position the conserved Cys124 by forming an array of hydrogen bonds formed with another two residues, Tyr78 and Thr73 (55). This role of the conserved His as a positioning lever for the active site cysteine was also found for other PTPs, such as Yersinia PTP or PTP1B (55-57). Thus, together with Met69 and Phe68, Tyr78 and Thr73 connect the variable insert region via several hydrogen bond interactions to His123, Arg125 and Arg130 in the catalytic active site of VHR. This could suggest a functional rather than a merely structural relationship between the catalytic activity of the active site and the function of the variable insert region. In Figure 3 we aligned the structure of VHR with those of DUSP13B, DUSP26 and DUSP27 showing that the overall fold of these A-DUSPs is highly similar. The catalytic core regions superimpose well. The more pronounced differences between VHR and each of the selected homologues phosphatases are discussed in the following.

The regions of the most prominent structural differences between VHR and DUSP27 are the N-terminal segment and the loop β3-α4 (54), which correspond to the “variable insert” segment in VHR (37). In VHR, the N-terminal helix is a part of the substrate-binding groove, while in DUSP27 it projects away from the active site. The crystal structure of DUSP27 showed that the protein crystallized as a dimer through swapping of the N-terminal helices, similar to VH1 (54,59). The dimerization mechanism likely involves the extended conformation of the N-terminal helix. Intriguingly, the surface charge distribution in DUSP27


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is reversed with respect to VHR (54,60). The aforementioned differences between VHR and DUSP27 could result in a different substrate portfolio (54), although similarly to VHR DUSP27 was reported to dephosphorylate ERK1/2 and p38MAPK (61,62). In DUSP26, the Cα-atoms of the β3-α3 region were found deviated by 10 Å compared to the equivalent positions in VHR or DUSP27 (63). The C-terminal helix α7 in DUSP26 projects away from the catalytic pocket. The crystal structure also revealed that the active site architecture in DUSP26 is somewhat distorted and the substrate-binding pocket is smaller than in VHR (63). The differences in the active site agree with the fact that DUSP27 and VHR show preferred activity toward pTyr, whereas DUSP26 was reported to dephosphorylate pSer (64). Furthermore, DUSP26 was inactive against pNPP, but showed basal activity against DIFMUP (63), which is bulkier than the pTyr-analog pNPP. Like DUSP27, DUSP26 was also found to dimerize in the crystal structure, although via C-terminal domain swapping (63). The most prominent structural differences between VHR and DUSP13B were observed in the α1-β1 loop, known as substrate recognition region in VHR, and in the β3-α4 loop, which is shorter in DUSP13B with the two residues that correspond to Phe68 and Met69 in VHR missing (37,65). The shorter β3-α4 loop in DUSP13B helps to create planar protein surface (65), unlike the deep and narrow substrate-binding pocket in VHR. VHR and DUSP13B exhibit different preferences for differentially phosphorylated peptides. Unlike VHR, DUSP13B shows no preference between pTyr- or pThr- residues. This feature can be explained by differences in the conformation of the α1-β1 loop, which in DUSP13B is wider and more planar than in VHR, thus enabling accommodation of pTyr- and pThr residues alike (65). Also, at the position of Tyr128 in VHR, DUSP13B contains Val142, which contributes to a wider entrance to the active site pocket by forming hydrophobic contacts with Leu43 of the α1-β1 loop. Similar to the interaction of Arg130 and Met69 in VHR, the P-loop in DUSP13B is connected by a hydrogen bond between the conserved Arg144 and Gln84 corresponding to His70 in VHR (65). DUSP13B also has a secondary phosphate-binding site, in a position similar to the functionally equivalent region in VHR (38). Substrates of DUSP13B are unknown. DUSP13B was found to dimerize in the crystal structure, but not in solution. In the DUSP13B crystal structure, the dimers have an interface of 841 Å2, which is less than 10% of the total surface area of a monomeric unit, making an inability to preserve selfassociation in solution plausible (65). Nevertheless, as with VHR, covalent chemical trapping This article is protected by copyright. All rights reserved.

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of the dimer in solution might prove transient dimerization of DUSP13B in solution. Thus, all four phosphatases have the propensity to dimerize, although through different modes of interaction.

Physiological substrates, interacting proteins and pathway involvements of VHR Only few physiological substrates of VHR have been reported and these include the MAPKs ERK1/2 (24,66), JNK (66,67) and p38 MAPK (38,62). Nevertheless, the physiological significance of the MAPK activity of VHR still remains controversial and will therefore be discussed in detail below. The substrates outside of the MAPK family include signal transducers and activators of transcription (STAT) 5 (68), EGFR and erythroblastic leukemia viral oncogene homolog 2 (ErbB2) (69). Figure 4 shows the reported substrates of VHR and their pathway involvements. In addition, VHR’s involvement and interacting proteins in DNA damage response (DDR) (72) and in PKC signalling (51,52) were reported recently.

