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

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

doi:10.1186/s12858-014-0027-0

Abstract

BackgroundThe mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function.ResultsIn this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P.ConclusionTaken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-014-0027-0) contains supplementary material, which is available to authorized users.

Highlights

The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development
dual specificity phosphatase-5 (DUSP5) protein was observed by Coomassie staining but, the DUSP5 protein band was again observed in the insoluble cell pellet (Figure 1C)
We switched the media from lysogeny broth (LB) to terrific broth (TB), and the expression was carried out at 16°C using an overnight induction with 0.05 mM and 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration (Figure 1F)

Summary

Introduction

The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. The mitogen-activated protein kinases (MAPKs) pathways are activated by dual phosphorylation of tyrosine and threonine residues in their activation loops [1]. To regulate the MAPKs, proteins that dephosphorylate at serine/threonine and tyrosine residues have evolved. One class of phosphatases that dephosphorylate serine/threonine and tyrosine residues is dual specificity phosphatases (DUSPs) [2]. Our laboratory has previously identified one of the members of the DUSP family, DUSP5, as critical for vascular. DUSP5 is predicted to have two globular domains; an N-terminal ERK binding domain (EBD) and a Cterminal phosphatase domain (PD) [2,6].

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