Membrane Binding Kinetics of Protein Kinase C βII Mediated by the C2 Domain

Abstract

Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca²⁺, respectively. This study investigates the mechanism of Ca²⁺-induced binding of PKC βII to anionic membranes mediated by the C2 domain. Stopped-flow fluorescence spectroscopy reveals that Ca²⁺-induced binding of the isolated C2 domain to anionic vesicles proceeds via at least two steps: (1) rapid binding of two or more Ca²⁺ ions to the free domain with relatively low affinity and (2) diffusion-controlled association of the Ca²⁺-occupied domain with vesicles. Ca²⁺ increases the affinity of the C2 domain for anionic membranes by both decreasing the dissociation rate constant (k_off) and increasing the association rate constant (k_on) for membrane binding. For binding to vesicles containing 40 mol % anionic lipid in the presence of 200 μM Ca²⁺, k_off and k_on are 8.9 s^-1 and 1.2 × 10¹⁰ M^-1 s^-1, respectively. The k_off value increases to 150 s^-1 when free Ca²⁺ levels are rapidly reduced, decreasing the average lifetime of the membrane-bound C2 domain (τ = k_off^-1) from 110 ms in the presence of Ca²⁺ to 6.7 ms when Ca²⁺ is rapidly removed. Experiments addressing the role of electrostatic interactions reveal that they stabilize either the initial C2 domain−membrane encounter complex or the high-affinity membrane-bound complex. Specifically, lowering the phosphatidylserine mole fraction or including MgCl₂ in the binding reaction decreases the affinity of the C2 domain for anionic vesicles by both reducing k_on and increasing k_off measured in the presence of 200 μM Ca²⁺. These species do not affect the k_off value when Ca²⁺ is rapidly removed. Studies with PKC βII reveal that Ca²⁺-induced binding to membranes by the full-length protein proceeds minimally via two kinetically resolvable steps: (1) a rapid bimolecular association of the enzyme with vesicles near the diffusion-controlled limit and, most likely, (2) subsequent conformational changes of the membrane-bound enzyme. As is the case for the C2 domain, k_off for full-length PKC βII increases when Ca²⁺ is rapidly removed, reducing τ from 11 s in the presence of Ca²⁺ to 48 ms in its absence. Thus, both the C2 domain and the slow conformational change prolong the lifetime of the PKC βII−membrane ternary complex in the presence of Ca²⁺, with rapid membrane release triggered by removal of Ca²⁺. These results provide a molecular basis for cofactor regulation of PKC whereby the C2 domain searches three-dimensional space at the diffusion-controlled limit to target PKC to relatively common anionic phospholipids, whereupon a two-dimensional search is initiated by the C1 domain for the more rare, membrane-partitioned DG.