Genes encoding MKPs are induced by different stimuli, such as mitogens, growth factors and different stress conditions, resulting in the expression of MKPs in a cell-specific way (20,24). Observations that the phosphorylation level of ERK1/2 was reduced sooner than the expression of inducible MKPs was detected indicated the involvement of a constitutively expressed and nuclear-localised PTP in ERK1/2 regulation (24). By trapping the interaction using a catalytically inactive mutant of VHR, this work identified VHR as a phosphatase dephosphorylating ERK1/2. Moreover, recombinant VHR demonstrated catalytic activity toward ERK1/2 in cell extracts of COS-1 cells in a concentration-dependent manner, and also with purified recombinant ERK2, where the activity depended on the structural integrity of ERK2. In addition, it was shown that VHR preferentially dephosphorylated pTyr185 in ERK1/2. The pTyr185 specificity was confirmed by another biochemical study (30), which also showed that the in vitro activity of VHR towards ERK1/2 is lower compared to that of MKP-3. Based on this biochemical data the authors concluded that it is unlikely that ERK1/2 is a physiological substrate of VHR (30). Nevertheless, VHR’s activity on ERK1/2 was demonstrated at the level of the endogenous protein in the other study (24). Upon


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immunodepletion of endogenous VHR in COS-1 cell extracts, elevated levels of pERK were detected over time. In addition, COS-1 cells in which VHR was either overexpressed or VHR knocked down showed a direct correlation between the protein level of VHR and the downregulation of ERK (24). Todd et al. observed only slight activity of VHR against p38 or JNK (24). However, the possible explanation for low activity of VHR against JNK was explained in a later study (67). Recombinant JNK2 was used as a substrate for recombinant VHR, yielding a second order rate constant that was comparable to the value obtained previously for ERK1/2 (24). Moreover, mouse fibroblast NIH3T3 cells, with almost no detectable amounts of endogenous VHR present, were used to stably transfect either the wild type human VHR or its catalytic mutant C124S (67). Monitoring levels of pJNK in response to various cellular stress conditions demonstrated an at least five-fold reduced JNK activation in cells expressing VHR, but not its catalytic mutant. The study was extrapolated to COS-1 cells, showing that downregulation of JNK activation positively correlates with the VHR protein level. JNK was also co-precipitated with a catalytically inactive VHR mutant, C124S, from stably transfected NIH3T3 cells, thus demonstrating direct association in cells. Interestingly, only a weak band of ERK1/2 was detected. In addition, this work of Todd et al. also helped to explain discrepancies with their previous study (24), addressing activity of VHR towards JNK. They showed that the ability of VHR to dephosphorylate JNK depends on JNK not being associated with the transcription factor c-Jun (67). In the presence of c-Jun, the second order rate constant was reduced about 6-fold, whereas the ability of VHR to dephosphorylate ERK was not affected, thus showing that c-Jun negatively affects the catalytic activity of VHR against JNK by binding to JNK. The authors proposed that VHR could act as a general MKP, and that its activity towards different MAPK substrates could be regulated by the accessibility of target phosphorylated sites, which could be sterically protected from VHR by complexes formed between a MAPK and an associating protein, as demonstrated was the case with c-Jun and JNK (67). VHR was observed to dephosphorylate the pTyr residue of the peptide corresponding to the p38-MAPK activation loop with a second order rate constant comparable to the value obtained for the bisphosphorylated peptides corresponding to the activation loops of ERK and JNK (38,45). Moreover, VHR also showed catalytic activity against purified recombinant This article is protected by copyright. All rights reserved.

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p38 in vitro (38). Further data demonstrating the MAPK activity of VHR included the observation of enzymatic activity of overexpressed VHR towards overexpressed ERK1/2, JNK and p38 in EGF- and sorbitol-stimulated COS-7 cells (62). Also, in NSCLC H1792 cells overexpression of VHR decreased the phosphorylation levels of ERK1/2 and JNK1/2, while p38 was not affected (73). Indirect downregulation of VHR via stable over-expression of the histone lysine demethylase KDM2A in NSCLC H460 cells upregulated phospho-ERK1/2 but not phospho-JNK1/2 levels (73). The opposite was observed in another study where, when stably expressing VHR in NSCLC H1299 cells, almost no effect on the phospho-levels of ERK1/2, JNK and p38 were detected following various cell stimulations (69). In human umbilical vein endothelial cells (HUVEC), VHR also did not appear to target MAPKs (51). In the recently published VHR knock out mouse, the authors did not see any effect of VHR deficiency on the activity of ERK1/2 and Jnk1/2 in B cells, T cells, macrophages and platelets (51). The authors noted that discrepancies between studies in cell culture and in knock out mice regarding DUSP substrates have been observed before, reflecting potentially a difference between acute or chronic loss of a protein. In the chronic loss, in this case the knock out model, the absence of a particular DUSP could also be compensated by other DUSPs. Until now, the knock out mouse has not revealed a clear VHR substrate, which may be due to compensation by another DUSP or other cellular mechanisms, particularly when processes as central and important as MAPK signaling are involved. Concerns about the physiological relevance of VHR’s activity against MAPKs thus remain. Unlike typical MKPs, VHR is not upregulated in response to diverse MAPK-activating stimuli and it does not contain a docking MKB-domain. It is therefore unclear how it targets its MAPK substrates, especially in the context of other MKPs present in cells. In vivo there must be mechanisms governing VHR’s activity towards MAPKs that overcome the lower catalytic efficiency in vitro and the missing MKB-domain. This could be achieved, for example, through scaffolding or associated proteins, which could bring together VHR and its MAPK substrate (35). It appears at the moment that VHR can function as an MKP under certain conditions or in certain cell types, and that it does not require an N-terminal kinase interaction motif to bind and dephosphorylate its MAPK substrates. STAT5 was identified as a VHR substrate outside of the MAPK family (68). STATs are a family of transcription factors that undergo phosphorylation-induced activation (71,74,75). This article is protected by copyright. All rights reserved.

