skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

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.

Authors:
 [1];  [1];  [2];  [1];  [3];  [3];  [1];  [1];  [4];  [3];  [2];  [5]
  1. Medical College of Wisconsin, Milwaukee, WI (United States)
  2. Marquette Univ., Milwaukee, WI (United States)
  3. Concordia Univ. of Wisconsin, Mequon, WI (United States)
  4. Argonne National Lab. (ANL), Argonne, IL (United States)
  5. Medical College of Wisconsin, Milwaukee, WI (United States); CRI Developmental Vascular Biology Program, Milwaukee, WI (United States)
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1201767
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
BMC Biochemistry (Online)
Additional Journal Information:
Journal Volume: 15; Journal Issue: 1; Journal ID: ISSN 1471-2091
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; DUSP5; mutation; vascular anomalies; protein purification; molecular modeling

Citation Formats

Nayak, Jaladhi, Gastonguay, Adam J., Talipov, Marat R., Vakeel, Padmanabhan, Span, Elise A., Kalous, Kelsey S., Kutty, Raman G., Jensen, Davin R., Pokkuluri, Phani Raj, Sem, Daniel S., Rathore, Rajendra, and Ramchandran, Ramani. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins. United States: N. p., 2014. Web. doi:10.1186/s12858-014-0027-0.
Nayak, Jaladhi, Gastonguay, Adam J., Talipov, Marat R., Vakeel, Padmanabhan, Span, Elise A., Kalous, Kelsey S., Kutty, Raman G., Jensen, Davin R., Pokkuluri, Phani Raj, Sem, Daniel S., Rathore, Rajendra, & Ramchandran, Ramani. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins. United States. https://doi.org/10.1186/s12858-014-0027-0
Nayak, Jaladhi, Gastonguay, Adam J., Talipov, Marat R., Vakeel, Padmanabhan, Span, Elise A., Kalous, Kelsey S., Kutty, Raman G., Jensen, Davin R., Pokkuluri, Phani Raj, Sem, Daniel S., Rathore, Rajendra, and Ramchandran, Ramani. 2014. "Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins". United States. https://doi.org/10.1186/s12858-014-0027-0. https://www.osti.gov/servlets/purl/1201767.
@article{osti_1201767,
title = {Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins},
author = {Nayak, Jaladhi and Gastonguay, Adam J. and Talipov, Marat R. and Vakeel, Padmanabhan and Span, Elise A. and Kalous, Kelsey S. and Kutty, Raman G. and Jensen, Davin R. and Pokkuluri, Phani Raj and Sem, Daniel S. and Rathore, Rajendra and Ramchandran, Ramani},
abstractNote = {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.},
doi = {10.1186/s12858-014-0027-0},
url = {https://www.osti.gov/biblio/1201767}, journal = {BMC Biochemistry (Online)},
issn = {1471-2091},
number = 1,
volume = 15,
place = {United States},
year = {Thu Dec 18 00:00:00 EST 2014},
month = {Thu Dec 18 00:00:00 EST 2014}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 10 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers
journal, February 2014


Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network
journal, January 1993


Regulation of the inducible nuclear dual-specificity phosphatase DUSP5 by ERK MAPK
journal, December 2009


ERK1/2 MAP kinases: Structure, function, and regulation
journal, August 2012


Mechanism of Cdc25B Phosphatase with the Small Molecule Substrate p -Nitrophenyl Phosphate from QM/MM-MFEP Calculations
journal, April 2009


Vascular tumors of infancy and childhood: beyond capillary hemangioma
journal, November 2006


Crystal structures of fusion proteins with large-affinity tags
journal, July 2003


Experimental Validation of the Docking Orientation of Cdc25 with Its Cdk2−CycA Protein Substrate
journal, December 2005


Dual-specificity MAP kinase phosphatases (MKPs) and cancer
journal, March 2008


Crystal structure of the catalytic domain of human DUSP5, a dual specificity MAP kinase protein phosphatase
journal, October 2006


Dusp-5 and Snrk-1 coordinately function during vascular development and disease
journal, January 2009


