C2 Domains of Protein Kinase C Isoforms α, β, and γ:  Activation Parameters and Calcium Stoichiometries of the Membrane-Bound State

Abstract

The independently folding C2 domain motif serves as a Ca(2+)-dependent membrane docking trigger in a large number of Ca(2+) signaling pathways. A comparison was initiated between three closely related C2 domains from the conventional protein kinase C subfamily (cPKC, isoforms alpha, beta, and gamma). The results reveal that these C2 domain isoforms exhibit some similarities but are specialized in important ways, including different Ca(2+) stoichiometries. In the absence of membranes, Ca(2+) affinities of the isolated C2 domains are similar (2-fold difference) while Hill coefficients reveal cooperative Ca(2+) binding for the PKC beta C2 domain but not for the PKC alpha or PKC gamma C2 domain (H = 2.3 +/- 0.1 for PKC beta, 0.9 +/- 0.1 for PKC alpha, and 0.9 +/- 0.1 for PKC gamma). When phosphatidylserine-containing membranes are present, Ca(2+) affinities range from the sub-micromolar to the micromolar (7-fold difference) ([Ca(2+)](1/2) = 0.7 +/- 0.1 microM for PKC gamma, 1.4 +/- 0.1 microM for PKC alpha, and 5.0 +/- 0.2 microM for PKC beta), and cooperative Ca(2+) binding is observed for all three C2 domains (Hill coefficients equal 1.8 +/- 0.1 for PKC beta, 1.3 +/- 0.1 for PKC alpha, and 1.4 +/- 0.1 for PKC gamma). The large effects of membranes are consistent with a coupled Ca(2+) and membrane binding equilibrium, and with a direct role of the phospholipid in stabilizing bound Ca(2+). The net negative charge of the phospholipid is more important to membrane affinity than its headgroup structure, although a slight preference for phosphatidylserine is observed over other anionic phospholipids. The Ca(2+) stoichiometries of the membrane-bound C2 domains are detectably different. PKC beta and PKC gamma each bind three Ca(2+) ions in the membrane-associated state; membrane-bound PKC alpha binds two Ca(2+) ions, and a third binds weakly or not at all under physiological conditions. Overall, the results indicate that conventional PKC C2 domains first bind a subset of the final Ca(2+) ions in solution, and then associate weakly with the membrane and bind additional Ca(2+) ions to yield a stronger membrane interaction in the fully assembled tertiary complex. The full complement of Ca(2+) ions is needed for tight binding to the membrane. Thus, even though the three C2 domains are 64% identical, differences in Ca(2+) affinity, stoichiometry, and cooperativity are observed, demonstrating that these closely related C2 domains are specialized for their individual functions and contexts.

A C2 domain protein kinase initiates phagocytosis in the protozoan parasite Entamoeba histolytica

“…2f,g). These findings are consistent with the properties of other C2-domain-containing proteins, in which binding to phosphatidylserine is mediated through the C2 domain 34,35 . We conclude from these results that EhCaBP1 binds lipids through EhC2PK and this interaction is lipid-specific and requires the presence of Ca 2 + .…”

Ca2+ Activation of the cPLA2 C2 Domain: Ordered Binding of Two Ca2+ Ions with Positive Cooperativity

“…The reduced magnitude of this electrostatic switch increases the relative importance of other activation mechanisms, particularly the direct or indirect coordination of the two Ca 2+ in the binding pocket by lipid headgroup oxygens upon membrane docking. Evidence supporting the essential roles of both the electrostatic switch activation mechanism and the coordinating lipid activation mechanism is now available for C2 domains, suggesting that both mechanisms contribute to Ca 2+ -activated membrane docking (5,6,14,(41)(42)(43)(44)(45).…”

“…The probability of the assumed order being correct is then given by the product of the probabilities of adding each successive proton. Using the partition function given by eq 4, the probability of correctness associated with a specific order (x1 → x2 → … → xN) can be easily derived as: (5) Since these probabilities map the magnitude of correctness onto the assumed orders of protonation and thereby onto different protonated conformers, they were subsequently used to weigh the pK a 's generated from using different protonated conformers.…”

Specific Translocation of Protein Kinase Cα to the Plasma Membrane Requires Both Ca2+and PIP2Recognition by Its C2 Domain

“…Best-fit analysis using the Hill equation (solid lines) yields [Ca 2ϩ ] 1/2 values of 2.9 Ϯ 0.05 M (ϩPIP 2 ) and 51 Ϯ 1.5 M (ϪPIP 2 ) for PKC␣C2 and 15 Ϯ 0.28 M (ϩPIP 2 ) and 13 Ϯ 0.29 M (ϪPIP 2 ) for cPLA 2 ␣C2. Hill coefficients ranged from 1.5 to 1.9 because of the activation of each C2 domain by multiple Ca 2ϩ ions (Kohout et al, 2002 plasma membrane targeting, we constructed another hybrid C2 domain containing the PKC␣C2 CBLs and ␤3-4 hairpin fused to a cPLA 2 C2 scaffold and coupled to CFP (CFPcPLA 2 /PKC_CBLsϩ␤3-4; Figure 1A). In response to Ca 2ϩ increase, CFP-cPLA 2 /PKC_CBLsϩ␤3-4 moved from the cytosol and colocalized completely with YFP-PKC␣C2 at apical puncta ( Figure 6C and Supplementary Figure 6Cvideo).…”

“…Ca 2ϩ binding in the pocket defined by the three CBLs of the PKC␣ C2 domain has long been recognized as critical for the translocation of PKC␣ and the isolated PKC␣ C2 domain to the plasma membrane in cells and for lipid bilayer binding in vitro (Medkova and Cho, 1998;Corbalan-Garcia et al, 1999;Verdaguer et al, 1999;Conesa-Zamora et al, 2001;Stahelin and Cho, 2001;Kohout et al, 2002Kohout et al, , 2003Murray and Honig, 2002). In vitro, and presumably in vivo as well, this translocation involves binding of the Ca 2ϩ -loaded protein to anionic PS headgroups on the membrane surface (Verdaguer et al, 1999;(Evans et al, 2004).…”