The C2 Domain of PKCα Is a Ca2+-dependent PtdIns(4,5)P2 Sensing Domain: A New Insight into an Old Pathway

ArticleinJournal of Molecular Biology 362(5):901-14 · November 2006with6 Reads
DOI: 10.1016/j.jmb.2006.07.093 · Source: PubMed
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.
    • "Unlike lipid binding PH domains [62], C2 lipid targeting often involves two recognition components, such as two lipids or a lipid/protein combination . For instance the protein kinase C (PKC) C2 domain uses its basic surface residues to bind plasma membrane PS and PI(4,5)P2 [63], while cytosolic phospholipase A2 (cPLA2) binds to the neutral lipid phosphatidylcholine (PC) and the anionic lipid ceramide-1-phos- phate (C1P) through C2 domain Ca 2+ site charged hydrophobic side chains and a basic cluster [64]. Synaptotagmin utilizes two C2 domains to bridge the vesicular and plasma membranes, with the C2A domain binding vesicular PS and SNARE, while the C2B domain binds plasma membrane PI(4,5)P2 and SNARE [65, 66]. "
    [Show abstract] [Hide abstract] ABSTRACT: EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation.
    Full-text · Article · Jun 2016
    • "This is probably due to the lower sensitivity of the NMR technique for this type of event. We hypothesize that the reason why POPS demixing by the addition of C2a was greatly increased by the presence of PIP 2 could be due to the great increase in the protein binding to the membrane that is observed when PIP 2 is added to a POPC/POPS membrane [15,18,27] and to the different docking of the C2a protein in the presence of PIP 2 , which has been shown to change with respect to a membrane without this phosphoinositide, by both ATR-infrared spectroscopy [41] and EPR site-directed spinlabeling and relaxation [42]. The physiological significance of the observed anionic lipid demixing can be suggested, since the concentrations of phospholipids used in these experiments are close to the concentrations molar ratio); POPC/POPC-d 31 /POPS/PIP 2 (35:40:20:5, molar ratio); POPC/ POPS-d 31 /PIP 2 (75:20:5, molar ratio and POPC/POPS-d 31 /PIP 2 (75:20:5, molar ratio) in the presence of C2a domain (40:1 phospholipid/protein molar ratio). "
    [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.
    Full-text · Article · Apr 2014
    • "One of the main sources of diacylglycerol in the plasma membrane following cell stimulation is PIP 2 which is hydrolyzed by phospholipase C to produce diacylglycerol and inositol 1,4,5-trisphosphate, which together activate protein kinase C for sustained cellular responses [2]. However, it has been shown that PIP 2 may also activate PKCa by direct binding to a polylysine motif located in strands b3 and b434567 and that can be considered a specific site for PIP 2 [8] (seeFig. 1). "
    [Show abstract] [Hide abstract] ABSTRACT: The C2 domain of PKCα possesses two different binding sites, one for Ca(2+) and phosphatidylserine and a second one that binds PIP2 with very high affinity. The enzymatic activity of PKCα was studied by activating it with large unilamellar lipid vesicles, varying the concentration of Ca(2+) and the contents of dioleylglycerol (DOG), phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphadidylserine (POPS) in these model membranes. The results showed that PIP2 increased the Vmax of PKCα and, when the PIP2 concentration was 5 mol% of the total lipid in the membrane, the addition of 2 mol% of DOG did not increase the activity. In addition PIP2 decreases K0.5 of Ca(2+) more than 3-fold, that of DOG almost 5-fold and that of POPS by a half. The K0.5 values of PIP2 amounted to only 0.11 µM in the presence of DOG and 0.39 in its absence, which is within the expected physiological range for the inner monolayer of a mammalian plasma membrane. As a consequence, PKCα may be expected to operate near its maximum capacity even in the absence of a cell signal producing diacylglycerol. Nevertheless, we have shown that the presence of DOG may also help, since the K0.5 for PIP2 notably decreases in its presence. Taken together, these results underline the great importance of PIP2 in the activation of PKCα and demonstrate that in its presence, the most important cell signal for triggering the activity of this enzyme is the increase in the concentration of cytoplasmic Ca(2+).
    Full-text · Article · Jul 2013
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