Learning the lipid language of plant signalling

Plant cells respond to different biotic and abiotic stresses by producing various uncommon phospholipids that are believed to play key roles in cell signalling. We can predict how they work because animal and yeast proteins have been shown to have specific lipid-binding domains, which act as docking sites. When such proteins are recruited to the membrane locations where these phospholipids are synthesized, the phospholipids activate them directly, by inducing a conformational change, or indirectly, by juxtaposing them with an activator protein. The same lipid-binding domains are present in Arabidopsis proteins. We believe that they represent an untapped well of information about plant lipid signalling.     

Probing phosphoinositide functions in signaling and membrane trafficking

The inositol phospholipids (PIs) comprise a family of eight species with different combinations of phosphate groups arranged around the inositol ring. PIs are among the most versatile signaling molecules known, with key roles in receptor-mediated signal transduction, actin remodeling and membrane trafficking. Recent studies have identified effector proteins and specific lipid-binding domains through which PIs signal. These lipid-binding domains can be used as probes to further our understanding of the spatial and temporal control of individual PI species. New layers of complexity revealed by the use of such probes include the occurrence of PIs at intracellular locations, the identification of phosphatidylinositol signaling hotspots and the presence of non-membrane pools of PIs in cell nuclei.     

Structure of the binding site for inositol phosphates in a PH domain.

Phosphatidylinositol bisphosphate has been found to bind specifically to pleckstrin homology (PH) domains that are commonly present in signalling proteins but also found in cytoskeleton. We have studied the complexes of the beta-spectrin PH domain and soluble inositol phosphates using both circular dichroism and nuclear magnetic resonance spectroscopy, and X-ray crystallography. The specific binding site is located in the centre of a positively charged surface patch of the domain. The presence of 4,5-bisphosphate group on the inositol ring is critical for binding. In the crystal structure that has been determined at 2.0 A resolution, inositol-1,4,5-trisphosphate is bound with salt bridges and hydrogen bonds through these phosphate groups whereas the 1-phosphate group is mostly solvent-exposed and the inositol ring has virtually no interactions with the protein. We propose a model in which PH domains are involved in reversible anchoring of proteins to membranes via their specific binding to phosphoinositides. They could also participate in a response to a second messenger such as inositol trisphosphate, organizing cross-roads in cellular signalling.     

Genome-wide functional annotation of dual-specificity protein- and lipid-binding modules that regulate protein interactions

Emerging evidence indicates that membrane lipids regulate protein networking by directly interacting with protein-interaction domains (PIDs). As a pilot study to identify and functionally annodate lipid-binding PIDs on a genomic scale, we performed experimental and computational studies of PDZ domains. Characterization of 70 PDZ domains showed that ~40% had submicromolar membrane affinity. Using a computational model built from these data, we predicted the membrane-binding properties of 2,000 PDZ domains from 20 species. The accuracy of the prediction was experimentally validated for 26 PDZ domains. We also subdivided lipid-binding PDZ domains into three classes based on the interplay between membrane- and protein-binding sites. For different classes of PDZ domains, lipid binding regulates their protein interactions by different mechanisms. Functional studies of a PDZ domain protein, rhophilin 2, suggest that all classes of lipid-binding PDZ domains serve as genuine dual-specificity modules regulating protein interactions at the membrane under physiological conditions.     

Signalling through phosphoinositide 3-kinases: the lipids take centre stage

Phosphoinositide 3-kinases (PI3Ks) phosphorylate inositol lipids at the 3' position of the inositol ring to generate the 3-phosphoinositides PI(3)P, PI(3,4) P2 and PI(3,4,5) P3. Recent research has shown that one way in which these lipids function in signal transduction and membrane trafficking is by interacting with 3-phosphoinositide-binding modules in a broad variety of proteins. Specifically, certain FYVE domains bind PI(3)P whereas certain pleckstrin homology domains bind PI(3,4) P2 and/or PI(3,4,5) P3. Also in 1998, PTEN - a major tumour suppressor in human cancer - was also shown to antagonise PI3K signalling by removing the 3-phosphate from 3-phosphoinositides.     

