CALPAIN CALPASTATIN INTERACTIONS IN EPIDERMOID CARCINOMA KB CELLS

We examined the activation of mu-calpain in human epidermoid carcinoma KB cells following a rise in intracellular calcium concentration usin g antibodies specifically recognizing different activation states of m u-calpain. KB cells possess calpastatin activity in large excess of ca lpain activity as analyzed by ion exchange HPLC. Stimulation of the ce lls with a calcium ionophore, ionomycin, caused production of the auto lytic intermediate form (M(r) = 78 K) of mu-calpain derived from the p reautolysis farm (80 k), while the fully autolyzed postautolysis form (76 k) remained below detectable levels at all times. The appearance o f the autolytic intermediate paralleled limited proteolysis of the mem brane-associated calpastatin fractions (110 k and 106 k); the resultin g fragments (68 k and 45 k) were released into the cytosol. Both the p roduction of the autolytic mu-calpain intermediate and the limited pro teolysis of calpastatin in cell lysates in the presence of calcium wer e inhibited by a synthetic calpastatin peptide, indicating that proteo lysis of calpastatin was indeed catalyzed by calpain and that the auto lytic intermediate may have exerted the proteolytic activity. Furtherm ore, mu-calpain autolysis and calpastatin degradation, upon ionomycin treatment, were both augmented by epidermal growth factor (EGF). These results suggest that calpastatin serves not only as an inhibitor but also as a substrate for calpain at cell membranes and that intracellul ar conditions associated with the cell cycle may affect the activation of mu-calpain.