PROTEIN-KINASE-C-EPSILON SUBCELLULAR-LOCALIZATION DOMAINS AND PROTEOLYTIC DEGRADATION SITES - A MODEL FOR PROTEIN-KINASE-C CONFORMATIONAL-CHANGES

Protein kinase C (PKC) epsilon has been found to have unique propertie s among the PKC isozymes in terms of its membrane association, oncogen ic potential, and substrate specificity, Recently we have demonstrated that PKC epsilon localizes to the Golgi network via its zinc finger d omain and that both the holoenzyme and its zinc finger region modulate Golgi function. To further characterize the relationship between the domain organization and the subcellular localization of PKC epsilon, a series of NIH 3T3 cell lines were created, each overexpressing a diff erent truncated version of PKC epsilon. The overexpressed proteins eac h were designed to contain an epsilon-epitope tag peptide at the COOH terminus to allow ready detection with an antibody specific for the ta g. The subcellular localization of the recombinant proteins was analyz ed by in vivo phorbol ester binding, immunocytochemistry, and cell fra ctionation followed by immunoblotting. Results revealed several region s of PKC epsilon that contain putative subcellular localization signal s. The presence either of the hinge region or of a 33-amino-acid regio n including the pseudosubstrate sequence in the recombinant proteins r esulted in association with the plasma membrane and cytoskeletal compo nents. The catalytic domain was found predominantly in the cytosolic f raction. The accessibility and thus the dominance of these localizatio n signals is likely to be affected by the overall conformation of the recombinant proteins. Regions with putative proteolytic degradation si tes also were identified. The susceptibility of the overexpressed prot eins to proteolytic degradation was dependent on the protein conformat ion. Based on these observations, a model depicting the interaction an d hierarchy of the suspected localization signals and proteolytic degr adation sites is presented.