Attracted to membranes: lipid-binding domains in plants

Membranes are essential for cells and organelles to function. As membranes are impermeable to most polar and charged molecules, they provide electrochemical energy to transport molecules across and create compartmentalized microenvironments for specific enzymatic and cellular processes. Membranes are also responsible for guided transport of cargoes between organelles and during endo- and exocytosis. In addition, membranes play key roles in cell signaling by hosting receptors and signal transducers and as substrates and products of lipid second messengers. Anionic lipids and their specific interaction with target proteins play an essential role in these processes, which are facilitated by specific lipid-binding domains. Protein crystallography, lipid-binding studies, subcellular localization analyses, and computer modeling have greatly advanced our knowledge over the years of how these domains achieve precision binding and what their function is in signaling and membrane trafficking, as well as in plant development and stress acclimation.

Lipid-binding domains represent essential motifs within proteins that allow them to bind specific lipids in membranes in a spatial and temporal manner for signaling and trafficking purposes.     

Real-time assay for monitoring membrane association of lipid-binding domains

The C2 domain is a common protein module which mediates calcium-dependent phospholipid binding. Several assays have previously been developed to measure membrane association. However, these assays either have technical drawbacks or are laborious to carry out. We now present a simple solution-based turbidity method for rapidly assaying membrane association of single lipid-binding domains in real time. We used the first C2 domain of synaptotagmin1 (C2A) as a model lipid-binding moiety. Our use of the common dimeric glutathione-S-transferase (GST) fusion tag allowed two C2A domains to be brought into close proximity. Consequently, calcium-triggered phospholipid binding by this artificially dimerized C2A resulted in liposomal aggregation, easily assayed by following absorbance of the solution at 350 nm. The assay is simple and sensitive and can be scaled up conveniently for use in a multiwell plate format, allowing high-throughput screening. In our screens, we identified nickel as a novel activator of synaptotagmin1 C2A domain membrane association. Finally, we show that the turbidity method can be applied to the study of other GST-tagged lipid-binding proteins such as epsin, protein kinase C-β, and synaptobrevin.     

Regulation of protein kinases by lipids

Membranes are sites of intense signaling activity within the cell, serving as dynamic scaffolds for the recruitment of signaling molecules and their substrates. The specific and reversible localization of these signaling molecules to membranes is critical for the appropriate activation of downstream signaling pathways. Phospholipid-binding domains, including C1, C2, PH, and PX domains, play critical roles in the membrane targeting of protein kinases. Recent structural studies have identified a new membrane association domain, the Kinase Associated 1 (KA1) domain, which targets a number of yeast and mammalian protein kinases to membranes containing acidic phospholipids. Despite an abundance of localization studies on lipid-binding proteins and structural studies of the isolated lipid-binding domains, the question of how membrane binding is coupled to the activation of the kinase catalytic domain has been virtually untouched. Recently, structural studies on protein kinase C (PKC) have provided some of the first structural insights into the allosteric regulation of protein kinases by lipid second messengers.     

植物信号传导中的磷脂酶

20世纪 80年代早期人们意识到构成细胞膜的磷脂不只是一道将细胞物质与外界隔开的屏障,而且是细胞对外界环境刺激作出应答的物质基础。磷脂酰肌醇（ｐｈｏｓｐｈｏｔｉｄｙｌｉｎｏｓｉｔｏｌ,ＰＩ）不但是构成细胞膜的重要组分（约占细胞膜组分的 10 %）,在细胞内外环境信号的传递方面也起着重要的作用［1］。磷脂酶（ｐｈｏｓｐｈｏｌｉｐａｓｅ）水解磷脂后产生的三磷酸肌醇 （ｉｎｏｓ ｉｔｏｌｔｒｉｓｐｈｏｓｐｈａｔｅ,ＩＰ3 ）／二酰基甘油（ｄｉａｃｙｌｇｌｙｃｅｒｏｌ,ＤＡＧ）、磷脂酸（ｐｈｏｓｐｈａｔｉｄｉｃａｃｉ…     

The yeast inositol monophosphatase is a lithium- and sodium-sensitive enzyme encoded by a non-essential gene pair

Inositol monophosphatases (IMPases) are lithium-sensitive enzymes that participate in the inositol cycle of calcium signalling and in inositol biosynthesis. Two open reading frames (YHR046c and YDR287w) with homology to animal and plant IMPases are present in the yeast genome. The two recombinant purified proteins were shown to catalyse inositol-1-phosphate hydrolysis sensitive to lithium and sodium. A double gene disruption had no apparent growth defect and was not auxotroph for inositol. Therefore, lithium effects in yeast cannot be explained by inhibition of IMPases and inositol depletion, as suggested for animal systems. Overexpression of yeast IMPases increased lithium and sodium tolerance and reduced the intracellular accumulation of lithium. This phenotype was blocked by a null mutation in the cation-extrusion ATPase encoded by the ENA1/PMR2A gene, but it was not affected by inositol supplementation. As overexpression of IMPases increased intracellular free Ca2+, it is suggested that yeast IMPases are limiting for the optimal operation of the inositol cycle of calcium signalling, which modulates the Ena1 cation-extrusion ATPase.     

