Diffusion-limited translocation mechanism of protein kinase C isotypes

The plasma membrane translocation. of protein kinase C (PKC) is driven by an as yet unidentified mechanism. We investigated the transport mechanisms of classical and novel PKCs coupled to fluorescent proteins. Classical PKCalpha and PKCbetaI half-maximally translocated 300-750 ms after receptor stimulation, whereas the novel isotypes PKCdelta and PKCepsilon were redistributed five to ten times more slowly. Although fluorescence recovery after photobleach experiments demonstrated that PKCs are freely diffusible in quiescent cells, the similar Stokes' radii of all fusion proteins indicated that diffusion velocities cannot account for the kinetic differences. Alternatively, active transport or different collisional coupling efficacies could explain the effects. A diffusion-driven translocation mechanism was demonstrated by confocal line-scan microscopy to follow subcellular PKC concentrations at high spatiotemporal resolution. The observation of a temporary subplasmalemmal depletion zone is direct evidence for a diffusion-limited binding process. Computer-assisted modeling revealed that Ca2+-bound PKCalpha molecules require only 2 or 3 collisions with the plasma membrane to achieve a binding event. Our data further demonstrate that the slower association kinetic of novel PKC isoenzymes relies on lower collisional. coupling efficacies. The superior collisional coupling of classical PKCs points to a role of Ca2+-binding C2-domains to electrostatically facilitate binding, an effect that is lacking in novel isoenzymes.