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Activated STATs form homo- or heterodimers. Dimerization is achieved by intermolecular association involving the Src homology (SH) 2 domain of one STAT monomer and a pTyr residue in the C-terminal region of the second STAT monomer. Activated STATs translocate to the nucleus to activate transcription of the target genes regulating diverse cellular processes, such as immune response, apoptosis or growth suppression (76). The mechanism of STAT5 dephosphorylation by VHR was found to proceed in two steps (68). First, it requires phosphorylation of Tyr138 in VHR, which acts as a binding site for the SH2 domain in STAT5. The second step involves dephosphorylation of Tyr694 in the displaced C-terminal end of STAT5. This mechanism was also confirmed recently by Jardin and Sticht who applied molecular modeling and dynamics approaches to profile structural requirements for STATs dephosphorylation by VH1 and VHR (76). The overall conclusion was that the specificity for dephosphorylating a particular form of STATs is derived from the nature of the residues located at the interface of the STAT-dimers. After the first report that VHR dephosphorylates different Tyr kinase receptors in an in vitro assay (36), it was shown that VHR can directly dephosphorylate EGFR in cells (69). VHR preferentially dephosphorylated Tyr992 of EGFR (Tyr1016 in the human canonical sequence; 41), and in an in vitro assay it also demonstrated activity against the tyrosines at positions 845 and 1068 (Tyr869 and 1092 in the human canonical sequence; 41). VHR-mediated dephosphorylation of Tyr992 had a suppressive effect on the activation of the PLCγ/PKC signaling pathway, whilst not affecting the activation of Src. The same study also identified ErbB2 as a novel substrate of VHR, in vitro and in cells, by using stably transfected non-small cell lung cancer (NSCLC) H1299 cells (69). Moreover, in the same study, the expression of VHR suppressed tumor formation in a mouse xenograft model, but the authors concluded that the effect was not likely due to VHR-mediated suppression of ErbB2, due to the low expression level of ErbB2 in NSCLC tissues. As with the MAPK substrates of VHR, inconsistencies are also noted here. In HUVEC and in NSCLC H1792 cells, EGFR was not observed as a substrate of VHR (51,73), arguing for cell-type specific actions of VHR. Because only few physiological substrates of VHR have been reported to date, the first extensive systematic profiling of the substrate specificity-determining factors was conducted recently by screening combinatorial peptide libraries (48). VHR was found to recognize two distinct classes of peptide sequences. The Class I resembles pTyr-motifs This article is protected by copyright. All rights reserved.

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derived from the known substrates of VHR including the MAPKs, whereas the substrates of the Class II contain a (Val/Ala)Pro(Ile/Leu/Met/Val/Phe) motif, preferentially at the Ntermini of the peptides, and would bind the phosphatase in the orientation opposite to the canonical binding mode. The report concludes with speculation that, although no intact proteins containing the N-terminal peptide corresponding to the Class II- motif were found in the PhosphoSite database search, one cannot exclude the possibility that such motif could be generated following the proteolytic cleavage of the proteins which do contain the aforementioned consensus sequence internally, thus leaving room for tracking down novel VHR substrates in vivo (48). A proteomics study by Panico and Forti (72) aimed to investigate the roles VHR might have in human cervical cancer cells. The study focused on the possible involvement of VHR in mediating DDR following stress triggered by exposure to UV or gamma radiation. The strategy involved employing GST-fused VHR as a bait in HeLa cell lysate. Out of 46 proteins which were pulled-down and identified by LC-MS/MS, six were validated biochemically. The best-validated protein hits from this study included nucleolin (NUCL), nucleophosmin (NPM) and heterogeneous nuclear ribonucleoprotein (HnRNP) C1/C2, all of which had been linked to DDR before (77-79). The results of the proteomics study were then used to construct VHR’s interacting networks in DDR. Lastly, the authors drew an interesting remark linking the newly identified VHR interactome to the data previously established for VH1 phosphatase. VH1 interferes with the antiviral machinery of the host by dephosphorylating Tyr701 of STAT1 in the interferon (IFN)-γ/STAT1 pathway (59,80,81). Viral proteins such as VH1 are known to localize in the nucleolus of their host. They help to provide more favorable conditions for viral replication and infectivity by interfering with host proteins, some of which are involved in cell cycle regulation and are also localized in the nucleolus. Based on the findings from the proteomics study in HeLa cells, VHR could also be involved in nucleolus-linked cellular processes. Recently, another study by the same group attempted to predict and identify nuclear targets of VHR by combining bioinformatics tools and their experimental validation (82). The focus of the investigation was to expand the portfolio of VHR substrates involved in DNA stability following genotoxic stress. It was established that VHR protein levels do not vary under genotoxic stress in HeLa cells. Unexpectedly, VHR was found to co-localize with a This article is protected by copyright. All rights reserved.

