Identification of a calcium-regulated insulinoma cell phosphoprotein as an islet cell keratin.

Abstract

This report describes the cytoskeleton nature of a 60,000-mol-wt protein, P60, previously shown to undergo Ca2+ influx-induced phosphorylation concomitant with insulin release in hamster insulinoma cells. Four lines of evidence suggest that P60 is an intermediate filament protein of the keratin class. (a) As previously described (Schubart, U.K., 1982, J. Biol. Chem. 257:12231-12238), Triton X-100-insoluble cytoskeletons are enriched for P60; (b) these cytoskeletons contain 7-11-nm filaments as determined by negative staining; (c) immunoblot analysis revealed that all proteins detected in the insulinoma cell cytoskeletons are recognized by a monoclonal antibody that interacts with a common determinant in all intermediate filament proteins; and (d) P60 was shown, by its identical migration on two-dimensional electrophoresis and by its immunologic relatedness, to be analogous to a known keratin present in HeLa cells. An antibody specific for P60, as judged by immunoblotting, was developed in a rabbit. In indirect immunofluorescence studies on insulinoma cells, this anti-P60 antibody produced a filamentous staining pattern. The antibody also permitted the identification of P60 in normal pancreatic islets as determined both by immunoblotting of hamster islet proteins resolved by two-dimensional electrophoresis and by indirect immunofluorescence microscopy on cryostat sections of hamster pancreas. In addition, the antibody recognized an antigen in the epithelial layer of pancreatic exocrine ducts, as determined by indirect immunofluorescence. The data have implications for the embryonic origin of pancreatic islets. Together with the phosphorylation data, these findings suggest that this islet cell cytokeratin may be involved in the regulation of insulin release.