title: C2 domains of protein kinase C isoforms alpha, beta, and gamma: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
authors.forename: Susy C
authors.initials: SC
authors.lastname: Kohout
authors.forename: Senena
authors.initials: S
authors.lastname: Corbalán-García
authors.forename: Alejandro
authors.initials: A
authors.lastname: Torrecillas
authors.forename: Juan C
authors.initials: JC
authors.lastname: Goméz-Fernandéz
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D018360:Crystallography, X-Ray; D005982:Glutathione Transferase / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014508:Urea
chemical_list: D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D014508:Urea; D005982:Glutathione Transferase; C087857:protein kinase C gamma; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D002118:Calcium