IDENTIFICATION AND SUBCELLULAR CHARACTERIZATION OF PROTEIN-KINASE-C ISOFORMS IN INSULINOMA BETA-CELLS AND WHOLE ISLETS

Protein kinase-C (PKC) represents a growing family of serine/ threonin e kinases, which include both Ca2+-dependent and Ca2+-independent memb ers. To evaluate the expression of PKC isoforms in insulin-secreting b eta-cells, purified beta-cells from a glucose-sensitive rat insulinoma were fractionated into cytosolic, crude membrane, and cytoskeletal/nu cleoskeletal fractions. Protein samples from each fraction were resolv ed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to nylon membranes. The blots were then analyzed with ant ibodies specific for the alpha, beta, gamma, epsilon, zeta, and delta isoforms. In addition, expression was analyzed in whole isolated rat i slets. Expression of all except the gamma isoform was detected in the insulinoma-derived beta-cells. Expression of the alpha, beta, and epsi lon isoforms was confined predominantly to the cytosolic fractions. Th e delta isoform could be detected in all three of the subcellular frac tions, whereas the zeta isoform was present in approximately equal amo unts in both the cytosolic and crude membrane fractions. The delta iso form could be eluted from the cytoskeletal/nucleoskeletal fraction wit h 1% Triton X-100. All of the isoforms detected in the insulinoma-deri ved beta-cells were also detected in whole isolated islets. It is conc luded that rat insulinoma beta-cells and whole islets express numerous isoforms of PKC, including both Ca2+-dependent and Ca2+-independent i soforms, which may be important in the various signal transduction pro cesses of insulin secretion, proinsulin biosynthesis, and insulin gene expression.