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. DOI:10.1371/journal.pone.0095973 · 3.53 Impact Factor
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ABSTRACT: Protein kinase C alpha (PKCα) is a member of the conventional family of protein kinase C isoforms (cPKCs) that regulate diverse cellular signaling pathways, share a common activation mechanism, and are linked to multiple pathologies. The cPKC domain structure is modular, consisting of an N-terminal pseudosubstrate peptide, two inhibitory domains (C1A and C1B), a targeting domain (C2), and a kinase domain. Mature, cytoplasmic cPKCs are inactive until switched on by a multi-step activation reaction that occurs largely on the plasma membrane surface. Often this activation begins with a cytoplasmic Ca2+ signal that triggers C2 domain targeting to the plasma membrane where it binds phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PIP2). Subsequently, the appearance of the signaling lipid diacylglycerol (DAG) activates the membrane-bound enzyme by recruiting the inhibitory pseudosubstrate and one or both C1 domains away from the kinase domain. To further investigate this mechanism, the present study has utilized single molecule TIRF microscopy to quantitate the binding and lateral diffusion of full-length PKCα and fragments missing specific domain(s) on supported lipid bilayers. Lipid binding events, and events inserting additional protein into the bilayer, were detected by their effects on the equilibrium bound particle density and the 2-D diffusion rate. In addition to the previously proposed activation steps, the findings reveal a major, undescribed, kinase-inactive intermediate. On bilayers containing PS or PS+PIP2, the full-length PKCα first docks to the membrane via its C2 domain, then its C1A domain embeds in the bilayer even before DAG appears. The resulting pre-DAG intermediate with membrane-bound C1A and C2 domains is the predominant state of PKCα while awaiting the DAG signal. The newly detected, membrane-embedded C1A domain of this pre-DAG intermediate confers multiple useful features including enhanced membrane affinity and longer bound state lifetime. The findings also identify the key molecular step in kinase activation: since C1A is already membrane-embedded in the kinase off-state, recruitment of C1B to the bilayer by DAG or phorbol ester is the key regulatory event that stabilizes the kinase on-state. More broadly, the study illustrates the power of single molecule methods to elucidate the activation mechanisms and hidden regulatory states of membrane-bound signaling proteins.Biochemistry 02/2014; 53(10). DOI:10.1021/bi4016082 · 3.38 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; 1838(7). DOI:10.1016/j.bbamem.2014.04.003 · 4.66 Impact Factor