Phosphoinositide switches in cell physiology - From molecular mechanisms to disease.

Phosphoinositides are amphipathic lipid molecules derived from phosphatidylinositol that represent low abundance components of biological membranes. Rather than serving as mere structural elements of lipid bilayers, they represent molecular switches for a broad range of biological processes, including cell signaling, membrane dynamics and remodeling, and many other functions. Here, we focus on the molecular mechanisms that turn phosphoinositides into molecular switches and how the dysregulation of these processes can lead to disease.

Mechanisms controlling membrane recruitment and activation of the autoinhibited SHIP1 inositol 5-phosphatase.

Signal transduction downstream of growth factor and immune receptor activation relies on the production of phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3) lipids by PI3K. Regulating the strength and duration of PI3K signaling in immune cells, Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) controls the dephosphorylation of PI(3,4,5)P3 to generate phosphatidylinositol-(3,4)-bisphosphate. Although SHIP1 has been shown to regulate neutrophil chemotaxis, B-cell signaling, and cortical oscillations in mast cells, the role that lipid and protein interactions serve in controlling SHIP1 membrane recruitment and activity remains unclear. Using single-molecule total internal reflection fluorescence microscopy, we directly visualized membrane recruitment and activation of SHIP1 on supported lipid bilayers and the cellular plasma membrane. We find that localization of the central catalytic domain of SHIP1 is insensitive to dynamic changes in PI(3,4,5)P3 and phosphatidylinositol-(3,4)-bisphosphate both in vitro and in vivo. Very transient SHIP1 membrane interactions were detected only when membranes contained a combination of phosphatidylserine and PI(3,4,5)P3 lipids. Molecular dissection reveals that SHIP1 is autoinhibited with the N-terminal Src homology 2 domain playing a critical role in suppressing phosphatase activity. Robust SHIP1 membrane localization and relief of autoinhibition can be achieved through interactions with immunoreceptor-derived phosphopeptides presented either in solution or conjugated to a membrane. Overall, this work provides new mechanistic details concerning the dynamic interplay between lipid-binding specificity, protein-protein interactions, and the activation of autoinhibited SHIP1.

PIP2 regulation of TRPC5 channel activation and desensitization.

Transient receptor potential canonical type 5 (TRPC5) ion channels are expressed in the brain and kidney and have been identified as promising therapeutic targets whose selective inhibition can protect against diseases driven by a leaky kidney filter, such as focal segmental glomerular sclerosis. TRPC5 channels are activated not only by elevated levels of extracellular Ca2+or lanthanide ions but also by G protein (Gq/11) stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipase C enzymes leads to PKC-mediated phosphorylation of TRPC5 channels and their subsequent desensitization. However, the roles of PIP2 in activation and maintenance of TRPC5 channel activity via its hydrolysis product diacyl glycerol (DAG), as well as the mechanism of desensitization of TRPC5 activity by DAG-stimulated PKC activity, remain unclear. Here, we designed experiments to distinguish between the processes underlying channel activation and inhibition. Employing whole-cell patch-clamp, we used an optogenetic tool to dephosphorylate PIP2 and assess channel-PIP2 interactions influenced by activators, such as DAG, or inhibitors, such as PKC phosphorylation. Using total internal reflection microscopy, we assessed channel cell surface density. We show that PIP2 controls both the PKC-mediated inhibition and the DAG- and lanthanide-mediated activation of TRPC5 currents via control of gating rather than channel cell surface density. These mechanistic insights promise to aid in the development of more selective and precise inhibitors to block TRPC5 channel activity and illuminate new opportunities for targeted therapies for a group of chronic kidney diseases for which there is currently a great unmet need.

Identification of Rac guanine nucleotide exchange factors promoting Lgl1 phosphorylation in glioblastoma.

The protein Lgl1 is a key regulator of cell polarity. We previously showed that Lgl1 is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN tumour suppressor loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl1 is phosphorylated by atypical protein kinase C that has been activated by binding to a complex of the scaffolding protein Par6 and active, GTP-bound Rac. The specific Rac guanine nucleotide exchange factors that generate active Rac to promote Lgl1 hyperphosphorylation in glioblastoma are unknown. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor, in patient-derived glioblastoma cells. Knockout cells had reduced Lgl1 phosphorylation, which was reversed by re-expressing PREX1. They also had reduced motility and an altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses identified sets of PREX1-regulated genes associated with cell motility and neuronal differentiation. PREX1 knockout in glioblastoma cells from a second patient did not affect Lgl1 phosphorylation. This was due to overexpression of a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in these PREX1 knockout cells reduced Lgl1 phosphorylation. These data show that PREX1 links aberrant PI 3-kinase signaling to Lgl1 phosphorylation in glioblastoma, but that TIAM1 is also to fill this role in a subset of patients. This redundancy between PREX1 and TIAM1 is only partial, as motility was impaired in PREX1 knockout cells from both patients.

