Association of calpastatin with inactive calpain: a novel mechanism to control the activation of the protease?

It is generally accepted that the Ca2-dependent interaction of calpain with calpastatin is the most relevant mechanism involved in the regulation of Ca2-induced proteolysis.Wenow report that a calpain-calpastatin association can occur also in the absence of Ca2 or at very low Ca2 concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4–7. This calpastatin region recognizes a calpain sequence located near the end of the DIIdomain. Interaction of calpain with calpastatins lacking these sequences becomes strictly Ca2-dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca2-free calpain and various recombinant calpastatin forms has been demonstrated by the following experimental results. Addition of calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when calpastatin was immunoprecipitated. The calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-calpastatin interaction was perturbed by protein kinaseCphosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-calpastatin interaction at a physiological concentration of Ca2 represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca2 influx.