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Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC-binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB-associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST-pull-down assay using GST-PKCalpha, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCalpha., (Copyright 2002 Wiley-Liss, Inc.)

The treatment of isolated cell nuclei with citric acid was described as a method for separating inner and outer nuclear membrane. Using cell nuclei from bovine cerebral cortex, we can show that citric acid does not cause a separation of the two nuclear membranes, but extracts a specific set of proteins from the nuclei. The extraction of proteins is not just an effect of damaging the nuclear membrane or destructing the cytoskeleton, but rather a specific effect of citric acid treatment. One of the extracted proteins, chosen as a marker for the putative outer nuclear membrane fraction, has an apparent molecular weight of 145 kDa and is located in the nucleoplasm as shown by immunofluorescence microscopy. By sequencing tryptic peptides it was identified as RNA helicase A, an abundant nuclear protein assumed to participate in the processing of mRNA.

Using Western blotting and immunofluorescence microscopy we detected the protein kinase C isoforms delta, epsilon and zeta in isolated cell nuclei from bovine cerebral cortex. Both protein kinase C (PKC) delta and PKC epsilon are present in higher concentrations in neuronal than in glial nuclei and are located inside the nucleus and at the nuclear envelope. There they give a punctate staining in immunofluorescence microscopy. PKC zeta is also present both in the nucleoplasm and at the nuclear envelope. PKC eta could not be detected in the cell nuclei and, even in the homogenate of cerebral cortex, this isoform is present only in very low concentrations. The antibody against PKC eta bound strongly to a nucleoplasmic protein with an apparent molecular mass of 99 kDa. The localization of non-conventional PKC isoforms at the cell nucleus strongly indicates that these isoforms are directly involved in the regulation of nuclear processes.