Transcriptome analysis reveals the mechanism of zinc ion-mediated plant resistance to TMV in Nicotiana benthamiana.

Zinc ions (Zn2+) are used to promote plant growth and treat multiple diseases. However, it is still unclear which pathways in plants respond to Zn2+. In this study, we found that supplying (CH3COO)2Zn can effectively delay tobacco mosaic virus (TMV) replication and movement in Nicotiana benthamiana. To further understand the regulatory mechanism of antiviral activity mediated by Zn2+, we examined the transcriptomic changes of leaves treated with Zn2+. Three days after treatment, 7575 differential expression genes (DEGs) were enriched in the Zn2+ treatment group compared with the control group. Through GO and KEGG analysis, the pathway of phosphatidylinositol signaling system and inositol phosphate metabolism were significantly enriched after treated with Zn2+, and a large number of ethylene-responsive transcription factors (ERFs) involved in inositol phosphate metabolism were found to be enriched. We identified ERF5 performed a positive effect on plant immunity. Our findings demonstrated that Zn2+-mediated resistance in N. benthamiana activated signal transduction and regulated the expression of resistance-related genes. The results of the study uncover a global view of mRNA changes in Zn2+-mediated cellular processes involved in the competition between plants and viruses.

Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A.

Phospholipase Cε (PLCε) is activated downstream of G protein-coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of ß-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.

Transforming growth factor ß (TGF-ß) receptor signaling regulates kinase networks and phosphatidylinositol metabolism during T-cell activation.

The cytokine content in tissue microenvironments shapes the functional capacity of a T cell. This capacity depends on the integration of extracellular signaling through multiple receptors, including the T-cell receptor (TCR), co-receptors, and cytokine receptors. Transforming growth factor ß (TGF-ß) signals through its cognate receptor, TGFßR, to SMAD family member proteins and contributes to the generation of a transcriptional program that promotes regulatory T-cell differentiation. In addition to transcription, here we identified specific signaling networks that are regulated by TGFßR. Using an array of biochemical approaches, including immunoblotting, kinase assays, immunoprecipitation, and flow cytometry, we found that TGFßR signaling promotes the formation of a SMAD3/4-protein kinase A (PKA) complex that activates C-terminal Src kinase (CSK) and thereby down-regulates kinases involved in proximal TCR activation. Additionally, TGFßR signaling potentiated CSK phosphorylation of the P85 subunit in the P85-P110 phosphoinositide 3-kinase (PI3K) heterodimer, which reduced PI3K activity and down-regulated the activation of proteins that require phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) for their activation. Moreover, TGFßR-mediated disruption of the P85-P110 interaction enabled P85 binding to a lipid phosphatase, phosphatase and tensin homolog (PTEN), aiding in the maintenance of PTEN abundance and thereby promoting elevated PtdIns(4,5)P2 levels in response to TGFßR signaling. Taken together, these results highlight that TGF-ß influences the trajectory of early T-cell activation by altering PI3K activity and PtdIns levels.

Interaction of the late endo-lysosomal lipid PI(3,5)P2 with the Vph1 isoform of yeast V-ATPase increases its activity and cellular stress tolerance.

The low-level endo-lysosomal signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), is required for full assembly and activity of vacuolar H+-ATPases (V-ATPases) containing the vacuolar a-subunit isoform Vph1 in yeast. The cytosolic N-terminal domain of Vph1 is also recruited to membranes in vivo in a PI(3,5)P2-dependent manner, but it is not known if its interaction with PI(3,5)P2 is direct. Here, using biochemical characterization of isolated yeast vacuolar vesicles, we demonstrate that addition of exogenous short-chain PI(3,5)P2 to Vph1-containing vacuolar vesicles activates V-ATPase activity and proton pumping. Modeling of the cytosolic N-terminal domain of Vph1 identified two membrane-oriented sequences that contain clustered basic amino acids. Substitutions in one of these sequences (231KTREYKHK) abolished the PI(3,5)P2-dependent activation of V-ATPase without affecting basal V-ATPase activity. We also observed that vph1 mutants lacking PI(3,5)P2 activation have enlarged vacuoles relative to those in WT cells. These mutants exhibit a significant synthetic growth defect when combined with deletion of Hog1, a kinase important for signaling the transcriptional response to osmotic stress. The results suggest that PI(3,5)P2 interacts directly with Vph1, and that this interaction both activates V-ATPase activity and protects cells from stress.

