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.