Investigating Interactions of the PKCα C1B Domain with Lipid Bilayers and Activators: An EPR Site-Directed Spin-Labeling and Relaxation Study

It has been previously shown that activation of PKCα on the leading edge membrane of a polarized chemotaxing cell contributes to a positive feedback loop that sustains leading edge formation and facilitates eukaryotic cell movement. As a result of its vital role in this process and other known signaling pathways, PKCα has been studied to better understand both normal cell function and the various diseases in which PKCα is an important player, including cancer. A recent single molecule study on PKCα has shown that, once bound to the membrane bilayer, the PKCα C1B domain plays a critical role in the final step of kinase activation on the target membrane. Specifically, C1B is recruited to the membrane by its binding to the activating lipid diacylglycerol (DAG) to activate the kinase. However, the key interactions of C1B with the lipid bilayer and its known lipid activators including DAG and phorbol esters are still under investigation. In this study, EPR site-directed spin labeling and power saturation measurements were employed to determine membrane insertion depth values for the PKCα C1B domain on simple lipid bilayers containing PC and PS in the presence and absence of known lipid activators DAG and PMA (the latter a phorbol ester). These depth values were then compared to protein-to-membrane depth values previously obtained through molecular dynamic (MD) simulations of the C1B domain by others, and to diffusion rates previously measured for C1B bound to the same lipid mixtures by others. All three approaches yielded a common trend in binding depths for C1B, indicating that C1B adopts a shallow docking depth when bound to PMA and a deeper docking depth when in a membrane-bound, activator-free state or bound to DAG on the lipid membrane bilayer. The present EPR-determined membrane depths provide the most direct measure of this shallower docking geometry on PMA-containing membranes. The shallow geometry may well play an important role in the higher affinity of C1B for PMA compared to DAG, where additional membrane penetration may have an energetic cost. The higher affinity for PMA superactivates PKCα and plays a central role in well established ability of phorbol esters to promote tumor formation