The C2 domain calcium-binding motif: structural and functional diversity.

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.     

The lipid-binding SEC14 domain

Protein-lipid interactions are important for protein targeting, signal transduction, lipid transport, lipid biosynthesis, lipid metabolism, and the maintenance of cellular compartments and membranes. Specific lipid-binding protein domains, such as PH, FYVE, PX, PHD, C2 and SEC14 homology domains, mediate interactions between proteins and specific phospholipids. Here we review the published literature, plus some of our most recent unpublished findings, regarding the biology of the SEC14 domain, also known as CRAL_TRIO domain.     

Homologous desensitization of signalling by the beta (beta) isoform of the human thromboxane A2 receptor

Thromboxane (TX) A(2) is a potent stimulator of platelet activation/aggregation and smooth muscle contraction and contributes to a variety of pathologies within the vasculature. In this study, we investigated the mechanism whereby the cellular responses to TXA(2) mediated through the TPbeta isoform of the human TXA(2) receptor (TP) are dynamically regulated by examining the mechanism of agonist-induced desensitization of intracellular signalling and second messenger generation by TPbeta. It was established that TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA(2) mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser(145) within intracellular domain (IC)(2) has been identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser(239) and Ser(357) within its IC(3) and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser(239) or Ser(357), through site directed mutagenesis, impaired desensitization while mutation of both Ser(239) and Ser(357) almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser(145) partially impairs. TPbeta signalling while GRK2/3 phosphorylation at both Ser(239) and Ser(357) within its IC(3) and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization. Thromboxane (TX) A(2) is a potent stimulator of platelet aggregation and smooth muscle contraction and contributes to a variety of vascular pathologies. Herein the mechanism whereby the cellular responses to TXA(2) mediated through the TPbeta isoform of the human TXA(2) receptor (TP) are dynamically regulated was investigated by examining the mechanism of its agonist-induced desensitization of intracellular signalling and second messenger generation. TPbeta is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA(2) mimetic U46619 and this occurs through two key mechanisms: TPbeta undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser(145) within intracellular domain (IC)(2) was identified as the key phospho-target. In addition, TPbeta also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled receptor kinase (GRK)2/3-phosphorylation of both Ser(239) and Ser(357) within its IC(3) and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser(239) or Ser(357), through site directed mutagenesis, impaired desensitization while mutation of both Ser(239) and Ser(357) almost completely abolished desensitization of signalling, GRK phosphorylation and beta-arrestin association, thereby blocking TPbeta internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser(145) partially impairs TPbeta signalling while GRK2/3 phosphorylation at both Ser(239) and Ser(357) within its IC(3) and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes beta-arrestin association and, in turn, facilitates TPbeta internalization.     

Epidermal growth factor alters metabolism of inositol lipids and activity of protein kinase C in mouse embryo palate mesenchyme cells

Epidermal growth factor (EGF) stimulated mouse embryo palate mesenchyme (MEPM) cells (1) to incorporate [32P]O4(3-) into phosphatidylinositol (PI), phosphatidylcholine, and phosphatidic acid over a period of 60 min; 2) to incorporate [32P]O4(3-) into polyphosphoinositides as a function of time; and 3) to incorporate [32P]O4(-3) into PI, only, as a function of concentration when the period of stimulation was kept short. EGF stimulated the release of radiolabeled inositol phosphates from MEPM cells that had been radiolabeled with [3H]myoinositol. The release of inositol 1-phosphate was sustained over a period of at least 60 min, whereas the release of inositol 1,4-bisphosphate and inositol trisphosphate peaked during the first 10 min of stimulation. EGF also stimulated phosphorylation of an Mr 80,000 protein whose pI, phosphopeptide map, and phosphoamino acid pattern were identical to those of an Mr 80,000 protein phosphorylated in response to phorbol 12-myristate 13-acetate. Mobilization or metabolism of arachidonic acid was not stimulated under the same conditions that permitted EGF to alter inositol lipid metabolism. We interpret these data to mean that 1) in contrast to the findings with some cell lines, alterations in inositol lipid metabolism may be part of the signalling mechanism for EGF in embryonic cells; 2) EGF is capable of activating inositol-dependent signalling pathways leading to activation of protein kinase C in MEPM cells; and 3) mobilization and metabolism of arachidonic acid are not an inherent part of this signalling mechanism.     

