The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway.
ABSTRACT The C2 domain is a targeting domain that responds to intracellular Ca2+ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P2 specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca2+ specifically binds to the Ca2+ -binding region, facilitating PtdIns(4,5)P2 access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P2 binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P2 binding are essential for the plasma membrane localization of PKCalpha when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P2 and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P2 is hydrolyzed to generate diacylglycerol and IP3, an important amount still remains in the membrane where it is available to activate PKCalpha. These findings entail revision of the currently accepted model of PKCalpha recruitment to the membrane and its activation.
- SourceAvailable from: Juan C Gómez-Fernández[Show abstract] [Hide abstract]
ABSTRACT: 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.PLoS ONE 04/2014; 9(4):e95973. · 3.53 Impact Factor
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ABSTRACT: C2 domains are membrane-binding modules that share a common overall fold: a single compact greek-key motif organized as an eight-stranded anti-parallel β-sandwich consisting of a pair of four-stranded β-sheets. A myriad of studies have demonstrated that in spite of sharing the common structural β-sandwich core, slight variations in the residues located in the interconnecting loops confer C2 domains with functional abilities to respond to different Ca(2+) concentrations and lipids, and to signal through protein-protein interactions as well. This review summarizes the main structural and functional findings on Ca(2+) and lipids interactions by C2 domains, including the discovery of the phosphoinositides-binding site located in the β3-β4 strands. The wide variety of functions, together with the different Ca(2+) and lipids affinities of these domains converts this superfamily in a crucial player in many functions in the cell and more to be discovered. This article is part of a Special Issue entitled: Membrane structure and function: Relevance in the cell's physiology, pathology and therapy.Biochimica et Biophysica Acta 01/2014; · 4.66 Impact Factor
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ABSTRACT: The C1 domains of novel PKCs mediate the diacylglycerol-dependent translocation of these enzymes. The four different C1B domains of novel PKCs (δ, ε, θ and η) were studied, together with different lipid mixtures containing acidic phospholipids and diacylglycerol or phorbol ester. The results show that either in the presence or in the absence of diacylglycerol, C1Bε and C1Bη exhibit a substantially higher propensity to bind to vesicles containing negatively charged phospholipids than C1Bδ and C1Bθ. The observed differences between the C1B domains of novel PKCs (in two groups of two each) were also evident in RBL-2H3 cells and it was found that, as with model membranes, in which C1Bε and C1Bη could be translocated to membranes by the addition of a soluble phosphatidic acid without diacylglycerol or phorbol ester, C1Bδ and C1Bθ were not translocated when soluble phosphatidic acid was added, and diacylglycerol was required to achieve a detectable binding to cell membranes. It is concluded that two different subfamilies of novel PKCs can be established with respect to their propensity to bind to the cell membrane and that these peculiarities in recognizing lipids may explain why these isoenzymes are specialized in responding to different triggering signals and bind to different cell membranes.Biochimica et Biophysica Acta 04/2014; · 4.66 Impact Factor