MRI: Acquisition of a Phosphorimager to Investigate Phosphorylation and Regulation of TACE

Kalamazoo College, Kalamazoo MI

Investigators

Abstract

A grant has been awarded to Kalamazoo College under the supervision of Dr. Moore,to acquire a phosphoimager, which will allow faculty to investigate phosphorylation and regulation of the TNF-alpha converting enzyme (TACE). TACE is a protein that works as an enzyme, catalyzing the cleavage of large, full-length precursors (known as substrates of TACE) into smaller fragments. Understanding how the activity of the TACE enzyme is regulated will provide insights as to how, mechanistically, these fragments are generated. Specific experiments are planned to determine the sites (specific amino acids) on TACE that are phosphorylated by PKC, and to determine whether the phosphorylation state of TACE directly influences its activity. The former involves the use of mutated versions of TACE that lack critical proposed phosphorylation sites. This experiment will determine where on TACE these phosphate groups are added. The latter also involves the use of the aforementioned mutant versions of TACE in cleavage assays to determine whether the cleavage of TACE substrates (other proteins that TACE acts on and cleaves) is affected by phosphorylation state. In addition to providing basic science information about the regulation of the TACE enzyme, the experiments described above will have a much broader impact. For example, much of what we learn about the TACE enzyme may be generalized to similar proteases that are in the same protein "family" as TACE. Furthermore, understanding the mechanisms whereby TACE is "turned on" will illuminate general strategies used by cells to "turn on" protein enzymes. The broader impact of this work lies in uncovering the complexities of cellular function: the more scientist know about how cells work normally, the better prepared scientists are to determine what goes wrong in malfunctioning cells in disease or injury states. Moreover, because these experiments will be done in conjunction with undergraduate student collaborators, an important educational experience will be provided. In addition several other faculty members in the college are anxious to learn application of the technique, and they will benefit from the presence of this piece of equipment on the campus. Both faculty and students from the biology and chemistry departments will be involved in research utilizing the equipment.