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Objective: Gαh (tissue transglutaminase; TGII), known as the α1B-adrenoceptor signal mediator, is a bifunctional enzyme possessing transglutaminase and guanosine triphosphatase activities. The microtubule-associated protein tau plays a role in formation of neurofibrillary tangles in Alzheimer's disease (AD) brain. Tau is also known as a substrate of transglutaminase. We aimed to better understand the correlation between α1B-adrenoceptor signaling and AD. Methods: We examined not only the cross-linking ability of TGII for tau, but also the expression level of tau as well as α1B-adrenoceptor signaling molecules, TGII and phospholipase C-δ1, in the human brain. Results: When the tau protein was assayed as a transglutaminase substrate of TGII, tau proteins formed cross-linked products. However, phospholipase C-δ1 inhibited transglutaminase activity in TGII to cross-link with tau in vitro. The amount of expressed mRNA in AD brain tissue was elevated 2~10 fold for tau and 3~20 fold for TGII. Consistent with these observations, the densities of expressed proteins in AD brain tissue also increased 9 fold for tau and 15 fold for TGII. Moreover, phospholipase C-δ1, which is a negative regulator for transglutaminase activity of TGII, also increased 2~25 fold for mRNA as well as 8 fold for protein in AD brain tissue. In contrast, expressed mRNA and protein activity for α1B-adrenoceptor were almost the same between AD and normal brains. Conclusion: These results suggest that the α1B-adrenoceptor signal mediator, TGII and tau, as a substrate of TGII might have a pathophysiological role in AD, but this correlation may be independent of the α1B-adrenoceptor signaling system, including α1B-adrenoceptor and phospholipase C-δ1. [ABSTRACT FROM AUTHOR]

Abstract: Protein activities are generally regulated by intramolecular allosteric interactions, by which spatially separated sites in a protein molecule communicate. Intramolecular allosteric interactions in the phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain were investigated by solution NMR spectroscopy for selectively [α-15N]Lys-labeled proteins. The results of NMR analyses indicated that the binding of inositol 1,4,5-trisphosphate (IP3) to the protein induces local environmental changes at all lysine residues, including residues such as Lys-43 spatially separated from the specific IP3 binding site consisting of Lys-30, Lys-32, and Lys-57. IP3 binding also induces conformational stabilization of a characteristic short α-helix (α2) from residues 82 to 87. Mutational analyses indicated that an interaction network mainly consisting of the side chains of Lys-30, Lys-32, and Lys-43 exists in the ligand-free protein, and it was therefore predicted that binding of IP3 to the specific site modifies the interaction network, resulting in formation of a new interaction network, in which the side chains of Lys-57 and Phe-87 contribute to stable IP3 binding. These results provide evidence for intramolecular interactions in the PLC-δ1 PH domain, the function of which could be allosterically regulated by modifications at sites spatially separated from the ligand-binding site through the intramolecular interaction network. [Copyright &y& Elsevier]

Abstract: The phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain has a characteristic short α-helix (α2) from residues 82 to 87. The contributions of the α2-helix toward the inositol 1,4,5-trisphosphate (IP3) binding activity and thermal stability of the PLC-δ1 PH domain were investigated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE analyses of gel migration shift induced by IP3 binding and of protein aggregation induced by heating indicated that disruption of the α-helical conformation by replacement of Lys86 with proline resulted in reduced affinity for IP3 and in thermal destabilization of the IP3-binding state. Although the mutant protein with replacement of Lys86 with alanine showed a slight reduction in thermal stability, the IP3-binding affinity was similar to that of the wild-type protein. Replacement of Phe87 with alanine, but not with tyrosine, also resulted in reduced affinity for IP3 and in thermal instability. These results indicated that the helical conformation of the α2-helix and the phenyl ring of Phe87 play important roles in the IP3-binding activity and thermal stability of the PLC-δ1 PH domain. Based on these results, the biological role of the α2-helix of the PLC-δ1 PH domain is discussed in terms of membrane binding. [Copyright &y& Elsevier]

