The goal of this project is to study the role of protein kinases in cellular metabolism and signaling using immunocytochemical methods along with biochemical techniques. Current studies and major findings are: 1) We showed for the first time that protein kinase C isozymes type 11 (beta) and type 111 (alpha) but not type 1 (gamma) can be found in rat optic nerve. Type 11 (beta) is localized in axons, while type 111 (alpha) is expressed exclusively by the astrocytes. Accordingly, in the retina type 11 (beta) isozyme was found in the ganglion cells and nerve fiber layer, type 111 (alpha) in the Muller cells and interneurons. Intracellularly the protein kinase C-like immunoreactivity was associated in addition with cell membranes with cytoskeletal structures such as glial filaments (alpha-isozyme), neurotubules and neurofilaments (beta-isozyme). 2) Comparative studies revealed that dilapidation of the specimens abolishes the protein kinase C-like, but not the raf protein kinase-like immunostaining in frozen and vibratome sections. Quantitative image analysis of immunostained fibroblast cultures revealed cell density dependent translocation of raf protein kinase from the cytoplasm into the nucleus. Protein kinase C did not show this distribution. In contrast, phorbol ester treatment caused nuclear translocation and down-regulation in a dose- and time-dependent manner of protein kinase C while such an effect of phorbol ester was very limited on raf protein kinase. 3) Translocation of raf protein kinase from cytoplasm into the nucleus was also observed in hippocampal neurons in experimental cerebral ischemia. 4) In preliminary immunocytochemical experiments the localization of GM1 ganglioside was examined with the use of cholera toxin B-subunit. In the brain the B-subunit binding sites were most dense in myelinated pathways, while in tissue cultures (fibroblast, neuroblastoma, embryonic spinal cord cell lines) the immunostaining suggested highest GM1 concentration in neuronal cells. An increase in """"""""B-subunit binding capacity"""""""" of the cellular membranes was also noted with increasing cell density.
