期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 371, 期 3, 页码 608-621出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2007.05.086
关键词
PKC; PtdIns(4,5)P-2; C2 domain; GFP; phosphatidylserine
C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca2+-dependent manner and thereby act as cellular Ca2+ effectors. Recent findings suggest that the C2 domain of PKC alpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P-2) through its lysine rich cluster, for which it shows higher affinity than for POPS. In this work, we compared the three C2 domains of classical PKCs. Isothermal titration calorimetry revealed that the C2 domains of PKC alpha and beta display a greater capacity to bind to PtdIns(4,5)P-2-containing vesicles than the C2 domain of PKC gamma. Comparative studies using lipid vesicles containing both POPS and PtdIns(4,5)P-2 as ligands revealed that the domains behave as PtdIns(4,5)P-2-binding modules rather than as POPS-binding modules, suggesting that the presence of the phosphoinositide in membranes increases the affinity of each domain. When the magnitude of PtdIns(4,5)P-2 binding was compared with that of other poly-phosphate phosphatidylinositols, it was seen to be 2 greater in both PKC beta- and PKC gamma-C2 domains. The concentration of Ca2+ required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P2 for all C2 domains, especially PKC alpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with 2, ATP demonstrated that, at limiting intracellular concentration of Ca2+, the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKC alpha persisting for the longest time in the plasma membrane, followed by PKC gamma and, finally, PKC beta, which probably reflects the different levels of Ca2+ needed by each domain and their different affinities for PtdIns(4,5)P-2. (c) 2007 Published by Elsevier Ltd.
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