calpastatin and Carcinoma--Squamous-Cell

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.

Topics: Amino Acid Sequence; Antibody Specificity; Autolysis; Binding Sites; Calcium; Calcium-Binding Proteins; Calpain; Carcinoma, Squamous Cell; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Molecular Sequence Data; Tumor Cells, Cultured

Little is known about the relative intracellular localizations of the calcium-dependent proteases, calpains, and their naturally occurring inhibitor, calpastatin. In the present study, the intracellular localization of mu-calpain, m-calpain, and calpastatin was studied at the light microscopic level in proliferating A431 cells. Highly specific antibodies against the three antigens revealed distinct staining patterns in interphase and mitotic cells. Most notably, calpastatin in interphase cells was localized near the nucleus in tube-like, or large granular structures, while the calpains were more uniformly distributed through the cytoplasm in either a fibrillar form (mu-calpain) or a diffuse or fine granular form (m-calpain). The distribution patterns of the two calpain isozymes were distinctly different during mitosis. m-Calpain was concentrated at the mitotic spindle poles and midbody, while mu-calpain appeared to accumulate at the cell membrane and the spindles. Four other human cell lines as well as normal human monocytes were examined to determine if the calpains-calpastatin segregation patterns are common to other cells or are unique to the A431 line. With the exception of abundant nuclear mu-calpain in the C-33A cervical carcinoma, the segregation of the proteins was similar to that of A431. These studies indicate that calpains may be localized at regions which are relatively poor in calpastatin content. Proteins at these sites may be susceptible to calpain-catalyzed cleavage.

Topics: Antibodies, Monoclonal; Blotting, Western; Calcium-Binding Proteins; Calpain; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cytoskeleton; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Interphase; Subcellular Fractions; Tumor Cells, Cultured