C2 Domains of Protein Kinase C Isoforms , , and <
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- 作者：Susy C. Kohout ; Senena Corbalá ; n-Garcí ; a ; Alejandro Torrecillas ; Juan C. Gomé ; z-Fernandé ; z ; and Joseph J. Falke
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：September 24, 2002
- 年：2002
- 卷：41
- 期：38
- 页码：11411 - 11424
- 全文大小：395K
- 年卷期：v.41,no.38(September 24, 2002)
- ISSN：1520-4995
文摘
The independently folding C2 domain motif serves as a Ca2+-dependent membrane dockingtrigger in a large number of Ca2+ signaling pathways. A comparison was initiated between three closelyrelated C2 domains from the conventional protein kinase C subfamily (cPKC, isoforms 
, 
, and 
). Theresults reveal that these C2 domain isoforms exhibit some similarities but are specialized in importantways, including different Ca2+ stoichiometries. In the absence of membranes, Ca2+ affinities of the isolatedC2 domains are similar (2-fold difference) while Hill coefficients reveal cooperative Ca2+ binding for thePKC C2 domain but not for the PKC or PKC C2 domain (H = 2.3 ± 0.1 for PKC, 0.9 ± 0.1 forPKC, and 0.9 ± 0.1 for PKC). When phosphatidylserine-containing membranes are present, Ca2+affinities range from the sub-micromolar to the micromolar (7-fold difference) ([Ca2+]1/2 = 0.7 ± 0.1 
Mfor PKC, 1.4 ± 0.1 M for PKC, and 5.0 ± 0.2 M for PKC), and cooperative Ca2+ binding isobserved for all three C2 domains (Hill coefficients equal 1.8 ± 0.1 for PKC, 1.3 ± 0.1 for PKC, and1.4 ± 0.1 for PKC). The large effects of membranes are consistent with a coupled Ca2+ and membranebinding equilibrium, and with a direct role of the phospholipid in stabilizing bound Ca2+. The net negativecharge of the phospholipid is more important to membrane affinity than its headgroup structure, althougha slight preference for phosphatidylserine is observed over other anionic phospholipids. The Ca2+stoichiometries of the membrane-bound C2 domains are detectably different. PKC and PKC each bindthree Ca2+ ions in the membrane-associated state; membrane-bound PKC binds two Ca2+ ions, and athird binds weakly or not at all under physiological conditions. Overall, the results indicate that conventionalPKC C2 domains first bind a subset of the final Ca2+ ions in solution, and then associate weakly with themembrane and bind additional Ca2+ ions to yield a stronger membrane interaction in the fully assembledtertiary complex. The full complement of Ca2+ ions is needed for tight binding to the membrane. Thus,even though the three C2 domains are 64% identical, differences in Ca2+ affinity, stoichiometry, andcooperativity are observed, demonstrating that these closely related C2 domains are specialized for theirindividual functions and contexts.
, , and ). Theresults reveal that these C2 domain isoforms exhibit some similarities but are specialized in importantways, including different Ca2+ stoichiometries. In the absence of membranes, Ca2+ affinities of the isolatedC2 domains are similar (2-fold difference) while Hill coefficients reveal cooperative Ca2+ binding for thePKC C2 domain but not for the PKC or PKC C2 domain (H = 2.3 ± 0.1 for PKC, 0.9 ± 0.1 forPKC, and 0.9 ± 0.1 for PKC). When phosphatidylserine-containing membranes are present, Ca2+affinities range from the sub-micromolar to the micromolar (7-fold difference) ([Ca2+]1/2 = 0.7 ± 0.1 Mfor PKC, 1.4 ± 0.1 M for PKC, and 5.0 ± 0.2 M for PKC), and cooperative Ca2+ binding isobserved for all three C2 domains (Hill coefficients equal 1.8 ± 0.1 for PKC, 1.3 ± 0.1 for PKC, and1.4 ± 0.1 for PKC). The large effects of membranes are consistent with a coupled Ca2+ and membranebinding equilibrium, and with a direct role of the phospholipid in stabilizing bound Ca2+. The net negativecharge of the phospholipid is more important to membrane affinity than its headgroup structure, althougha slight preference for phosphatidylserine is observed over other anionic phospholipids. The Ca2+stoichiometries of the membrane-bound C2 domains are detectably different. PKC and PKC each bindthree Ca2+ ions in the membrane-associated state; membrane-bound PKC binds two Ca2+ ions, and athird binds weakly or not at all under physiological conditions. Overall, the results indicate that conventionalPKC C2 domains first bind a subset of the final Ca2+ ions in solution, and then associate weakly with themembrane and bind additional Ca2+ ions to yield a stronger membrane interaction in the fully assembledtertiary complex. The full complement of Ca2+ ions is needed for tight binding to the membrane. Thus,even though the three C2 domains are 64% identical, differences in Ca2+ affinity, stoichiometry, andcooperativity are observed, demonstrating that these closely related C2 domains are specialized for theirindividual functions and contexts.