Analysis of the subcellular distribution of protein kinase C alpha using PKC-GFP fusion proteins

One important factor for the determination of the specific functions of pro tein kinase C (PKC) isoforms is their specific subcellular localization. In NTH 3T3 fibroblasts phorbol esters induce translocation of PKC alpha to th e plasma membrane and the nucleus. In order to investigate PKC alpha's subc ellular distribution and especially its nuclear accumulation in more detail we used fusion proteins consisting of PKC alpha and the green fluorescent protein (GFP). Purified GFP-PKC alpha from baculovirus-infected insect cell s undergoes nuclear accumulation without any further stimuli in digitonin-p ermeabilized cells. Interestingly, permeabilization appears to be a trigger for PKC alpha's nuclear translocation, since the fusion protein also trans locates to the nucleus in transiently transfected cells following permeabil ization. This suggests that PKC alpha has a high nuclear binding capacity e ven in the case of large protein amounts. In contrast to endogenous PKC alp ha, overexpressed GFP-PKC alpha as well as overexpressed PKC alpha itself t ranslocates mainly to the plasma membrane and only to a smaller extent to t he nucleus following stimulation with phorbol ester. Use of fusion proteins of GFP and different mutants of PKC alpha enabled determination of motifs involved PKC alpha's subcellular distribution: A25E and K368R point mutatio ns of PKC alpha showed enhanced affinity for the plasma membrane, whereas s equences within the regulatory domain probably confer PKC alpha's nuclear a ccumulation. (C) 2000 Academic Press.