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marker for DNA double-stranded breaks, histone H2Ax (Ser139), and was also pulled down with phospho-H2Ax-containing complex from Hela cells. Furthermore, 121 nuclear proteins containing the Thr-X-Tyr motif, found in the MAPK substrates of VHR (24) were identified as potential nuclear substrates of VHR by bioinformatics analysis (82). In addition, confocal immunofluorescence experiments on HeLa cells after gamma irradiation showed colocalization of VHR with other proteins involved in DDR, further strengthening the involvement of VHR in the activities of the DNA damage repair machinery. Another interacting protein of VHR is VHR itself. Through site-selective incorporation of the photo-crosslinkable amino acid para-benzoylphenylalanine (pBPA) by unnatural amino acid mutagenesis, it was demonstrated that VHR can dimerize in vitro and in cells, and that the Phe68 residue of the “variable insert” segment is involved in dimerization (53). Two recent studies implicate VHR in PKC pathways (51,52), with the involved substrate(s) remaining elusive. In HUVEC cells, VHR is involved in FGF-induced angiogenesis through the PLCγ/PKC activation pathway in a MAKPs-independent manner, as VHR deficiency led to hyper-phosphorylation of PKC under basal and FGF-stimulated conditions (51). In platelets from knock-out mice, the authors found that VHR is involved in GPVI and CLEC-2 signaling (52). VHR deficiency impaired tyrosine phosphorylation of the PTK Syk including the activatory residue Tyr-525/526, subsequently reducing the phosphorylation of PLCγ2 and calcium fluxes (52). Syk is activated by its recruitment to the GPVI/Fc receptor chain (FcRγ) complex upon phosphorylation of FcRγ-associated immunoreceptor tyrosine-based activation motifs (ITAMs) by Src family kinases (SRKs), and subsequent autophosphorylation (83). Syk recruitment in VHR-deficient platelets is impaired due to a reduced phosphorylation level of the ITAMs, but SRKs were not aberrantly activated (52).

Regulation of VHR It is well acknowledged that PTPs in general are susceptible to inactivation through oxidation of the catalytic cysteine (7,8,84-86). PTPs are targets for reactive oxygen species (ROSs) such as hydrogen peroxide (H2O2) because catalytic cysteine exists in the form of a thiolate ion under physiological conditions, which enables it to function as a nucleophile,


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but it also renders it sensitive to oxidation (85,87,88). PTPs have different predispositions towards oxidative inactivation of their catalytic domains (84,89,90). For oxidation to represent a general regulatory mechanism of PTPs, the oxidation of the catalytic cysteine must be reversible, which means that it must not proceed further than sulfenic acid (SOH-) (7,88,91,92). Oxidations to sulfinic (SO2H-) and sulfonic acid (SO3H-) are considered irreversible, but there are several mechanisms for PTPs to prevent this excessive oxidation (7,90,93-95). For VHR it was shown that already low concentrations of H2O2 (45 µM) were sufficient to completely inactivate the enzyme, in a reversible manner, which was consistent with the idea of the redox-based regulation of the PTPs (96). Intriguingly, VHR was resistant to harsh oxidizing conditions, such as excess H2O2 and dissolved atmospheric oxygen at 0.27 mM. These results indicated that in VHR a mechanism must exist that would protect the catalytic cysteine from irreversible oxidative damage (96). A possible explanation for this resistance was discovered recently (53). Evidence suggested that the aforementioned dimer formation of VHR could block the active site and render it less exposed to the solvent, thus potentially protecting is from over-oxidation. Moreover, this mechanism could explain the finding that dimer formation reduced the catalytic activity of VHR. In addition, the extensive hydrogen bonding between the active site and the residues involved in the dimerization of VHR in the variable insert region (Figure 2B) could also hint toward an influence of VHR dimerization on its catalytic activity. Thus, VHR dimerization could be a mechanism for the negative regulation of its enzymatic activity, likely in response to oxidative stress as shown in the report (53). Self-association of VHR to form dimers would result in a high local concentration of the enzymatically active form, which could act rapidly to elicit a specific cellular response after the complex is disassembled into monomers. Nevertheless, the detailed mechanism by which oxidative stress would be the causal or stabilizing effect in the dimeric association of VHR in vivo is yet to be investigated. VHR expression is not triggered in response to various mitogenic stimuli (24). Related to this, it was reported that inactivation of ERK1/2 in EGF-stimulated cells did not cease after treatment with cycloheximide, which inhibits protein synthesis. This was noted to be in accordance with the earlier reports pointing towards a constitutively expressed phosphatase in the ERK1/2 pathway. Accordingly, downregulation of ERK1/2 occurred This article is protected by copyright. All rights reserved.

