Subnuclear localization of protein kinase C delta in beta cells.
Our laboratory has previously shown that beta cells express multiple isoforms of protein kinase C (PKC) and that some isoforms are located to multiple pools within the cell, including the cytoskeletal elements. In this study we analyzed the localization of the delta, epsilon, zeta, beta, and alpha isoforms of PKC to the nucleus. Nuclei were isolated from insulinoma beta cells and fractionated by centrifugation to give the nuclear soluble fraction, nuclear membrane fraction, and the insoluble matrix. The nuclear pellet was enriched in DNA and contained less than 5% of the total cellular nucleotidase activity. The nuclear membrane contained less than 2% of the total cellular nucleotidase activity, suggesting negligible plasma membrane contamination. Analysis of cellular fractions by immunoblotting with isoform-specific anti-PKC antibodies showed that PKC alpha, beta, zeta, and epsilon could be detected in the soluble fraction of the cell but could not be detected in the nucleus. Only PKC delta could be detected in the nucleus and was mostly present in the nuclear membrane fraction. There was light staining in the nucleocytosol and the nuclear matrix but the enzyme in the nuclear membrane represented approximately 76% of the total nuclear enzyme. Nuclear PKC delta constituted approximately 9% of the total cellular enzyme. Phorbol ester (1 microM, 15 min) increased the levels associated with the nuclear membrane approximately threefold but not to the nuclear matrix or nucleocytosol. Inhibition of PKC with MDL 29152 increased levels of preproinsulin mRNA relative to beta-actin mRNA levels, while chronic phorbol ester treatment led to a slight decrease. Taken together, these data suggest that PKC is constitutively active in the nucleus and may be important in modulating preproinsulin mRNA levels.
Knutson
K L
KL
College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602, USA.
Hoenig
M
M
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology*; D007328:Insulin; D007515:Islets of Langerhans / Q000201:enzymology* / Q000648:ultrastructure; D007527:Isoenzymes / Q000032:analysis*; D008297:Male; D011384:Proinsulin / Q000235:genetics; D011493:Protein Kinase C / Q000032:analysis*; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007328:Insulin; D007527:Isoenzymes; D011498:Protein Precursors; D012333:RNA, Messenger; C013737:preproinsulin; D011384:Proinsulin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate