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.
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ABSTRACT: The C2 domain of PKCalpha is a Ca(2+)-dependent membrane-targeting module involved in the plasma membrane localization of the enzyme. Recent findings have shown an additional area located in the beta3-beta4 strands, named the lysine-rich cluster, which has been demonstrated to be involved in the PtdIns(4,5)P(2)-dependent activation of the enzyme. Nevertheless, whether other anionic phospholipids can bind to this region and contribute to the regulation of the enzyme's function is not clear. To study other possible roles for this cluster, we generated double and triple mutants that substituted the lysine by alanine residues, and studied their binding and activation properties in a Ca(2+)/phosphatidylserine-dependent manner and compared them with the wild-type protein. It was found that some of the mutants exerted a constitutive activation independently of membrane binding. Furthermore, the constructs were fused to green fluorescent protein and were expressed in fibroblast cells. It was shown that none of the mutants was able to translocate to the plasma membrane, even in saturating conditions of Ca(2+) and diacylglycerol, suggesting that the interactions performed by this lysine-rich cluster are a key event in the subcellular localization of PKCalpha. Taken together, the results obtained showed that these lysine residues might be involved in two functions: one to establish an intramolecular interaction that keeps the enzyme in an inactive conformation; and the second, once the enzyme has been partially activated, to establish further interactions with diacylglycerol and/or acidic phospholipids, leading to the full activation of PKCalpha.Journal of Molecular Biology 02/2004; 335(4):1117-29. · 3.91 Impact Factor
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ABSTRACT: The C2 domain is a Ca(2+)-binding motif of approximately 130 residues in length originally identified in the Ca(2+)-dependent isoforms of protein kinase C. Single and multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid-second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 domains are regulated by Ca2+, suggesting that some C2 domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 domain and provides a novel sequence alignment of 65 C2 domain primary structures. This alignment predicts that C2 domains form two distinct topological folds, illustrated by the recent crystal structures of C2 domains from synaptotagmin 1 and phosphoinositide-specific phospholipase C-delta 1, respectively. The alignment highlights residues that may be critical to the C2 domain fold or required for Ca2+ binding and regulation.Protein Science 01/1997; 5(12):2375-90. · 2.74 Impact Factor
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ABSTRACT: Infrared spectroscopy (IR) and differential scanning calorimetry (DSC) were used to study the biophysical properties of the PKCepsilon-C2 domain, a C2 domain that possess special characteristics as it binds to acidic phospholipids in a Ca2+-independent manner and no structural information about it is available to date. When the secondary structure was determined by IR spectroscopy in H2O and D2O buffers, beta sheet was seen to be the major structural component. Spectroscopic studies of the thermal denaturation in D2O showed a broadening in the amide I' band starting at 45 degrees C. Curve fitting analysis of the spectra demonstrated that two components appear upon thermal denaturation, one at 1623 cm(-1) which was assigned to aggregation and a second one at 1645 cm(-1), which was assigned to unordered or open loop structures. A lipid binding assay has demonstrated that PKCepsilon-C2 domain has preferential affinity for PIP2 although it exhibits maximal binding activity for phosphatidic acid when 100 mol% of this negatively charged phospholipid was used. Thus, phosphatidic acid containing vesicles were used to characterize the effect of lipid binding on the secondary structure and thermal stability. These experiments showed that the secondary structure did not change upon lipid binding and the thermal stability was very high with no significant changes occurring in the secondary structure after heating. DSC experiments demonstrated that when the C2-protein was scanned alone, it showed a Tm of 49 degrees C and a calorimetric denaturation enthalpy of 144.318 kJ x mol(-1). However, when phoshatidic acid vesicles were included in the mixture, the transition disappeared and further IR experiments demonstrated that the protein structure was not modified under these conditions.European Journal of Biochemistry 03/2001; 268(4):1107-17. · 3.58 Impact Factor