Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K7, PIP5K8, and PIP5K9 are redundantly involved in root growth adaptation to osmotic stress

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P₂), a signaling phospholipid critical for various cellular processes in eukaryotes. The Arabidopsis thaliana genome encodes 11 PIP5K genes. Of these, three type B PIP5K genes, PIP5K7, PIP5K8, and PIP5K9, constitute a subgroup highly conserved in land plants, suggesting that they retain a critical function shared by land plants. In this study, we comprehensively investigated the biological functions of the PIP5K7–9 subgroup genes. Reporter gene analyses revealed their preferential expression in meristematic and vascular tissues. Their YFP-fusion proteins localized primarily to the plasma membrane in root meristem epidermal cells. We selected a mutant line that was considered to be null for each gene. Under normal growth conditions, neither single mutants nor multiple mutants of any combination exhibited noticeable phenotypic changes. However, stress conditions with mannitol or NaCl suppressed main root growth and reduced proximal root meristem size to a greater extent in the pip5k7pip5k8pip5k9 triple mutant than in the wild type. In root meristem epidermal cells of the triple mutant, where plasma membrane localization of the PtdIns(4,5)P₂ marker P24Y is impaired to a large extent, brefeldin A body formation is retarded compared with the wild type under hyperosmotic stress. These results indicate that PIP5K7, PIP5K8, and PIP5K9 are not required under normal growth conditions, but are redundantly involved in root growth adaptation to hyperosmotic conditions, possibly through the PtdIns(4,5)P₂ function promoting plasma membrane recycling in root meristem cells.     

Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).     

Phosphatidylinositol phosphate 5-kinase Ibeta recruits AP-2 to the plasma membrane and regulates rates of constitutive endocytosis

Overexpression of phosphatidylinositol phosphate 5-kinase (PIP5KI) isoforms alpha, beta, or gamma in CV-1 cells increased phosphatidylinositol 4,5-bisphosphate (PIP2) levels by 35, 180, and 0%, respectively. Endocytosis of transferrin receptors, association of AP-2 proteins with membranes, and the number of clathrin-coated pits at the plasma membrane increased when PIP2 increased. When expression of PIP5KIbeta was inhibited with small interference RNA in HeLa cells, expression of PIP5KIalpha was also reduced slightly, but PIP5KIgamma expression was increased. PIP2 levels and internalization of transferrin receptors dropped 50% in these cells; thus, PIP5KIgamma could not compensate for loss of PIP5KIbeta. When expression of PIP5KIalpha was reduced, expression of both PIP5KIbeta and PIP5KIgamma increased and PIP2 levels did not change. A similar increase of PIP5KIalpha and PIP5KIbeta occurred when PIP5KIgamma was inhibited. These results indicate that constitutive endocytosis in CV-1 and HeLa cells requires (and may be regulated by) PIP2 produced primarily by PIP5KIbeta.     

用快速琼脂糖层析纯化磷酸甘油酸激酶及磷酸甘油醛脱氢酶

研究了用快速琼脂糖离子交换层析(DEAE—Fast Flow sepharose)结台PEG 4000／Reppal PES双水相体系从黄豆中分离纯化磷酸甘油酸激酶(PGK)及磷酸甘油醛脱氢酶(GAPDH)。控制床层高度(10～20cm),径向放大具有压降低的优点．设计多点进料取代传统的中心管进料,解决了径向流场不均匀的问题。GAPr)H的总收率及纯化倍数分别为58％和144,PGK的总收率及纯化倍数分别为41％和44。工艺成本为2．92美元／ku GPADH,具有一定的实用价值。     

Phosphatidylinositol 4,5-Bisphosphate Homeostasis Regulated by Nir2 and Nir3 Proteins at Endoplasmic Reticulum-Plasma Membrane Junctions

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP₂) at the plasma membrane (PM) constitutively controls many cellular functions, and its hydrolysis via receptor stimulation governs cell signaling. The PI transfer protein Nir2 is essential for replenishing PM PIP₂ following receptor-induced hydrolysis, but key mechanistic aspects of this process remain elusive. Here, we demonstrate that PI at the membrane of the endoplasmic reticulum (ER) is required for the rapid replenishment of PM PIP₂ mediated by Nir2. Nir2 detects PIP₂ hydrolysis and translocates to ER-PM junctions via binding to phosphatidic acid. With distinct phosphatidic acid binding abilities and PI transfer protein activities, Nir2 and its homolog Nir3 differentially regulate PIP₂ homeostasis in cells during intense receptor stimulation and in the resting state, respectively. Our study reveals that Nir2 and Nir3 work in tandem to achieve different levels of feedback based on the consumption of PM PIP₂ and function at ER-PM junctions to mediate nonvesicular lipid transport between the ER and the PM.     

Transmembrane Helix Packing of ErbB/Neu Receptor in Membrane Environment: A Molecular Dynamics Study

Abstract

Dimerization or oligomerization of the ErbB/Neu receptors are necessary but not sufficient for initiation of receptor signaling. The two intracellular domains must be properly oriented for the juxtaposition of the kinase domains allowing trans-phosphorylation. This suggests that the transmembrane (TM) domain acts as a guide for defining the proper orientation of the intracellular domains.