Abstract: Phospholipase C-δ1 (PLCδ1) is a widely expressed highly active PLC isoform, modulated by Ca2+ that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLCδ1 modulated expression of the pro-inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLCδ1 was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLCδ1 knockdown enhanced expression IL-1β and tumor necrosis factor-α (TNF-α) mRNA by at least 100 fold after 4h of LPS stimulation compared to control siRNA treatment. PLCδ1 knock down caused persistently high Nfκb levels at 4h of LPS stimulation compared to control siRNA-treated cells. PLCδ1 knockdown was also associated with elevated nuclear levels of c-Jun after 30min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1β and TNF-α mRNA’s induced by PLCδ1 knockdown. Our results show that loss of PLCδ1 enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nfκb pathway. Our findings are consistent with the idea that PLCδ1 is a suppressor of PKC activity. [Copyright &y& Elsevier]

Abstract: Phospholipase C-δ1 (PLCδ1) is the most fundamental form of the eukaryotic PLC and thought to play important roles in the regulation of cells. We previously reported that PLCδ1 shuttles between the cytoplasm and nucleus, and an influx of Ca2+ triggers the nuclear import of PLCδ1 via Ca2+-dependent interaction with importin β1, although the physiological meaning of this is unclear. Here we have examined the distribution of PLCδ1 using primary cultures of rat hippocampal neurons. Treatment of 7DIV neurons with ionomycin or thapsigargin caused the nuclear localization of PLCδ1 as has been observed in other cell lines. Similar results were obtained with neurons treated with glutamate, suggesting that the nuclear localization of PLCδ1 plays some roles in excitotoxicity associated with ischemic stress. Generally, cells undergoing ischemic or hypoxic cell death show nuclear shrinkage. We confirmed that a massive influx of Ca2+ caused similar results. Furthermore, overexpression of GFP-PLCδ1 facilitated ionomycin-induced nuclear shrinkage in embryonic fibroblasts derived from PLCδ1 gene-knockout mice (PLCδ1KO-MEF). By contrast, an E341A mutant that cannot bind with importin β1 and be imported into the nucleus by ionomycin and also lacks enzymatic activity did not cause nuclear shrinkage in PLCδ1KO-MEF. Nuclear translocation and the PLC activity of PLCδ1, therefore, may regulate the nuclear shape by controlling the nuclear scaffold during stress-induced cell death caused by high levels of Ca2+. [Copyright &y& Elsevier]

Abstract: The effects of geometric properties of membranes on the structure of the phospholipase C-δ1 (PLC-δ1) pleckstrin homology (PH) domain were investigated using solid state 13C NMR spectroscopy. Conformations of the PLC-δ1 PH domain at the surfaces of multilamellar vesicles (MLV), small unilamellar vesicles (SUV), and micelles were examined to evaluate the effects of membrane curvature on the PH domain. An increase in curvature of the water–hydrophobic layer interface hinders membrane-penetration of the amphipathic α2-helix of the PH domain that assists the membrane-association of the PH domain dominated by the phosphatidylinositol 4,5-bisphosphate (PIP2) specific lipid binding site. The solid state 13C NMR signal of Ala88 located at the α2-helix indicates that the conformation of the α2-helix at the micelle surface is similar to the solution conformation and significantly different from those at the MLV and SUV surfaces which were characterized by membrane-penetration and re-orientation. The signal of Ala112 which flanks the C-terminus of the β5/β6 loop that includes the α2-helix, showed downfield displacement with decrease in the interface curvature of the micelles, SUV and MLV. This reveals that the conformation of the C-terminus of the β5/β6 loop connecting the β-sandwich core containing the PIP2 binding site and the amphipathic α2-helix is sensitive to alterations of the curvature of lipid bilayer surface. It is likely that these alterations in the conformation of the PLC-δ1 PH domain contribute to the regulatory mechanisms of the intracellular localization of PLC-δ1 in a manner dependent upon the structure of the molecular complex containing PIP2. [Copyright &y& Elsevier]

