SUBNUCLEAR LOCALIZATION OF PROTEIN-KINASE-C-DELTA IN BETA-CELLS

Our laboratory has previously shown that beta cells express multiple i soforms of protein kinase C (PKC) and that some isoforms are located t o multiple pools within the cell, including the cytoskeletal elements. In this study we analyzed the localization of the delta, epsilon, zet a, beta, and alpha isoforms of PKC to the nucleus. Nuclei were isolate d from insulinoma beta cells and fractionated by centrifugation to giv e the nuclear soluble fraction, nuclear membrane fraction, and the ins oluble matrix. The nuclear pellet was enriched in DNA and contained le ss than 5% of the total cellular nucleotidase activity. The nuclear me mbrane contained less than 2% of the total cellular nucleotidase activ ity, suggesting negligible plasma membrane contamination. Analysis of cellular fractions by immunoblotting with isoform-specific anti-PKC an tibodies showed that PKC alpha, beta, zeta, and epsilon could be detec ted in the soluble fraction of the cell but could not be detected in t he nucleus. Only PKC delta could be detected in the nucleus and was mo stly present in the nuclear membrane fraction. There was light stainin g in the nucleocytosol and the nuclear matrix but the enzyme in the nu clear membrane represented approximate to 76% of the total nuclear enz yme. Nuclear PKC delta constituted approximate to 9% of the total cell ular enzyme. Phorbol ester (1 mu M, 15 min) increased the levels assoc iated with the nuclear membrane approximately threefold but not to the nuclear matrix or nucleocytosol. Inhibition of PKC with MDL 29152 inc reased levels of preproinsulin mRNA relative to beta-actin mRNA levels , while chronic phorbol ester treatment led to a slight decrease. Take n together, these data suggest that PKC is constitutively active in th e nucleus and may be important in modulating preproinsulin mRNA levels . (C) 1997 Academic Press.