Comparison of simple potential functions for simulating liquid water
journal, July 1983


Structural Mechanism of Oxidative Regulation of the Phosphatase Cdc25B via an Intramolecular Disulfide Bond ,
journal, April 2005


Flipped Out:  Structure-Guided Design of Selective Pyrazolylpyrrole ERK Inhibitors
journal, March 2007


Increasing the precision of comparative models with YASARA NOVA-a self-parameterizing force field
journal, May 2002


Dual-specificity MAP kinase phosphatases (MKPs): Shaping the outcome of MAP kinase signalling
journal, August 2012


Making optimal use of empirical energy functions: Force-field parameterization in crystal space
journal, January 2004


ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids
journal, May 2010


Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: Four approaches that performed well in CASP8
journal, January 2009


Signal-dependent repression of DUSP5 by class I HDACs controls nuclear ERK activity and cardiomyocyte hypertrophy
journal, May 2013


PROSESS: a protein structure evaluation suite and server
journal, May 2010


A smooth particle mesh Ewald method
journal, November 1995


Pathogenesis of Vascular Anomalies
journal, January 2011


Solution Structure of the MAPK Phosphatase PAC-1 Catalytic Domain
journal, February 2003


A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations
journal, October 2003


Structure and regulation of MAPK phosphatases
journal, July 2004


Mechanistic Basis for Catalytic Activation of Mitogen-activated Protein Kinase Phosphatase 3 by Extracellular Signal-regulated Kinase
journal, March 2000


In Vitro Enzymatic Assays of Protein Tyrosine Phosphatase 1B
journal, May 2001


A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations
journal, October 2003


Increasing the precision of comparative models with YASARA NOVA-a self-parameterizing force field
journal, May 2002


Making optimal use of empirical energy functions: Force-field parameterization in crystal space
journal, January 2004


Dual-specificity MAP kinase phosphatases (MKPs) and cancer
journal, March 2008


Structure and regulation of MAPK phosphatases
journal, July 2004


Regulation of the inducible nuclear dual-specificity phosphatase DUSP5 by ERK MAPK
journal, December 2009


ERK1/2 MAP kinases: Structure, function, and regulation
journal, August 2012


Activation Mechanism of the MAP Kinase ERK2 by Dual Phosphorylation
journal, September 1997


Solution Structure of ERK2 Binding Domain of MAPK Phosphatase MKP-3
journal, February 2001


Structural Mechanism of Oxidative Regulation of the Phosphatase Cdc25B via an Intramolecular Disulfide Bond
journal, April 2005


Flipped Out:  Structure-Guided Design of Selective Pyrazolylpyrrole ERK Inhibitors
journal, March 2007


Mechanism of Cdc25B Phosphatase with the Small Molecule Substrate p -Nitrophenyl Phosphate from QM/MM-MFEP Calculations
journal, April 2009


Comparison of simple potential functions for simulating liquid water
journal, July 1983


Signal-dependent repression of DUSP5 by class I HDACs controls nuclear ERK activity and cardiomyocyte hypertrophy
journal, May 2013


Mechanistic Basis for Catalytic Activation of Mitogen-activated Protein Kinase Phosphatase 3 by Extracellular Signal-regulated Kinase
journal, March 2000


T-cell Development and Function Are Modulated by Dual Specificity Phosphatase DUSP5
journal, April 2008


Spatiotemporal Regulation of ERK2 by Dual Specificity Phosphatases
journal, July 2008


Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding
journal, August 2008


ZDOCK server: interactive docking prediction of protein-protein complexes and symmetric multimers
journal, February 2014


PROSESS: a protein structure evaluation suite and server
journal, May 2010


ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids
journal, May 2010


Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network
journal, January 1993


Dual-specificity MAP kinase phosphatases (MKPs): Shaping the outcome of MAP kinase signalling
journal, August 2012


ERKs in Cancer: Friends or Foes?
journal, January 2014


Dusp-5 and Snrk-1 coordinately function during vascular development and disease
journal, January 2009


Works referencing / citing this record: