Endocrinology, Vol 135, 881-886, Copyright © 1994 by Endocrine Society

ARTICLES

Identification and subcellular characterization of protein kinase-C isoforms in insulinoma beta-cells and whole islets

KL Knutson and M Hoenig
Department of Physiology and Pharmacology, University of Georgia College of Veterinary Medicine, Athens 30602.

Protein kinase-C (PKC) represents a growing family of serine/threonine kinases, which include both Ca(2+)-dependent and Ca(2+)-independent members. To evaluate the expression of PKC isoforms in insulin- secreting beta-cells, purified beta-cells from a glucose-sensitive rat insulinoma were fractionated into cytosolic, crude membrane, and cytoskeletal/nucleoskeletal fractions. Protein samples from each fraction were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to nylon membranes. The blots were then analyzed with antibodies specific for the alpha, beta, gamma, epsilon, zeta, and delta isoforms. In addition, expression was analyzed in whole isolated rat islets. Expression of all except the gamma isoform was detected in the insulinoma-derived beta-cells. Expression of the alpha, beta, and epsilon isoforms was confined predominantly to the cytosolic fractions. The delta isoform could be detected in all three of the subcellular fractions, whereas the zeta isoform was present in approximately equal amounts in both the cytosolic and crude membrane fractions. The delta isoform could be eluted from the cytoskeletal/nucleoskeletal fraction with 1% Triton X-100. All of the isoforms detected in the insulinoma-derived beta-cells were also detected in whole isolated islets. It is concluded that rat insulinoma beta-cells and whole islets express numerous isoforms of PKC, including both Ca(2+)-dependent and Ca(2+)-independent isoforms, which may be important in the various signal transduction processes of insulin secretion, proinsulin biosynthesis, and insulin gene expression.

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