Protein Expression, Characterization and Activity Comparisons of Wild Type and Mutant DUSP5 Proteins

Authors

Jaladhi Nayak, Medical College of Wisconsin
Adam J. Gastonguay, Medical College of Wisconsin
Marat R. Talipov, Marquette UniversityFollow
Padmanabhan Vakeel, Medical College of Wisconsin
Elise A. Span, Concordia University - Wisconsin
Kelsey S. Kalous, Concordia University - Wisconsin
Raman G. Kutty, Medical College of Wisconsin
David R. Jensen, Medical College of Wisconsin
Phani Raj Pokkuluri, Argonne National Laboratory
Daniel S. Sem, Marquette UniversityFollow
Rajendra Rathore, Marquette University
Ramani Ramchandran, Medical College of Wisconsin

Document Type

Article

Publication Date

12-2014

Source Publication

BMC Biochemistry

Source ISSN

1471-2091

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.

Comments

Published version. BMC Biochemistry, Vol. 15, No. 1 (December 2014). DOI. © BioMed Central 2014.

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Recommended Citation

Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, David 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" (2014). Chemistry Faculty Research and Publications. 384.
https://epublications.marquette.edu/chem_fac/384