Calcium activation and membrane docking of C2 domains for conventional isoforms of protein kinase C.

摘要

Calcium-regulated signaling pathways are central to a variety of cellular functions. These pathways are triggered by optimized Ca2+ fluxes that activate an array of Ca2+ binding proteins. The most common Ca2+ binding motifs used to regulate Ca2+, signaling pathways are the EF hand and the C2 domain. The EF hand is a helix-loop-helix motif which undergoes conformational changes upon Ca2+ binding, typically docking to other proteins. The C2 domain is a β-sandwich motif that is activated by Ca2+ binding, typically docking to intracellular membranes. To understand how C2 domains are tuned, the Ca2+ activation and membrane docking parameters of three C2 domains isolated from conventional protein kinase C isoforms α, β and γ (cPKCα, β and γ) were compared. The PKCγ C2 domain displays the highest Ca2+ affinity either in the presence or absence of membranes while PKCβ C2 domain displays the lowest, suggesting that these C2 domain isoforms may be specialized to respond to different amplitude Ca2+ signals. When complexed with the membrane, the PKCα C2 domain binds two Ca2+ ions, while the PKCβ and PKCγ C2 domains bind three Ca2+ ions. All three C2 domains appear to require membranes to bind the full number of Ca2+ ions. The sensitivity of the full length PKCs to small changes in intracellular Ca 2+, concentrations may be influenced by these differences observed for the isolated C2 domain. The membrane docking interface of the PKCα C2 domain was mapped out to resolve two models for the membrane docking orientation. Using site-directed mutagenesis, cysteine residues were introduced at scattered positions across the surface of the domain for use as attachment sites for fluorescence and EPR spin probes. The largest perturbations of the fluorescent probe were localized to the Ca2+ binding loops. Similarly, the nitroxide spin probe penetrated into the membrane only at positions on the Ca2+ binding loops. The evidence indicates that direct membrane contacts are limited to the Ca2+ binding loops, favoring a membrane docking model where the β-strands of the domain are oriented at an oblique angle relative to the membrane surface. Overall, these studies expand the general understanding of how C2 domains bind Ca 2+ and dock to membranes.