Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

Nayak, Jaladhi; Gastonguay, Adam; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, P.R.; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

doi:10.1186/s12858-014-0027-0

Cited by 11 publications

(41 citation statements)

References 38 publications

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“…Indeed, we identified a clinically relevant serine to proline mutation (S147P) that is associated with vascular defects [ 2 ]. This mutation has been shown previously by our group to interfere with the dephosphorylating activity of DUSP5 protein [ 8 ], and makes the protein hypoactive. However, the direct causal role of S147P in vascular anomaly progression is yet to be established.…”

Section: Introductionmentioning

confidence: 86%

“…A molecular model based on the crystal structures of the human DUSP5 PD and ERK2, and a homology model of the DUSP5 EBD, illustrates the bivalent nature of the interaction between the two domains of DUSP5 and the ERK2 substrate (Fig. 1 ) [ 8 ]. The current study has identified small molecule inhibitors that occupy the active site pocket on the DUSP5 PD, and act as inhibitors of the phosphatase enzymatic activity.…”

Section: Introductionmentioning

confidence: 99%

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Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets

et al. 2015

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BackgroundDual-specificity phosphatase-5 (DUSP5) plays a central role in vascular development and disease. We present a p-nitrophenol phosphate (pNPP) based enzymatic assay to screen for inhibitors of the phosphatase domain of DUSP5.MethodspNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds the DUSP5 phosphatase domain with a Km of 7.6 ± 0.4 mM. Docking followed by inhibitor verification using the pNPP assay identified a series of polysulfonated aromatic inhibitors that occupy the DUSP5 active site in the region that is likely occupied by the dual-phosphorylated ERK2 substrate tripeptide (pThr-Glu-pTyr). Secondary assays were performed with full length DUSP5 with ERK2 as substrate.ResultsThe most potent inhibitor has a naphthalene trisulfonate (NTS) core. A search for similar compounds in a drug database identified suramin, a dimerized form of NTS. While suramin appears to be a potent and competitive inhibitor (25 ± 5 μM), binding to the DUSP5 phosphatase domain more tightly than the monomeric ligands of which it is comprised, it also aggregates. Further ligand-based screening, based on a pharmacophore derived from the 7 Å separation of sulfonates on inhibitors and on sulfates present in the DUSP5 crystal structure, identified a disulfonated and phenolic naphthalene inhibitor (CSD3_2320) with IC50 of 33 μM that is similar to NTS and does not aggregate.ConclusionsThe new DUSP5 inhibitors we identify in this study typically have sulfonates 7 Å apart, likely positioning them where the two phosphates of the substrate peptide (pThr-Glu-pTyr) bind, with one inhibitor also positioning a phenolic hydroxyl where the water nucleophile may reside. Polysulfonated aromatic compounds do not commonly appear in drugs and have a tendency to aggregate. One FDA-approved polysulfonated drug, suramin, inhibits DUSP5 and also aggregates. Docking and modeling studies presented herein identify polysulfonated aromatic inhibitors that do not aggregate, and provide insights to guide future design of mimics of the dual-phosphate loops of the ERK substrates for DUSPs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0048-3) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets

et al. 2015

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“…Of the different Dusp genes, five have been reported to prefer p38 and JNK over ERK1/2, including DUSP1, DUSP4, DUSP8, DUSP10, and DUSP16. Three cytoplasmic DUSPs: DUSP6, DUSP7, and DUSP9 were reported to be ERK1/2 specific ( Dickinson and Keyse, 2006 ), and one inducible nuclear DUSP, the DUSP5, was shown to be nuclear ERK1/2 specific ( Nayak et al, 2014 ). However, both DUSP7 and DUSP9 may also be able to inactivate p38 ( Dickinson and Keyse, 2006 ).…”

Section: Mitogen Activated Protein Kinase (Mapk) Pathwaymentioning

confidence: 99%

Extracellular signal-regulated kinases 1/2 as regulators of cardiac hypertrophy

Mutlak

Kehat

2015

Front. Pharmacol.

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Cardiac hypertrophy results from increased mechanical load on the heart and through the actions of local and systemic neuro-humoral factors, cytokines and growth factors. These mechanical and neuroendocrine effectors act through stretch, G protein–coupled receptors and tyrosine kinases to induce the activation of a myriad of intracellular signaling pathways including the extracellular signal-regulated kinases 1/2 (ERK1/2). Since most stimuli that provoke myocardial hypertrophy also elicit an acute phosphorylation of the threonine-glutamate-tyrosine (TEY) motif within the activation loops of ERK1 and ERK2 kinases, resulting in their activation, ERKs have long been considered promotors of cardiac hypertrophy. Several mouse models were generated in order to directly understand the causal role of ERK1/2 activation in the heart. These models include direct manipulation of ERK1/2 such as overexpression, mutagenesis or knockout models, manipulations of upstream kinases such as MEK1 and manipulations of the phosphatases that dephosphorylate ERK1/2 such as DUSP6. The emerging understanding from these studies, as will be discussed here, is more complex than originally considered. While there is little doubt that ERK1/2 activation or the lack of it modulates the hypertrophic process or the type of hypertrophy that develops, it appears that not all ERK1/2 activation events are the same. While much has been learned, some questions remain regarding the exact role of ERK1/2 in the heart, the upstream events that result in ERK1/2 activation and the downstream effector in hypertrophy.

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“…We mutated E264 to leucine (L) (E264L) and glutamine (Q) (E264Q) and R214 to glutamine (Q) (R214Q) using site-directed mutagenesis. We generated GST-tagged versions of these proteins in bacteria as described in our previous work 14 and purified them on a GST column. Upon expression of these point mutants in bacteria, we noticed three bands on the SDS-PAGE gel for E264Q and R214Q compared to four distinct bands (E264L1-E264L4, from top to bottom, respectively) in the E264L preparations (in Figure 5B, compare lanes E264L and E264Q).…”

Section: Site-directed Mutational Analysis Confirms the Importance Ofmentioning

confidence: 99%

“…Generation and Characterization of the DUSP5 Point Mutant Proteins Protein expression, GST pull-down assays, and purification protocols were similar to those reported in our previous work on GST-DUSP5 protein. 14 Modifications to the protocol included transfecting final sequenced plasmids into expression competent cells such as Shuffle cells. Shuffle cells are an Escherichia coli strain that allows for the production of proteins with disulfide bonds.…”

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confidence: 99%

Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs

et al. 2016

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DUSP5 is an inducible nuclear dual-specificity phosphatase that specifically interacts with and deactivates extracellular signal-regulated kinases ERK1 and ERK2, which are responsible for cell proliferation, differentiation, and survival. The phosphatase domain (PD) of DUSP5 has unique structural features absent from other nuclear DUSPs, such as the presence of a secondary anion-binding site in the proximity of the reaction center and a glutamic acid E264 positioned next to the catalytic cysteine C263, as well as a remote intramolecular disulfide linkage. The overall 400 ns molecular dynamics simulations indicate that the secondary binding site of DUSP5 PD acts as an allosteric regulator of the phosphatase activity of DUSP5. Our studies have identified E264 as a critical constituent of the dual binding pocket, which regulates the catalytic activity of DUSP5 by forming a salt bridge with arginine R269. Molecular dynamics studies showed that initial occupation of the secondary binding pocket leads to the breakage of the salt bridge, which then allows the occupation of the active site. Indeed, biochemical analysis using the pERK assay on mutant E264Q demonstrated that mutation of glutamic acid E264 leads to an increase in the DUSP5 catalytic activity. The role of the secondary binding site in assembling the DUSP5-pERK pre-reactive complex was further demonstrated by molecular dynamics simulations that showed that the remote C197-C219 disulfide linkage controls the structure of the secondary binding pocket based on its redox state (i.e., disulfide/dithiol) and, in turn, the enzymatic activity of DUSP5.

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Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

Cited by 11 publications

References 38 publications

Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets

Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets

Extracellular signal-regulated kinases 1/2 as regulators of cardiac hypertrophy

Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs

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