Protein kinase C (PKC) plays an important role in many signal transduction processes and in the regulation of proliferation and differentiation. Support is growing for the concept that the functions of the different PKC isoforms are realized by specific localization in cellular compartments. Since in many cases a stimulation-dependent change of localization can be observed, PKC can be seen as a shuttle molecule for cellular signalling. Therefore the general topic of my investigations is the regulation of the function of PKC by its compartmentation. I focus on the PKC-mediated signal transduction to the nucleus and the function of PKC in this compartment. 

For long-term changes of the physiological state of cells, changes in gene expression must occur and to this purpose signals must be transmitted into the nucleus. It has recently become apparent that PKC is active not only in the cytoplasm, but in the cell nucleus as well. On the one hand it can be translocated there upon stimulation; on the other hand it can be constitutively present and be activated in the nucleus. 
 

The hypothesis that PKC - either present in the nucleus or translocated there upon stimulation – is also involved in mechanisms of neuronal plasticity is supported by our observation that in cultured hippocampal neurons stimulation via the excitatory transmitter glutamate leads to a translocation of PKC-alpha into the nucleus. In the course of our investigations on the localization of PKC isoforms in primary neurons (performed during a research stay in R.Nixon’s lab, McLean Hospital, Harvard Medical School, Boston) we also observed that glutamate (via an increase in the intracellular calcium concentration) and phorbol ester have different effects on the distribution of PKC-alpha and
PKC-gamma. The investigations are currently being carried out in cooperation with G. Ahnert-Hilger (Inst. f. Anatomie, Charite, Berlin). 

To gain insight into the function of PKC in the nucleus, we began to look for its  substrates in the nucleus. So far, some proteins have been identified as in vitro substrates; we are currently investigating whether these are also PKC substrates in intact cells. The identification and characterization of PKC substrates is done using molecular biological techniques and protein-chemical methods such as MALDI mass spectrometry and chemical micro-sequencing. 

(3) While investigating the mechanism of PKC translocation into the nucleus, we found that it differs in several respects from the mechanism by which proteins carrying a classical nuclear localization signal are transported across the nuclear envelope. Therefore we postulate a completely novel transport mechanism. We try to identify the PKC regions which – most likely by interacting with other proteins – mediate translocation to the nucleus. After transient transfection with constructs coding for a fusion protein of PKC-alpha and green fluorescent protein (GFP) and transfection with constructs generated by site-directed mutagenesis we narrowed down the regions that govern transport into the nucleus.