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Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins. BMC Biochem 2014 Dec 18;15:27

Date

12/19/2014

Pubmed ID

25519881

Pubmed Central ID

PMC4299175

DOI

10.1186/s12858-014-0027-0

Abstract

BACKGROUND: 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. 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.

RESULTS: In 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.

CONCLUSION: Taken 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.

Author List

Nayak J, Gastonguay AJ, Talipov MR, Vakeel P, Span EA, Kalous KS, Kutty RG, Jensen DR, Pokkuluri PR, Sem DS, Rathore R, Ramchandran R

Author

Ramani Ramchandran PhD Professor in the Pediatrics department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Amino Acid Substitution
Catalytic Domain
Dual-Specificity Phosphatases
Extracellular Signal-Regulated MAP Kinases
Humans
Molecular Dynamics Simulation
Molecular Sequence Data
Protein Binding
Protein Biosynthesis
jenkins-FCD Prod-484 8aa07fc50b7f6d102f3dda2f4c7056ff84294d1d