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already 15 min after stimulation and independently of the de novo protein synthesis, whilst the transcript for the inducible MKPs was present no sooner than 30 min after the stimulation. The authors proposed a model according to which VHR could be kept inactive via the transiently oxidized nucleophilic Cys124, due to the production of H2O2 as a part of “normal” receptor-mediated signaling events (24). It is interesting to note that the activity of VHR against ERK1/2 was shown to be decreased in cultured human intestinal epithelial cells and in murine epithelia in vivo as a result of microbiota-mediated generation of ROSs (97). The benefits of commensal bacteria on homeostasis of the intestine have been acknowledged. The aforementioned study helped to elucidate a mechanism behind commensal bacteria-modulated manipulation of signaling in intestinal epithelial cells. According to this, mRNA and protein levels of VHR were upregulated upon stimulation with bacterial N-formyl-peptides. In addition, activation of the formyl peptide receptor (FPR), which recognizes bacterial N-formyl-peptides, results in the transient generation of ROS. This enables a feedback loop between the ERK1/2 mediated proliferation stimulative effect and transient downregulation of the catalytic activity of the ERK1/2-specific phosphatase VHR (97). Furthermore, it was demonstrated that the activity of VHR against ERK1/2 could be enhanced by Vaccinia-related kinase (VRK) 3 (98). In immortalized mouse hippocampal (HT22) cells, VRK3 imposed a negative effect on the activation of ERK1/2 by interacting with VHR, but independently of its kinase activity. The catalytic activity of VHR against pNPP and purified recombinant pERK2 was increased when it was overexpressed with VRK3. VRK3 was shown to be a negative regulator of ERK1/2 signaling, while JNK and p38 MAPKs were not affected (98). VHR is tyrosine-phosphorylated by the protein tyrosine kinase (PTK) ZAP-70 in activated Tcells (99). T-cell activation is characterized by an increase in the cellular content of tyrosinephosphorylated proteins (100). ZAP-70 is amongst the protein kinases catalyzing these reactions that eventually lead to the activation of many signaling pathways including the MAPK pathway (101). VHR suppresses T-cell antigen receptor (TCR)-induced activation of the MAPKs ERK and JNK (66,99). VHR was found to re-localize upon T-cell activation, from a diffused cytoplasmic distribution to the sites of the T-cell and antigen-presenting cell (APC)


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contact (99). The time course of re-localization coincided with the time after which the downregulation of ERK1/2 and JNK was observed. Stimulation of normal T-cells also showed that VHR becomes tyrosine-phosphorylated. The same modification was detected when endogenous phosphatases were inhibited by vanadate treatment, leading to the conclusion that VHR is in contact with TCR-associated PTKs also in resting T-cells. ZAP-70 was profiled as the key PTK responsible for tyrosine-phosphorylation of VHR in cells and in vitro (99). Based on structural information (37), the Tyr138 residue in VHR was marked as the likely site to be modified by ZAP-70 and in vitro phosphorylation experiments with purified ZAP-70 and GST-fused different variants of VHR confirmed that Tyr138 was the major phosphorylation site (99). Small levels of pTyr38 were also detected. Moreover, the effect of VHR on the MAPKs ERK and JNK was suppressed in case of the Tyr138Phe mutant. The authors tried to address the question if phosphorylation of Tyr138 in VHR directly activates its phosphatase activity, but they were not able to reach any final conclusions due to complex experimental conditions needed to address this question: when higher concentrations of VHR were used in an in vitro kinase assay with ZAP-70, the kinase was dephosphorylated and inactivated by VHR itself. Considering the localization of Tyr138 on the face opposite to the active site, the authors concluded that phosphorylation of this residue is more likely involved in regulating protein-protein interactions of VHR, subcellular localization or targeting to the substrate than in the activation of its phosphatase activity. Phosphorylation of Tyr138 in VHR was also shown to be important for the dephosphorylation of STAT5 (68), with the Tyr138Phe mutant failing to exert the same effect, which was consistent with the effect observed for this dominant negative mutant in the context of MAPK-suppression in activated T-cells discussed above (99). Phosphorylation of Tyr138 by ZAP-70 and tyrosine kinase (Tyk)-2 did not affect the catalytic activity of VHR in vitro, suggesting it has likely a regulatory role as a docking site for the SH2 domain of STAT5 (68). Tyr138 phosphorylation was detected only in the catalytically inactive form of VHR, most likely indicating trans- and not auto-dephosphorylation of wild type (wt) VHR, due to the already indicated localization of Tyr138 opposite to the active site pocket. Recently, the regulation of VHR through phosphorylation of Tyr138 was also addressed by using unnatural amino acid mutagenesis (102). Sulfotyrosine was incorporated at the positions of Tyr128 and Tyr138 in VHR by amber-codon suppression, thus creating mutant This article is protected by copyright. All rights reserved.

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forms of proteins mimicking the differentially phosphorylated states. Those sites were regarded as most likely to be modified by phosphorylation (37,102). Incorporation of the phosphate-mimic at the position of Tyr138 yielded a protein with slightly increased enzymatic activity compared to the native protein, whereas the mutant with sulfotyrosine introduced at position 128 showed low activity, leading the authors to propose that Tyr138 is indeed the key residue in VHR where modification through phosphorylation is likely to occur. A cancer-related study identified VHR as target gene of the histone lysine demethylase KDM2A (73). In H1792 and H1975 NSCLC cells, the authors showed that VHR expression was notably up-regulated by KDM2A knockdown and that as a transcriptional corepressor, KDM2A directly represses the DUSP3 gene by specific demethylation of H3K36me2 at the proximal promoter and the 5′ end of the DUSP3 gene. Aside from this pathological context, it is unclear if this repression is also a physiological mechanism of VHR regulation (73).

