The C2 domains of classical PKCs are specific PtdIns(4,5)P2-sensing domains with different affinities for membrane binding |
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Authors: | Guerrero-Valero Marta Marín-Vicente Consuelo Gómez-Fernández Juan C Corbalán-García Senena |
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Affiliation: | Departamento de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30100-Murcia, Spain. |
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Abstract: | 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 Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha 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 PKCalpha and beta display a greater capacity to bind to PtdIns(4,5)P(2)-containing vesicles than the C2 domain of PKCgamma. 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 polyphosphate phosphatidylinositols, it was seen to be greater in both PKCbeta- and PKCgamma-C2 domains. The concentration of Ca(2+) required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P(2) for all C2 domains, especially PKCalpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with ATP demonstrated that, at limiting intracellular concentration of Ca(2+), the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKCalpha persisting for the longest time in the plasma membrane, followed by PKCgamma and, finally, PKCbeta, which probably reflects the different levels of Ca(2+) needed by each domain and their different affinities for PtdIns(4,5)P(2). |
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Keywords: | dDHPE, N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine ECFP, enhanced cyan fluorescent protein FRET, fluorescence resonance of energy transfer HBS, Hepes buffer saline ITC, isothermal titration calorimetry PKC, protein kinase C POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine PtdIns(4,5)P2, phosphatidylinositols 4,5-bisphosphate SUV, small unilamellar vesicle |
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