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.
  
  
  CONCLUSIONS
  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.affiliation: Department of Pediatrics, Medical College of Wisconsin, CRI Developmental Vascular Biology Program, P.O. Box 26509, C3420, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. jnayak@mcw.edu.
authors.forename: Jaladhi
authors.initials: J
authors.lastname: Nayak
authors.affiliation: Department of Pediatrics, Medical College of Wisconsin, CRI Developmental Vascular Biology Program, P.O. Box 26509, C3420, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. agastong@gmail.com.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Gastonguay
authors.affiliation: Department of Chemistry, Marquette University, Wehr Chemistry Building, P.O. Box 1881, 535 N. 14th Street, Milwaukee, WI, 53201, USA. marat.talipov@marquette.edu.
authors.forename: Marat R
authors.initials: MR
authors.lastname: Talipov
authors.affiliation: Department of Pediatrics, Medical College of Wisconsin, CRI Developmental Vascular Biology Program, P.O. Box 26509, C3420, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. pvakeel@mcw.edu.
authors.forename: Padmanabhan
authors.initials: P
authors.lastname: Vakeel
authors.affiliation: Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, Concordia University of Wisconsin, Mequon, WI, 53097, USA. elise.span@gmail.com.
authors.forename: Elise A
authors.initials: EA
authors.lastname: Span
authors.affiliation: Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, Concordia University of Wisconsin, Mequon, WI, 53097, USA. kekalous@mcw.edu.
authors.forename: Kelsey S
authors.initials: KS
authors.lastname: Kalous
authors.affiliation: Department of Pediatrics, Medical College of Wisconsin, CRI Developmental Vascular Biology Program, P.O. Box 26509, C3420, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. rkutty@mcw.edu.
authors.forename: Raman G
authors.initials: RG
authors.lastname: Kutty
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Children's Research Institute (CRI) Developmental Vascular Biology Program, Translational and Biomedical Research Center, 8701 Watertown Plank Road, P.O. Box 26509, Milwaukee, WI, 53226, USA. djensen@mcw.edu.
authors.forename: Davin R
authors.initials: DR
authors.lastname: Jensen
authors.affiliation: Biosciences Division, Argonne National Laboratory, 9700 S. Cass Avenue Lemont, Argonne, IL, 60439, USA. rajp@anl.gov.
authors.forename: Phani Raj
authors.initials: PR
authors.lastname: Pokkuluri
authors.affiliation: Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, Concordia University of Wisconsin, Mequon, WI, 53097, USA. Daniel.Sem@cuw.edu.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Sem
authors.affiliation: Department of Chemistry, Marquette University, Wehr Chemistry Building, P.O. Box 1881, 535 N. 14th Street, Milwaukee, WI, 53201, USA. rajendra.rathore@marquette.edu.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Rathore
authors.affiliation: Department of Pediatrics, Medical College of Wisconsin, CRI Developmental Vascular Biology Program, P.O. Box 26509, C3420, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. rramchan@mcw.edu.|Department of Obstetrics and Gynecology, CRI Developmental Vascular Biology Program, P.O. Box 26509, C3420, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. rramchan@mcw.edu.
authors.forename: Ramani
authors.initials: R
authors.lastname: Ramchandran
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D054637:Dual-Specificity Phosphatases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis
chemical_list: D048049:Extracellular Signal-Regulated MAP Kinases; C442320:DUSP5 protein, human; D054637:Dual-Specificity Phosphatases