Role of Phosphorylation in Regulating Calpain Activity

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DESCRIPTION (provided by applicant): The long-term goal of this research is to learn how activity of the "ubiquitous calpains", u- and m-calpain, is regulated in living cells. The calpains are Ca2+-dependent proteases found in every vertebrate cell. Disruption of the gene encoding the 28-kDa subunit common to u- and m-calpain is embryonical lethal. Inappropriate calpain degradation is implicated in many tissue pathologies ranging from loss of muscle mass in the muscular dystrophies, to crystalline degradation and cataract formation, to Alzheimer's disease, to tissue damage in ischemic areas near a blocked blood vessel (stroke or myocardial infarction), to traumatic spinal cord injury, etc. Calpain activity in these pathologies is triggered by elevated, intracellular [Ca2+]; however, Ca2+ requirement of the calpains in in vitro assays is 3-50 uM (u-calpain) or 300-500 uM (m- calpain), much higher than intracellular free [Ca2+] is, even near ischemic areas. Cells, therefore, have a mechanism to reduce the [Ca2+] required for calpain activity. This mechanism evidently is altered in a way that activates the calpains during ischemic events. The same mechanism must regulate calpain activity during normal cell function. We have found that both u- and m-calpain are phosphorylated at multiple sites, and that dephosphorylated calpain is proteolytically inactive. Studies in this application test the hypothesis that phosphorylation regulates calpain activity in cells. There are five objectives. 1. Measure phosphorylation status and activities of calpains isolated from cells that have been incubated with phosphatase/kinase inhibitors to alter the level of calpain phosphorylation in these cells. 2. Determine the phosphorylation status and activities of calpains isolated from cells that have been treated to activate the calpains. 3. Determine the effects of in vitro phosphorylation by selected kinases on the activities of u- and m-calpain that have been dephosphorylated. 4. Use site-directed mutation of specific phosphorylation sites, chosen on the basis of results in aims 1 and 2, and expression of rat m-calpain to determine the effects of such mutations on activities of m-calpain. 5. Determine the effects of phosphorylation of u- or m-calpain on the calpain/ calpastatin interaction. Changes in calpain phosphorylation will be compared with changes in catalytic properties and the Ca2+ requirement of the calpains. The studies use mass spectrometry and Western analysis to monitor calpain phosphorylation and enzyme assays to monitor catalytic properties of the calpains.