Distinct substrate specificities and functional roles for the 78- and 76-kDa forms of μ-calpain in human platelets

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Abstract

The intracellular thiol protease μ-calpain exists as a heterodimeric proenzyme, consisting of a large 80-kDa catalytic subunit and a smaller 30- kDa regulatory subunit. Activation of μ-calpain requires calcium influx across the plasma membrane and the subsequent auto-proteolytic conversion of the 80-kDa large subunit to a 78-kDa 'intermediate' and a 76-kDa fully autolyzed form. Currently, there is limited information on the substrate specificities and functional roles of these distinct active forms of μ- calpain within the cell. Using antibodies that can distinguish among the 80- , 78-, and 76-kDa forms of μ-calpain, we have demonstrated a close correlation between the autolytic generation of the 78-kDa enzyme and the proteolysis of the non-receptor tyrosine phosphatase, PTP-1B, in ionophore A23187-stimulated platelets. Time course studies revealed that pp60(c-src) proteolysis lagged well behind that of PTP-1B and correlated closely with the generation of the fully proteolyzed form of μ-calpain (76 kDa). In vitro proteolysis experiments with purified μ-calpain and immunoprecipitated PTP- 1B or pp60(c-src) confirmed selective proteolysis of pp60(c-src) by the 76- kDa enzyme, whereas PTP-1B cleavage was mediated by both the 76- and 78-kDa forms of μ-calpain. Studies using selective pharmacological inhibitors against the different autolytic forms of μ-calpain have demonstrated that the initial conversion of the μ-calpain large subunit to the 78-kDa farm is responsible for the reduction in platelet-mediated clot retraction, whereas complete proteolytic activation of μ-calpain (76 kDa) is responsible for the shedding of procoagulant-rich membrane vesicles from the cell surface. These studies demonstrate the existence of multiple active forms of μ-calpain within the cell, that have unique substrate specificities and distinct functional roles.

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Schoenwaelder, S. M., Kulkarni, S., Salem, H. H., Imajoh-Ohmi, S., Yamao-Harigaya, W., Saido, T. C., & Jackson, S. P. (1997). Distinct substrate specificities and functional roles for the 78- and 76-kDa forms of μ-calpain in human platelets. Journal of Biological Chemistry, 272(40), 24876–24884. https://doi.org/10.1074/jbc.272.40.24876

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