Purification and Analysis of the Three isoforms of Mitogen Activated Protein Kinase-Interacting Serine/Threonine-Protein Kinase 1 (MKNK1)
Disciplines
Biochemistry
Abstract (300 words maximum)
Cellular responses to stimuli are essential to the proper functioning of all organisms. The specific protein interactions that pass along a message that induce cellular changes are numerus and include the extensive p38 MAPK pathway known to be activated by environmental stressors such as inflammatory cytokines, DNA damage, or oxidative stress. Dysregulation and improper responses to these conditions have been linked to various cancers and immune disorders, making members of the pathway highly desirable drug targets. We are focused on Mitogen Activated Protein Kinase-Interacting Serine/Threonine-Protein Kinase 1 (MKNK1) which has three isoforms, long, primary and short; long has never been studied but primary and short are known to be involved in translational regulation (via binding to eIF4G and phosphorylation of eIF4E), though the overall function and role of MKNK 1 is not entirely known. Thus far, we have successfully expressed and purified all isoforms using plasmids that enabled creation of gst-tagged fusion proteins. Further purification via tag removal was conducted to prepare the proteins for metal analysis, predicted in the cysteine containing loop of MKNK1s. Kinase assay was also performed to detect MKNK1 phosphorylation by p38, using a coupled assay with kemptide as the final substrate. Further analysis is underway to explore the functional differences of the isoforms.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Carol Chrestensen
Purification and Analysis of the Three isoforms of Mitogen Activated Protein Kinase-Interacting Serine/Threonine-Protein Kinase 1 (MKNK1)
Cellular responses to stimuli are essential to the proper functioning of all organisms. The specific protein interactions that pass along a message that induce cellular changes are numerus and include the extensive p38 MAPK pathway known to be activated by environmental stressors such as inflammatory cytokines, DNA damage, or oxidative stress. Dysregulation and improper responses to these conditions have been linked to various cancers and immune disorders, making members of the pathway highly desirable drug targets. We are focused on Mitogen Activated Protein Kinase-Interacting Serine/Threonine-Protein Kinase 1 (MKNK1) which has three isoforms, long, primary and short; long has never been studied but primary and short are known to be involved in translational regulation (via binding to eIF4G and phosphorylation of eIF4E), though the overall function and role of MKNK 1 is not entirely known. Thus far, we have successfully expressed and purified all isoforms using plasmids that enabled creation of gst-tagged fusion proteins. Further purification via tag removal was conducted to prepare the proteins for metal analysis, predicted in the cysteine containing loop of MKNK1s. Kinase assay was also performed to detect MKNK1 phosphorylation by p38, using a coupled assay with kemptide as the final substrate. Further analysis is underway to explore the functional differences of the isoforms.