Abstract: START-GAP2, also termed as DLC2, is a START domain-containing RhoGAP and a negative regulator of RhoA and Cdc42. Although it was reported as a tumor suppresser gene product, the molecular basis for function of START-GAP2 remains to be clarified. Here, we demonstrate that START-GAP2 is localized in focal adhesions through a “FAT (focal adhesion targeting)” region in the N-terminal half. START-GAP2 competes with START-GAP1/DLC1, another START domain-containing RhoGAP, in focal adhesion targeting. Moreover, the C-terminus of tensin2, one of focal adhesion components and reported to bind START-GAP1, also directly interacts with START-GAP2. These results suggest that START-GAP2 and START-GAP1 share the same molecular mechanism in targeting to focal adhesions. [Copyright &y& Elsevier]

The membrane binding affinity of the pleckstrin homology (PH) domain of phospholipase C (PLC)-δ1 was investigated using a vesicle coprecipitation assay and the structure of the membrane-associated PH domain was probed using solid-state 13C NMR spectroscopy. Twenty per cent phosphatidylserine (PS) in the membrane caused a moderate but significant reduction of the membrane binding affinity of the PH domain despite the predicted electrostatic attraction between the PH domain and the head groups of PS. Solid-state NMR spectra of the PH domain bound to the phosphatidylcholine (PC)/PS/phosphatidylinositol 4,5-bisphosphate (PIP2) (75 : 20 : 5) vesicle indicated loss of the interaction between the amphipathic α2-helix of the PH domain and the interface region of the membrane which was previously reported for the PH domain bound to PC/PIP2 (95 : 5) vesicles. Characteristic local conformations in the vicinity of Ala88 and Ala112 induced by the hydrophobic interaction between the α2-helix and the membrane interface were lost in the structure of the PH domain at the surface of the PC/PS/PIP2 vesicle, and consequently the structure becomes identical to the solution structure of the PH domain bound tod- myo-inositol 1,4,5-trisphosphate. These local structural changes reduce the membrane binding affinity of the PH domain. The effects of PS on the PH domain were reversed by NaCl and MgCl2, suggesting that the effects are caused by electrostatic interaction between the protein and PS. These results generally suggest that the structure and function relationships among PLCs and other peripheral membrane proteins that have similar PH domains would be affected by the local lipid composition of membranes. [ABSTRACT FROM AUTHOR]

Ischemia and simulated ischemic conditions cause intracellular Ca2+ overload in the myocardium. The relationship between ischemia injury and Ca2+ overload has not been fully characterized. The aim of the present study was to investigate the expression and characteristics of PLC isozymes in myocardial infarction-induced cardiac remodeling and heart failure. In normal rat heart tissue, PLC-δ1 (about 44 ng/mg of heart tissue) was most abundant isozymes compared to PLC-γ1 (6.8 ng/mg) and PLC-β1 (0.4 ng/mg). In ischemic heart and hypoxic neonatal cardiomyocytes, PLC-δ1, but not PLC-β1 and PLC-γ1, was selectively degraded, a response that could be inhibited by the calpain inhibitor, calpastatin, and by the caspase inhibitor, zVAD-fmk. Overexpression of the PLC-δ1 in hypoxic neonatal cardiomyocytes rescued intracellular Ca2+ overload by ischemic conditions. In the border zone and scar region of infarcted myocardium, and in hypoxic neonatal cardiomyocytes, the selective degradation of PLC-δ1 by the calcium sensitive proteases may play important roles in intracellular Ca2+ regulations under the ischemic conditions. It is suggested that PLC isozyme-changes may contribute to the alterations in calcium homeostasis in myocardial ischemia. [Copyright &y& Elsevier]