HIV gp41 C-terminal heptad repeat contains multifunctional domains. Relation to mechanisms of action of anti-HIV peptides

T20 (Fuzeon), a novel anti-human immunodeficiency virus (HIV) drug, is a peptide derived from HIV-1 gp41 C-terminal heptad repeat (CHR). Its mechanism of action has not yet been defined. We applied Pepscan strategy to determine the relationship between functional domains and mechanisms of action of five 36-mer overlapping peptides with a shift of five amino acids (aa): CHR-1 (aa 623-658), C36 (aa 628-663), CHR-3 (aa 633-668), T20 (aa 638-673), and CHR-5 (aa 643-678). C36 is a peptide with addition of two aa to the N terminus of C34. Peptides CHR-1 and C36 contain N-terminal heptad repeat (NHR)- and pocket-binding domains. They inhibited HIV-1 fusion by interacting with gp41 NHR, forming stable six-helix bundles and blocking gp41 core formation. Peptide T20 containing partial NHR- and lipid-binding domains, but lacking pocket-binding domain, blocked viral fusion by binding its N- and C-terminal sequences with gp41 NHR and cell membrane, respectively. Peptide CHR-3, which is located in the middle between C36 and T20, overlaps >86% of the sequences of these two peptides, and lacks pocket- and lipid-binding domains, exhibited marginal anti-HIV-1 activity. These results suggest that T20 and C36 contain different functional domains, through which they inhibit HIV-1 entry with distinct mechanisms of action. The multiple functional domains in gp41 CHR and their binding partners may serve as targets for rational design of new anti-HIV-1 drugs and vaccines.     

Identification of 3- and 4-phosphorylated phosphoinositides and inositol phosphates in stomatal guard cells

Components of the polyphosphoinositide signalling pathway have been identified in stomatal guard cells of Commelina communis L., one of the few plant systems shown unequivocally to be capable of responding to release of inositol 1,4,5-trisphosphate in the cytoplasm by increase in cytoplasmic Ca²⁺. 'Isolated' epidermal strips of C. communis (in which all cells other than guard cells have been killed by treatment at low pH) were radiolabelled with myo -[2n-³H]inositol or [³²P]orthophosphate for 17–18 h. The phosphoinositides and inositol phosphates were extracted. Phosphoinositides were deacylated and the head groups resolved by HPLC. The water-soluble products generated by mild periodate cleavage of HPLC-purified, deacylated lipid fractions were examined. The resulting biochemical analysis led to the identification of: PtdIns, PtdIns3 P , PtdIns4 P , PtdIns(3,4) P ₂ and PtdIns(4,5) P ₂. Thex inositol phosphates were resolved by HPLC. Preliminary analysis of HPLC-purified putative inositol phosphate fractions resulted in the identification of each inositol phosphate class, that is, Ins P , Ins P ₂, Ins P ₃, Ins P ₄, Ins P ₅ and Ins_P6. Many of these inositol phosphates occurred in different isomeric forms. The presence of 3-phosphorylated phosphoinositides suggests that they may have a role in signalling in stomatal guard cells.     

Novel PSI Domains in Plant and Animal H+‐Inositol Symporters

Inositols are indispensable components of cellular signaling molecules, and impaired cytoplasmic inositol concentrations affect cellular development. Although most cells can synthesize inositol de novo, plasma membrane‐localized inositol uptake systems are indispensable for normal development. Here, we present in‐depth functional analyses of plasma membrane‐localized H⁺‐inositol symporters from human and from the higher plant Arabidopsis thaliana. Sequence comparisons, structural and phylogenetic analyses revealed that these transporters possess conserved extracellular loop domains that represent homologs of plexins/semaphorin/integrin (PSI) domains from animal type I receptors. In these receptors, PSI domains modulate intracellular signaling via extracellular protein–protein interactions. Comparisons of H⁺‐inositol symporters with wild type, mutated and truncated PSI domains in different expression systems showed that removal of the entire loop domain increased the V_max of inositol uptake. Finally, we show that the PSI domains are targets for Ni⁺⁺ ions that cause a complete loss of transport activity. A possible role of Ni⁺⁺‐binding to PSI domains in Ni⁺⁺‐induced carcinogenicity is discussed.