Phosphatidylinositol-4,5-bisphosphate enhances anionic lipid demixing by the C2 domain of PKCα

• Published in: PloS one Vol. 9; no. 4; p. e95973
• Main Authors: Egea-Jiménez, Antonio L, Fernández-Martínez, Ana M, Pérez-Lara, Ángel, de Godos, Ana, Corbalán-García, Senena, Gómez-Fernández, Juan C
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 24-04-2014; Public Library of Science (PLoS)
• Subjects: Biology and Life Sciences; Calcium; Calcium - chemistry; Cations, Divalent; Demixing; Deuteration; Enzymes; Fluorescence; Kinases; Lecithin; Lipid rafts; Lipids; Membranes; Membranes, Artificial; NMR; Nuclear magnetic resonance; Phosphatidylcholine; Phosphatidylcholines - chemistry; Phosphatidylethanolamines - chemistry; Phosphatidylinositol 4,5-diphosphate; Phosphatidylinositol 4,5-Diphosphate - chemistry; Phosphatidylserine; Phospholipids; Physical Sciences; Protein kinase C; Protein Kinase C-alpha - chemistry; Protein Structure, Tertiary; Proteins; Quenching; Transition temperature; Transition temperatures; Spain
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0095973

The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca2+, which is interpreted as the demixing of phosphatidylserine from a mixture of this phospholipid with phosphatidylcholine. Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP2) was present in the membrane. When PIP2 was the labeled phospholipid, in the form of TopFluor-PIP2, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine. An independent indication of the phospholipid demixing effect given by the C2α domain was obtained by using 2H-NMR, since a shift of the transition temperature of deuterated phosphatidylcholine was observed as a consequence of the addition of the C2α domain, but only in the presence of PIP2. The demixing induced by the C2α domain may have a physiological significance since it means that the binding of PKCα to membranes is accompanied by the formation of domains enriched in activating lipids, like phosphatidylserine and PIP2. The formation of these domains may enhance the activation of the enzyme when it binds to membranes containing phosphatidylserine and PIP2.