VHR in disease and VHR inhibitors Studies on the involvement of DUSPs, including VHR, in cancer have been reviewed recently (5,103,104), therefore this subject will be only briefly addressed here. Aside from cancer, VHR was very recently reported to play a role in thrombosis (52). VHR has been linked to breast cancer (105). It was shown that expression of VHR was decreased upon overexpression of breast cancer 1 (BRCA1)-IRIS (operationally termed ‘inframe reading of BRCA1 intron 11 splice variant’) (106). BRCA1-IRIS was found overexpressed in different breast and ovarian cancer cell lines and was observed to promote cell proliferation during S-phase of the cell cycle (106). Its expression positively correlated with the expression of cyclin D1, which is implicated in cell growth regulation (105). Cyclin D1 expression is induced by binding of a transcription factor called activating protein (AP) 1, composed of c-Jun and Fos proteins, to the promoter region of cyclin D1. CJun is activated by JNK, which is reportedly downregulated by VHR (67). In addition, it was observed that overexpression of VHR in human mammary epithelial (HME) cells reduced expression of cyclin D1, thus opening up possibilities for VHR activators in the development


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of therapeutics targeting BRCA1-IRIS-mediated effects on cell proliferation (105). In agreement with the hypothesis of VHR as a tumor suppressor, another study demonstrated that mRNA levels of VHR were reduced in cancer tissues derived from NSCLC patients, and that nude mice injected with H1299 cells overexpressing VHR developed smaller tumours, thus linking VHR expression to suppression of cancer cell proliferation in NSCLC cells (69). In contrast to the above findings, VHR was shown to be overexpressed in prostate cancer as opposed to the normal prostate (107). This effect was linked to its ability to downregulate JNK; thus preventing JNK-mediated apoptosis. VHR was also found overexpressed in several cervix cancer cell lines (108). In those cell lines, VHR was found in the nucleus and in the cytoplasm, whereas in normal cervix cells, it was localised in the cytoplasm. Increased levels of VHR were the result of increased protein stability because mRNA levels of VHR in normal and cancer cervix cells were comparable. In the light of the previously reported demonstration that VHR loss by siRNA caused cell senescence (49), it was postulated that increased protein level of VHR in cervix cancer cells would enable the cells to proliferate (108). VHR’s function as pro-angiogenic A-DUSP could also imply a role in angiogenesis in cancer (51). Tumors resulting from the injection of Lung Lewis Carcinoma (LLC) cells subcutaneously into VHR-deficient mice had reduced haemoglobin content compared to the wild type mice, whereas the tumor size in these mice was not significantly reduced. These results showed that the tumor-induced angiogenic response was defective in the mutant mice. The healthy phenotype of the VHR-deficient mice created and used for the study of VHR in angiogenesis (51) makes VHR a promising drug target based on fewer expected side effects upon VHR inhibition. The knock-out mice are also a good model to investigate the role of VHR in different diseases, which is evident through the most recent study that showed that VHR deficiency through knock-out or chemical inhibition limits platelet activation and arterial thrombosis (52). Compared to wild type mice, VHR-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism. Upon ferric chlorideinduced carotid artery injury, they exhibited severely impaired thrombus formation, but bleeding times were not changed in these mice, suggesting together that VHR could be a promising drug target for the treatment or prevention of arterial thrombosis (52).


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The development of small-molecule inhibitors of VHR is appealing for therapeutic development and for their use as tools to manipulate and study physiological roles of VHR (109). Nevertheless, only few inhibitors were reported to date (52,110-120), and we discuss here some of the most potent VHR inhibitors as examples. The compound RK-682 (Figure 5) was discovered as an inhibitor of PTPs (113), inhibiting VHR’s phosphatase activity in vitro with an IC50 value of 2.0 µM. Although initially described as a non-competitive inhibitor, a later study from the same group showed that RK-682 is a competitive inhibitor (110). RK-682 served as a template for further derivatization and structure-affinity relationship (SAR) studies (110). Kinetic data obtained for RK-682 derivatives indicated that two molecules of RK-682 acted to inhibit a single VHR molecule. Therefore, a model of VHR in complex with a dimeric RK-682 was first constructed, followed by the synthesis of a dimeric inhibitor molecule, which indeed inhibited VHR with an IC50 value in the low µM range. Moreover, the dimeric derivative showed no inhibitory effect on PTP-S2 up to a concentration of 100 µM. A binding model for (RK-682)2 was designed summarizing kinetic and SAR data, proposing that Arg158 is an important residue for inhibition due to its interaction with the tetronic acid anion. Although this residue is conserved in many PTPs (37), its surrounding is markedly different in VHR, thereby showing that a promising aspect in VHR selective inhibitor development is to explore its unique structural features. Shi et al. docked over 80,000 compounds into the active site of VHR to isolate the most promising candidates (111). This approach was combined with highly sensitive diffusionedited NMR spectroscopy. (Glucosamine-aminoethoxy) triphenyltin (GATPT; Figure 5) was identified as a competitive inhibitor with a low-µM IC50 value. Molecular docking further suggested a binding model of GATPT to VHR. Accordingly, the glucosamine ring of GATPT was bound at the bottom of the VHR’s active site cleft through hydrogen bonds involving a number of conserved residues of the P-loop, including catalytically important Cys124, Asp92 and Arg130. In addition to charge-based interactions and hydrogen bonds, the model also suggested the importance of hydrophobic interactions occurring between three phenyl rings of GATPT and hydrophobic residues in VHR for the efficient binding of VHR to GATPT. GATPT efficiently suppressed the deactivation of ERK1/2 and JNK by VHR in HeLa cells and also caused cell cycle arrest at the G1-S phase. The effect was not visible when VHR expression This article is protected by copyright. All rights reserved.

