THE ETA-ISOFORM OF PROTEIN-KINASE-C IS LOCALIZED ON ROUGH ENDOPLASMIC-RETICULUM

The eta isoform of protein kinase C, isolated from a cDNA library of m ouse skin, has unique tissue and cellular distributions. It is predomi nantly expressed in epithelia of the skin, digestive tract, and respir atory tract in close association with epithelial differentiation. We r eport here that this isoform is localized on the rough endoplasmic ret iculum in transiently expressing COS1 cells and constitutively express ing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluo rescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron m icroscopic examination, electron-dense signals were located on the rou gh endoplasmic reticulum and on the outer nuclear membrane which is co ntinuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasm ic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treat ment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunob lotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions , all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localizat ion signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chai m, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12: 1304-1311, 1992).