T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols.

T-cell receptor (TCR) signaling strength is a dominant factor regulating T-cell differentiation, thymic development, and cytokine signaling. The molecular mechanisms by which TCR signal strength is transduced to downstream signaling networks remains ill-defined. Using computational modeling, biochemical assays, and imaging flow cytometry, we found here that TCR signal strength differentially generates phosphatidylinositol species. Weak TCR signals generated elevated phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and reduced phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, whereas strong TCR signals reduced PI(4,5)P2 and elevated PIP3 levels. A proteomics screen revealed that focal adhesion kinase bound PI(4,5)P2, biochemical assays disclosed that focal adhesion kinase is preferentially activated by weak TCR signals and is required for optimal Treg induction, and further biochemical experiments revealed how TCR signaling strength regulates AKT activation. Low PIP3 levels generated by weak TCR signals were sufficient to activate phosphoinositide-dependent kinase-1 to phosphorylate AKT on Thr-308 but insufficient to activate mTOR complex 2 (mTORC2), whereas elevated PIP3 levels generated by a strong TCR signal were required to activate mTORC2 to phosphorylate Ser-473 on AKT. Our results provide support for a model that links TCR signaling to mTORC2 activation via phosphoinositide 3-kinase signaling. Together, the findings in this work establish that T cells measure TCR signal strength by generating different levels of phosphatidylinositol species that engage alternate signaling networks to control cell fate decisions.

Neuronal ER-plasma membrane junctions organized by Kv2-VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis.

The association of plasma membrane (PM)-localized voltage-gated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER-PM junctions in mammalian brain neurons. Here, we used proteomics to identify proteins associated with Kv2/VAP-containing ER-PM junctions. We found that the VAP-interacting membrane-associated phosphatidylinositol (PtdIns) transfer proteins PYK2 N-terminal domain-interacting receptor 2 (Nir2) and Nir3 specifically associate with Kv2.1 complexes. When coexpressed with Kv2.1 and VAPA in HEK293T cells, Nir2 colocalized with cell-surface-conducting and -nonconducting Kv2.1 isoforms. This was enhanced by muscarinic-mediated PtdIns(4,5)P2 hydrolysis, leading to dynamic recruitment of Nir2 to Kv2.1 clusters. In cultured rat hippocampal neurons, exogenously expressed Nir2 did not strongly colocalize with Kv2.1, unless exogenous VAPA was also expressed, supporting the notion that VAPA mediates the spatial association of Kv2.1 and Nir2. Immunolabeling signals of endogenous Kv2.1, Nir2, and VAP puncta were spatially correlated in cultured neurons. Fluorescence-recovery-after-photobleaching experiments revealed that Kv2.1, VAPA, and Nir2 have comparable turnover rates at ER-PM junctions, suggesting that they form complexes at these sites. Exogenous Kv2.1 expression in HEK293T cells resulted in significant differences in the kinetics of PtdIns(4,5)P2 recovery following repetitive muscarinic stimulation, with no apparent impact on resting PtdIns(4,5)P2 or PtdIns(4)P levels. Finally, the brains of Kv2.1-knockout mice had altered composition of PtdIns lipids, suggesting a crucial role for native Kv2.1-containing ER-PM junctions in regulating PtdIns lipid metabolism in brain neurons. These results suggest that ER-PM junctions formed by Kv2 channel-VAP pairing regulate PtdIns lipid homeostasis via VAP-associated PtdIns transfer proteins.

G protein ßγ subunits directly interact with and activate phospholipase Cϵ.

Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP2) and regulate Ca2+ and protein kinase signaling in virtually all mammalian cell types. Chronic activation of the PLCϵ isoform downstream of G protein-coupled receptors (GPCRs) contributes to the development of cardiac hypertrophy. We have previously shown that PLCϵ-catalyzed hydrolysis of Golgi-associated phosphatidylinositol 4-phosphate (PI4P) in cardiac myocytes depends on G protein ßγ subunits released upon stimulation with endothelin-1. PLCϵ binds and is directly activated by Ras family small GTPases, but whether they directly interact with Gßγ has not been demonstrated. To identify PLCϵ domains that interact with Gßγ, here we designed various single substitutions and truncations of WT PLCϵ and tested them for activation by Gßγ in transfected COS-7 cells. Deletion of only a single domain in PLCϵ was not sufficient to completely block its activation by Gßγ, but blocked activation by Ras. Simultaneous deletion of the C-terminal RA2 domain and the N-terminal CDC25 and cysteine-rich domains completely abrogated PLCϵ activation by Gßγ, but activation by the GTPase Rho was retained. In vitro reconstitution experiments further revealed that purified Gßγ directly interacts with a purified fragment of PLCϵ (PLCϵ-PH-RA2) and increases PIP2 hydrolysis. Deletion of the RA2 domain decreased Gßγ binding and eliminated Gßγ stimulation of PIP2 hydrolysis. These results provide first evidence that Gßγ directly interacts with PLCϵ and yield insights into the mechanism by which ßγ subunits activate PLCϵ.

Phosphoinositide conversion in endocytosis and the endolysosomal system.

Phosphoinositides (PIs) are phospholipids that perform crucial cell functions, ranging from cell migration and signaling to membrane trafficking, by serving as signposts of compartmental membrane identity. Although phosphatidylinositol 4,5-bisphosphate, 3-phosphate, and 3,5-bisphosphate are commonly considered as hallmarks of the plasma membrane, endosomes, and lysosomes, these compartments contain other functionally important PIs. Here, we review the roles of PIs in different compartments of the endolysosomal system in mammalian cells and discuss the mechanisms that spatiotemporally control PI conversion in endocytosis and endolysosomal membrane dynamics during endosome maturation and sorting. As defective PI conversion underlies human genetic diseases, including inherited myopathies, neurological disorders, and cancer, PI-converting enzymes represent potential targets for drug-based therapies.

Induction of autophagy by PI3K/MTOR and PI3K/MTOR/BRD4 inhibitors suppresses HIV-1 replication.

In this study, we investigated the effects of the dual phosphatidylinositol 3-kinase/mechanistic target of rapamycin (PI3K/MTOR) inhibitor dactolisib (NVP-BEZ235), the PI3K/MTOR/bromodomain-containing protein 4 (BRD4) inhibitor SF2523, and the bromodomain and extra terminal domain inhibitor JQ1 on the productive infection of primary macrophages with human immunodeficiency type-1 (HIV). These inhibitors did not alter the initial susceptibility of macrophages to HIV infection. However, dactolisib, JQ1, and SF2523 all decreased HIV replication in macrophages in a dose-dependent manner via degradation of intracellular HIV through autophagy. Macrophages treated with dactolisib, JQ1, or SF2523 displayed an increase in LC3B lipidation combined with SQSTM1 degradation without inducing increased cell death. LC3B-II levels were further increased in the presence of pepstatin A suggesting that these inhibitors induce autophagic flux. RNA interference for ATG5 and ATG7 and pharmacological inhibitors of autophagosome-lysosome fusion and of lysosomal hydrolases all blocked the inhibition of HIV. Thus, we demonstrate that the mechanism of PI3K/MTOR and PI3K/MTOR/BRD4 inhibitor suppression of HIV requires the formation of autophagosomes, as well as their subsequent maturation into autolysosomes. These data provide further evidence in support of a role for autophagy in the control of HIV infection and open new avenues for the use of this class of drugs in HIV therapy.

Membrane curvature allosterically regulates the phosphatidylinositol cycle, controlling its rate and acyl-chain composition of its lipid intermediates.

Signaling events at membranes are often mediated by membrane lipid composition or membrane physical properties. These membrane properties could act either by favoring the membrane binding of downstream effectors or by modulating their activity. Several proteins can sense/generate membrane physical curvature (i.e. shape). However, the modulation of the activity of enzymes by a membrane's shape has not yet been reported. Here, using a cell-free assay with purified diacylglycerol kinase ϵ (DGKϵ) and liposomes, we studied the activity and acyl-chain specificity of an enzyme of the phosphatidylinositol (PI) cycle, DGKϵ. By systematically varying the model membrane lipid composition and physical properties, we found that DGKϵ has low activity and lacks acyl-chain specificity in locally flat membranes, regardless of the lipid composition. On the other hand, these enzyme properties were greatly enhanced in membrane structures with a negative Gaussian curvature. We also found that this is not a consequence of preferential binding of the enzyme to those structures, but rather is due to a curvature-mediated allosteric regulation of DGKϵ activity and acyl-chain specificity. Moreover, in a fine-tuned interplay between the enzyme and the membrane, DGKϵ favored the formation of structures with greater Gaussian curvature. DGKϵ does not bear a regulatory domain, and these findings reveal the importance of membrane curvature in regulating DGKϵ activity and acyl-chain specificity. Hence, this study highlights that a hierarchic coupling of membrane physical property and lipid composition synergistically regulates membrane signaling events. We propose that this regulatory mechanism of membrane-associated enzyme activity is likely more common than is currently appreciated.

PI signal transduction and ubiquitination respond to dehydration stress in the red seaweed Gloiopeltis furcata under successive tidal cycles.

BACKGROUND: Intermittent dehydration caused by tidal changes is one of the most important abiotic factors that intertidal seaweeds must cope with in order to retain normal growth and reproduction. However, the underlying molecular mechanisms for the adaptation of red seaweeds to repeated dehydration-rehydration cycles remain poorly understood. RESULTS: We chose the red seaweed Gloiopeltis furcata as a model and simulated natural tidal changes with two consecutive dehydration-rehydration cycles occurring over 24 h in order to gain insight into key molecular pathways and regulation of genes which are associated with dehydration tolerance. Transcription sequencing assembled 32,681 uni-genes (GC content = 55.32%), of which 12,813 were annotated. Weighted gene co-expression network analysis (WGCNA) divided all transcripts into 20 modules, with Coral2 identified as the key module anchoring dehydration-induced genes. Pathways enriched analysis indicated that the ubiquitin-mediated proteolysis pathway (UPP) and phosphatidylinositol (PI) signaling system were crucial for a successful response in G. furcata. Network-establishing and quantitative reverse transcription PCR (qRT-PCR) suggested that genes encoding ubiquitin-protein ligase E3 (E3-1), SUMO-activating enzyme sub-unit 2 (SAE2), calmodulin (CaM) and inositol-1,3,4-trisphosphate 5/6-kinase (ITPK) were the hub genes which responded positively to two successive dehydration treatments. Network-based interactions with hub genes indicated that transcription factor (e.g. TFIID), RNA modification (e.g. DEAH) and osmotic adjustment (e.g. MIP, ABC1, Bam1) were related to these two pathways. CONCLUSIONS: RNA sequencing-based evidence from G. furcata enriched the informational database for intertidal red seaweeds which face periodic dehydration stress during the low tide period. This provided insights into an increased understanding of how ubiquitin-mediated proteolysis and the phosphatidylinositol signaling system help seaweeds responding to dehydration-rehydration cycles.

Maternal and Post-weaning High-Fat Diets Produce Distinct DNA Methylation Patterns in Hepatic Metabolic Pathways within Specific Genomic Contexts.

Calorie-dense high-fat diets (HF) are associated with detrimental health outcomes, including obesity, cardiovascular disease, and diabetes. Both pre- and post-natal HF diets have been hypothesized to negatively impact long-term metabolic health via epigenetic mechanisms. To understand how the timing of HF diet intake impacts DNA methylation and metabolism, male Sprague-Dawley rats were exposed to either maternal HF (MHF) or post-weaning HF diet (PHF). At post-natal week 12, PHF rats had similar body weights but greater hepatic lipid accumulation compared to the MHF rats. Genome-wide DNA methylation was evaluated, and analysis revealed 1744 differentially methylation regions (DMRs) between the groups with the majority of the DMR located outside of gene-coding regions. Within differentially methylated genes (DMGs), intragenic DNA methylation closer to the transcription start site was associated with lower gene expression, whereas DNA methylation further downstream was positively correlated with gene expression. The insulin and phosphatidylinositol (PI) signaling pathways were enriched with 25 DMRs that were associated with 20 DMGs, including PI3 kinase (Pi3k), pyruvate kinase (Pklr), and phosphodiesterase 3 (Pde3). Together, these results suggest that the timing of HF diet intake determines DNA methylation and gene expression patterns in hepatic metabolic pathways that target specific genomic contexts.

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane.

Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability.

The binding of activated Gαq to phospholipase C-ß exhibits anomalous affinity.

Upon activation by the Gq family of Gα subunits, Gßγ subunits, and some Rho family GTPases, phospholipase C-ß (PLC-ß) isoforms hydrolyze phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-ß isoforms also function as GTPase-activating proteins, potentiating Gq deactivation. To elucidate the mechanism of this mutual regulation, we measured the thermodynamics and kinetics of PLC-ß3 binding to Gαq FRET and fluorescence correlation spectroscopy, two physically distinct methods, both yielded Kd values of about 200 nm for PLC-ß3-Gαq binding. This Kd is 50-100 times greater than the EC50 for Gαq-mediated PLC-ß3 activation and for the Gαq GTPase-activating protein activity of PLC-ß. The measured Kd was not altered either by the presence of phospholipid vesicles, phosphatidylinositol 4,5-bisphosphate and Ca2+, or by the identity of the fluorescent labels. FRET-based kinetic measurements were also consistent with a Kd of 200 nm We determined that PLC-ß3 hysteresis, whereby PLC-ß3 remains active for some time following either Gαq-PLC-ß3 dissociation or PLC-ß3-potentiated Gαq deactivation, is not sufficient to explain the observed discrepancy between EC50 and Kd These results indicate that the mechanism by which Gαq and PLC-ß3 mutually regulate each other is far more complex than a simple, two-state allosteric model and instead is probably kinetically determined.

Structural features of human inositol phosphate multikinase rationalize its inositol phosphate kinase and phosphoinositide 3-kinase activities.

Human inositol phosphate multikinase (HsIPMK) critically contributes to intracellular signaling through its inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 3-kinase activities. This catalytic profile is not conserved; orthologs from Arabidopsis thaliana and Saccharomyces cerevisiae are predominantly Ins(1,4,5)P3 6-kinases, and the plant enzyme cannot phosphorylate PtdIns(4,5)P2 Therefore, crystallographic analysis of the yeast and plant enzymes, without bound inositol phosphates, do not structurally rationalize HsIPMK activities. Here, we present 1.6-Å resolution crystal structures of HsIPMK in complex with either Ins(1,4,5)P3 or PtdIns(4,5)P2 The Ins(1,4,5)P3 headgroup of PtdIns(4,5)P2 binds in precisely the same orientation as free Ins(1,4,5)P3 itself, indicative of evolutionary optimization of 3-kinase activities against both substrates. We report on nucleotide binding between the separate N- and C-lobes of HsIPMK. The N-lobe exhibits a remarkable degree of conservation with protein kinase A (root mean square deviation = 1.8 Å), indicating common ancestry. We also describe structural features unique to HsIPMK. First, we observed a constrained, horseshoe-shaped substrate pocket, formed from an α-helix, a 310 helix, and a recently evolved tri-proline loop. We further found HsIPMK activities rely on a preponderance of Gln residues, in contrast to the larger Lys and Arg residues in yeast and plant orthologs. These conclusions are supported by analyzing 14 single-site HsIPMK mutants, some of which differentially affect 3-kinase and 6-kinase activities. Overall, we structurally rationalize phosphorylation of Ins(1,4,5)P3 and PtdIns(4,5)P2 by HsIPMK.

Integrated Analysis of Transcriptomic and Proteomics Data Reveals the Induction Effects of Rotenoid Biosynthesis of Mirabilis himalaica Caused by UV-B Radiation.

Mirabilis himalaica (Edgew.) Heimerl is one of the most important genuine medicinal plants in Tibet, in which the special plateau habitat has been associated with its excellent medicinal quality and efficacy. However, the mechanisms by which environmental factors affect biosynthesis of secondary metabolic components remain unclear in this species. In this study, RNA sequencing and iTRAQ (isobaric Tags for Relative and Absolute Quantification) techniques were used to investigate the critical molecular "events" of rotenoid biosynthesis responding to UV-B radiation, a typical plateau ecological factor presented in native environment-grown M. himalaica plants. A total of 3641 differentially expressed genes (DEGs) and 106 differentially expressed proteins (DEPs) were identified in M. himalaica between UV-B treatment and control check (CK). Comprehensive analysis of protein and transcript data sets resulted in 14 and 7 DEGs from the plant hormone signal transduction and phosphatidylinositol signaling system pathways, respectively, being significantly enriched. The result showed that the plant hormone signal transduction and phosphatidylinositol signaling system might be the key metabolic strategy of UV-B radiation to improve the biosynthesis of rotenoid in M. himalaica. At same time, most of the DEGs were associated with auxin and calcium signaling, inferring that they might drive the downstream transmission of these signal transduction pathways. Regarding those pathways, two chalcone synthase enzymes, which play key roles in the biosynthesis of rotenoid that were thought as the representative medicinal component of M. himalaica, were significantly upregulated in UV-B radiation. This study provides a theoretical basis for further exploration of the adaptation mechanism of M. himalaica to UV-B radiation, and references for cultivation standardization.