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was suppressed by siRNA. The screening approach was suggested as promising to increase the structural space of potential VHR inhibitors (111). Mustelin and Tautz used chemical library screening to identify VHR inhibitors (112). Starting from 50,000 drug-like molecules, 56 compounds which inhibited >90 % of VHR’ phosphatase activity were found, yet only two were selective against other PTPs. Docking of the most potent compound referred to as SA3 showed that its sulfonic acid group (Figure 5) acts as a phosphate-mimetic, binding into the P-loop of VHR and forming hydrogen bonds with the conserved residues of the VHR’s active site cleft. SA3 exhibited an IC50 value of 74 nM, with at least an order of magnitude lower potency for the additional PTPs screened. Detailed analysis of the analogues of the lead compound SA3 suggested that three hydrophobic patches proximal to VHR’s active site could be used for structural optimization of the inhibitors, which was also demonstrated by the crystal structure of VHR in complex with SA3. Importantly, these hydrophobic areas are unique for VHR and are comprised in part of the residues of the “recognition region” and “variable insert” segments in VHR (37). SA3 was efficient in decreasing proliferation rates of the cervix cancer cell lines HeLa and CaSki. The study showed that VHR could be exploited as a pharmaceutical target for treating cervix cancer (112). In the study that identified VHR’s therapeutic potential in thrombosis, the authors noted that previously described inhibitors, including their own (112), were not useful for the application in human platelets (52). Therefore, they screened 291,018 drug-like molecules using a pNPP-based high throughput format. After hit verification and inspection two molecules with different backbones were picked for further SAR studies. While one backbone series was terminated because no further hits were found, the other led to the discovery of compound MLS-0437605 (Figure 5), showing an IC50 of 3.7 µM and a good to excellent selectivity toward VHR over other PTPs. Importantly, this compound inhibited VHR in human platelets, and it blocked platelet aggregation in wild type mice but only minimally in VHR-deficient mice (52). Finally, the observed reduction of VHR catalytic activity through dimerization (53) suggests that stabilization of VHR dimerization could be a new mode of action for VHR inhibitors.


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Conclusion While VHR was originally used to characterize small DSPs, it is now clear that it plays important roles in cell cycle regulation, DNA damage response, angiogenesis, platelet activation and MAPK signaling. Its involvement in different pathways appears to be highly cell-type specific. Although its roles and mechanisms in cancer depend on the cancer type and require further investigation, it is already clear that its inhibition can reduce cancerrelated phenotypes in cancer cell lines, making it a promising target for future therapeutic efforts. The recent discovery of VHR’s role in arterial thrombosis demonstrates that this phosphatase is also involved and drug-targetable in diseases other than cancer. Future studies should aim at uncovering and validating more physiological substrates of VHR and at establishing links to a specific signaling outcome. New methodologies such as unnatural amino acid mutagenesis have already provided a deeper insight into VHR regulation, and could help, together with exploiting structurally unique features of VHR for drug design, to establish VHR as a drug target in the future. Lastly, the VHR knock-out mice should further help to elucidate the involvement and mechanisms of VHR in physiological and pathological processes, thus enabling the development of new therapeutic approaches.

Acknowledgements K.P. and M.K. would like to thank the German Science Foundation (Deutsche Forschungsgemeinschaft, DFG) for support within the Emmy-Noether program.


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Table and Table caption

Table 1 Summary of A-DUSPs in the human genome (7,9,12,14). The table summarizes their gene and protein names, their synonyms, the uniprot accession numbers and domain compositions. Protein, Synonyms# DUPD1, DUSP27 PIR1

Domain composition PTP domain PTP domain PTP domain – Cys-rich zinc-binding DUSP12 YVH1 Q9UNI6 domain (C-Zn) PTP domain DUSP13A MDSP Q6B8I1 PTP domain DUSP13B TMDP, SKRP4 Q9UII6 PTP domain DUSP14 MKP-6, MKP-L O95147 myristoylation site – PTP domain DUSP15 VHY Q9H1R2 PTP domain DUSP18 LMW-DSP20 Q8NEJ0 PTP domain DUSP19 SKRP1, TS-DSP1 Q8WTR2 PTP domain DUSP21 LMW-DSP21 Q9H596 myristoylation site – PTP domain DUSP22 JSP-1, LMW-DSP2 Q9NRW4 PTP domain DUSP23 VHZ, LDP-3 Q9BVJ7 PTP domain DUSP26 MKP-8, SKRP3 Q9BV47 PTP domain DUSP28 DUSP28 Q4G0W2 PTP domain DUSP3 VHR P51452 carbohydrate-binding domain EPM2A Laforin O95278 (CBD) – PTP domain PTP domain PTPMT1 PTPMT1 Q8WUK0 PTP domain – guanyltransferase RNGTT HCE O60942 domain (GTD) pseudo PTP domain STYX STYX Q8WUJ0 CDC25-homology domain (CH2) – STYXL1 DUSP24, MKSTYX Q9Y6J8 pseudo PTP domain # Abbreviations: DUSP, dual-specificity phosphatase; DUPD1, dual-specific protein phosphatase domain containing 1; HCE, human mRNA-capping enzyme; JSP-1, c-Jun Nterminal kinase stimulatory phosphatase; LDP-3, low molecular mass DUSP3; LMW-DUSP2, 20 and -21, low molecular weight DUSP2, -20, and -21; MDSP, muscle-restricted DUSP; MKP6, -8 and -L, mitogen activated protein kinase phosphatase member 6, -8 and –L; MKSTYX, mitogen activated protein kinase-like protein; PIR1, protein that interacts with RNA/RNP complex 1; PTPMT1, protein tyrosine phosphatase mitochondrial 1; SKRP1, -3 and -4, stressactivated protein kinase pathway-regulating phosphatase 1, -3 and -4; STYX, serine/ threonine/ tyrosine-interacting protein; TMDP, testis and skeletal muscle specific DUSP; VHR, Vaccinia H1-related; VHY and -Z, Vaccinia H1-related Y and -Z; YVH1, yeast VH1 homolog Gene DUPD1 DUSP11

