Intellectual merit. MAPKAP kinase 2 (MK2) is an important downstream target of the stress activated p38 MAP kinase pathway. This pathway is critical for sensing stress in the cellular environment. MK2 has been linked to many different and important cellular processes, including regulation of the cell cycle, cellular migration, and initiation of inflammation. MK2 has two known splice variants; both are expressed and catalytically active in cells. MK2-isoform 1 (NM 004759.3) ends with 17 distinct amino acids while MK2-isoform 2 (NM 032960.2) ends with 47 distinct amino acids. This research will investigate novel mechanisms of MK2 regulation. In particular, this project aims to determine if direct modification through oxidation has the ability to regulate MK2 in the cellular environment and to determine how isoform-1 is regulated differentially from isoform-2. The more studied and longer isoform-2 has well characterized nuclear export (NES) and nuclear localization signals (NLS) contained within the 47 distinct amino acids. These signaling sequences enable shuttling of isoform-2 between the nucleus and cytoplasm. Isoform-1 lacks these signal sequences, and this project will determine the localization of isoform-1. Additionally, a portion of the NLS in isoform-2 creates a binding site for p38, which enables phosphorylation and activation of isoform-2. This project will test how loss of that binding site impacts both the activation profile of isoform-1 and the ability of isoform-1 to interact with other proteins, particularly substrates. This research will identify if oxidative modification has the ability to regulate MK2 and will begin to clarify if the two isoforms of MK2 have different roles in regulating important cellular processes.
Broader impacts. This project will provide undergraduate students with a multitude of research experiences, including bacterial expression of proteins, protein purification, mammalian tissue culture, transfection, probing protein-protein interactions and kinase assays. Due to Kennesaw State University?s diverse student population - 25% minority and 55% female in the College of Science and Mathematics ? this project will have a large pool of students from which to recruit minorities and women to this research project and ultimately to STEM (Science, Technology, Engineering, Mathematics) careers. In addition to hands-on training in the lab, students will learn to critically analyze papers and will gain experience in scientific writing and making presentations to colleagues. Dissemination of the results at meetings and in peer reviewed manuscripts will provide a more detailed understanding of how the regulation of MK2 isoforms may impact cellular processes (e.g. the cell cycle, cellular migration). This project will strengthen the research environment at this predominantly teaching institution for faculty and students alike, providing essential resources for the Principal Investigator and research experiences for undergraduate students.
This award catalyzed a vibrant predominately undergraduate based research program on the oxidative regulation of MK2 and began the process of understanding the different roles of the two MK2 isoforms. During the period of this award a novel isoform specific MK2 antibody was developed, a number of MK2 variants were created and several sites of oxidation were detected. Inhibition of MK2 due to oxidation was found to be predominately due to oxidative modification at the activation loop cysteine. Localization studies of isoform 1 were undertaken and the two isoforms localize very differently in the cell, we continue to pursue the location of isoform 1. Excitingly this work created another entire avenue of collaborative research. Along with others at Kennesaw we discovered the interaction between MAP kinases (p38 & ERK) and endothelial nitric oxide synthase (eNOS) occurs through the pentabasic region in the autoinhibitory loop of eNOS. Further, we have also discovered novel eNOS phosphorylation sites and shown that phosphorylation at S602 by ERK is inhibitory. This project significantly strengthened the research environment at Kennesaw State University. It enabled the purchase of key instrumentation that is shared by other faculty leading undergraduate driven research programs, including a refrigerated shaking bacterial incubator, a new ultracentrifuge and SW-40Ti rotor, and a tissue culture incubator workstation that allows control of both oxygen and carbon dioxide levels. These will continue to be critical for the future work on this and many other project at Kennesaw. This project resulted in the intensive training of eight undergraduate students (6 women, 2 men, 3 under-represented minorities, and one international student) as researchers on the project. These students were immersed in the science and learned a variety of critical biochemical techniques, including bacterial expression of proteins, protein purification, mammalian tissue culture, transfection, confocal microscopy, analysis of protein-protein interactions as well as kinase assays. All of these students presented their work in poster format, some are or will be authors in peer-reviewed publications. All have gone on to work in STEM fields. One student is in medical school, 5 are in, or recently graduated from post-graduate programs, including one who is a Ph.D. candidate at Emory. Two are working as bench scientists, one at Emory and the other at a biotech company.
