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 using antibodies specifically recognizing different activation states of mu-calpain. KB cells possess calpastatin activity in large excess of calpain activity as analyzed by ion exchange HPLC. Stimulation of the cells with a calcium ionophore, ionomycin, caused production of the autolytic intermediate form (M(r) = 78 k) of mu-calpain derived from the preautolysis form (80 k), while the fully autolyzed postautolysis form (76 k) remained below detectable levels at all times. The appearance of the autolytic intermediate paralleled limited proteolysis of the membrane-associated calpastatin fractions (110 k and 106 k); the resulting fragments (68 k and 45 k) were released into the cytosol. Both the production of the autolytic mu-calpain intermediate and the limited proteolysis of calpastatin in cell lysates in the presence of calcium were inhibited by a synthetic calpastatin peptide, indicating that proteolysis of calpastatin was indeed catalyzed by calpain and that the autolytic intermediate may have exerted the proteolytic activity. Furthermore, 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 intracellular conditions associated with the cell cycle may affect the activation of mu-calpain.
Nagao
S
S
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science.
Saido
T C
TC
Akita
Y
Y
Tsuchiya
T
T
Suzuki
K
K
Kawashima
S
S
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001329:Autolysis; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; C033668:calpastatin; D002154:Calpain; D002118:Calcium