UniProt AC Q68J44 O75319


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Accepted Article

Figure 1. Evolutionary conservation analysis for (VHR) DUSP3 by ConSurf (39) (A) and sequence alignment of DUSP3 (VHR), DUSP13B, DUSP26 and DUSP27 including the secondary structure of VHR (B). (A) Residues were colored according to their ConSurf scores, the residues with higher ConSurf scores are more conserved. The evolutionary rate (ConSurf


Accepted Article

score) for VHR (DUSP3) was estimated in ConSurf by considering the evolutionary relatedness between VHR (DUSP3) and its homologues, and the similarity between amino acids as reflected in the substitutions matrix (39). The ConSurf analysis was based on 150 homologous sequences for VHR from UniRef90 (40) detected by PSI-BLAST with the E-value cutoff of 0.0001. (B) DUSP sequences were retrieved from UniProt (41); accession numbers: VHR (DUSP3): P51452, DUSP13B: Q9UII6, DUSP26: Q9BV47, DUSP27: Q68J44. T-Coffee (42) was used in the sequence alignment with accurate mode and BioEdit (43) was used to visualize the alignment result. The DUSPs crystal structures were downloaded from the PDB (44); entry numbers: DUSP3: 3F81, DUSP13B: 2PQ5, DUSP26: 2E0T, DUSP27: 2Y96. Different residues were colored according to their side chain polarity, and secondary structure elements were shown with different geometric shapes under the sequence alignment (helix: arrow; strand: round square; others: line).


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Figure 2. Crystal structure of VHR (PDB: 1VHR) shown as a combination of ribbon and surface representation (A) and zoom-in on interactions between the active site and the variable insert region (B). (A) Surface transparency was set to 60%. The sulfate ion bound in the active site is shown as stick and is colored in blue. Secondary structure elements are numbered. The following structural elements are indicated: “substrate recognition” segment in light blue (α1-β1; residues Gly19-Pro29), “variable insert” segment in red (β3-β7; residues Asn61-Ile83, with the following residues pointed out Phe68, Met69, His70, Thr73 and Tyr78), WPD-loop in magenta (Ala89-Asp92, with Asp92 pointed out) and the P-loop in yellow (His123-Ser131, with the following residues pointed out His123, Cys124, Arg125 and Arg130). (B) The crystal structure of VHR (PDB: 1VHR) is shown as a surface representation and is colored in wheat. The sulfate ion bound in the active site is shown as stick and is colored in blue. The following hydrogen bonds are indicated as dotted lines: Met69-Arg130, Phe68-Arg125 and His70-Arg125. The distance between the atoms associated through hydrogen-bonding is indicated and the atoms are colored as indicated: carbons in light blue, hydrogens in wheat, nitrogens in dark blue, oxygens in red and sulfur in yellow. The structures were generated using Pymol.


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Figure 3 Ribbon structure (A,B) for VHR (DUSP3) and structural superposition for DUSPs (C,D). In (A,B) different secondary structural elements were colored separately and some residues and regions were highlighted according to their mentioning in the main text. In (C,D) four DUSP crystal structures (DUSP3/13B/26/27) were compared using iterative magic fit considering carbon alpha (CA) only in DeepView (58). Different proteins were colored differently as indicated in the legend and shown as ribbon structures. (A) and (B) represent the same VHR (DUSP3) crystal structure from different angles, similarly the structural superposition for the DUSPs is shown from different angles in (C) and (D).


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Figure 4 Reported physiological substrates of VHR (STAT5, ERK1/2, JNK1/2, p38, EGFR, ErbB2) and their involvement in different pathways (simplified, schematic). Information about the pathways was gathered from the following sources and references: 23,70,71, KEGG PATHWAY: ErbB signaling pathway, MAPK signaling pathway, JAK-STAT signaling pathway. The question marks signify that it is unclear if VHR acts on the MAPKs ERK1/2, JNK1/2 and p38 also in the cytoplasm in addition to in the nucleus. L = hormones, growth factors, cytokines, interleukins, stress stimuli, lypopolysaccharide, mitogens, GPCR activation (corresponding to the receptors); ILR = interleukin receptor; TNFR = tumor necrosis factor receptor; GPCR = G-protein coupled receptor; CR = cytokine receptor; JAK = janus kinase; Raf = rapidly accelerated fibrosarcoma.


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Figure 5 Examples of small-molecule inhibitors of VHR. Details are discussed in the main text.


VHR is a pro-angiogenic atypical dual-specificity phosphatase.

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