Two structural models, with the two helices either in left-handed or in right-handed coiling have been proposed as the TM domain structure of the active receptor. Because experimental data do not distinguish clearly helix-helix packing, molecular dynamics (MD) simulations are used to investigate the energetic factors that drive Neu TM-TM interactions of the wild and the oncogenic receptor (Val664/Glu mutation) in DMPC or in POPC environments. MD results indicate that helix-lipid interactions in the bilayer core are extremely similar in the two environments and raise the role of the juxtamembrane residues in helix insertion and helix-helix packing. The TM domain shows a greater propensity to adopt a left-handed structure in DMPC, with helices in optimal position for strong inter-helical Hbonds induced by the Glu mutation. In POPC, the right-handed structure is preferentially formed with the participation of water in inter-helical Hbonds. The two structural arrangements of the NeuTM helices both with GG4 residue motif in close contact at the interface are permissible in the membrane environment. According to the hypothesis of a monomer-dimer equilibrium of the proteins it is likely that the bilayer imposes structural constraints that favor dimerization- competent structure responsible of the proper topology necessary for receptor activation.     

Lipid Membrane Structure and Dynamics Studied by All-Atom Molecular Dynamics Simulations of Hydrated Phospholipid Bilayers

Abstract

The structure and dynamics of phosphatidylcholine bilayers are examined by reviewing the results of several nanoseconds of molecular dynamics simulations on a number of bilayer and monolayer models. The lengths of these simulations, the longest single one of which was 2 nanoseconds, were sufficiently long to effectively sample many of the longer-scale motions governing the behaviour of biomembranes. These simulations reproduce many experimental observables well and provide a degree of resolution currently unavailable experimentally.     

Interactions of the EphA2 Kinase Domain with PIPs in Membranes: Implications for Receptor Function

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A Conspicuous Connection: Structure Defines Function for the Phosphatidylinositol-Phosphate Kinase Family

ABSTRACT

The phosphatidylinositol phosphate (PIP) kinases are a unique family of enzymes that generate an assortment of lipid messengers, including the pivotal second messenger phosphatidylinositol 4,5-bisphosphate (PI4,5P₂). While members of the PIP kinase family function by catalyzing a similar phosphorylation reaction, the specificity loop of each PIP kinase subfamily determines substrate preference and partially influences distinct subcellular targeting. Specific protein-protein interactions that are unique to particular isoforms or splice variants play a key role in targeting PIP kinases to appropriate subcellular compartments to facilitate the localized generation of PI4,5P₂ proximal to effectors, a mechanism key for the function of PI4,5P₂ as a second messenger. This review documents the discovery of the PIP kinases and their signaling products, and summarizes our current understanding of the mechanisms underlying the localized generation of PI4,5P₂ by PIP kinases for the regulation of cellular events including actin cytoskeleton dynamics, vesicular trafficking, cell migration, and an assortment of nuclear events.     

Membrane association and selectivity of the antimicrobial peptide NK‐2: a molecular dynamics simulation study

In an effort to better understand the initial mechanism of selectivity and membrane association of the synthetic antimicrobial peptide NK‐2, we have applied molecular dynamics simulation techniques to elucidate the interaction of the peptide with the membrane interfaces. A homogeneous dipalmitoylphosphatidylglycerol (DPPG) and a homogeneous dipalmitoylphosphatidylethanolamine (DPPE) bilayers were taken as model systems for the cytoplasmic bacterial and human erythrocyte membranes, respectively. The results of our simulations on DPPG and DPPE model membranes in the gel phase show that the binding of the peptide, which is considerably stronger for the negatively charged DPPG lipid bilayer than for the zwitterionic DPPE, is mostly governed by electrostatic interactions between negatively charged residues in the membrane and positively charged residues in the peptide. In addition, a characteristic distribution of positively charged residues along the helix facilitates a peptide orientation parallel to the membrane interface. Once the peptides reside close to the membrane surface of DPPG with the more hydrophobic side chains embedded into the membrane interface, the peptide initially disturbs the respective bilayer integrity by a decrease of the order parameter of lipid acyl chain close to the head group region, and by a slightly decrease in bilayer thickness. We found that the peptide retains a high content of helical structure on the zwitterionic membrane‐water interface, while the loss of α‐helicity is observed within a peptide adsorbed onto negatively charged lipid membranes. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Towards an Understanding of DNA Recognition by the Methyl-CpG Binding Domain 1

Abstract

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations.

As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be ?3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.     

A novel PIP2 binding of epsilonPKC and its contribution to the neurite induction ability

Protein kinase C-epsilon (epsilonPKC) induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the epsilonPKC-induced neurite outgrowth is not fully understood. Therefore, we investigated the ability of phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding of epsilonPKC and its correlation with the neurite extension. We found that full length epsilonPKC bound to PIP(2) in a 12-omicron-tetradecanoylphorbol-13-acetate dependent manner, while the regulatory domain of epsilonPKC (epsilonRD) bound to PIP(2) without any stimulation. To identify the PIP(2) binding region, we made mutants lacking several regions from epsilonRD, and examined their PIP(2) binding activity. The mutants lacking variable region 1 (V1) bound to PIP(2) stronger than intact epsilonRD, while the mutants lacking pseudo-substrate or common region 1 (C1) lost the binding. The PIP(2) binding ability of the V3-deleted mutant was weakened. Those PIP(2) bindings of epsilonPKC, epsilonRD and the mutants well correlated to their neurite induction ability. In addition, a chimera of pleckstrin homology domain of phospholipase Cdelta and the V3 region of epsilonPKC revealed that PIP(2) binding domain and the V3 region are sufficient for the neurite induction, and a first 16 amino acids in the V3 region was important for neurite extension. In conclusion, epsilonPKC directly binds to PIP(2) mainly through pseudo-substrate and common region 1, contributing to the neurite induction activity.     

Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP(2)): a study of channel rundown and reactivation

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6. 2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K(ATP) channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP(2)), and phosphorylation. Upon excision of inside-out patches into a Ca(2+)- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6. 2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca(2+) accelerated this process, suggesting a role for PIP(2) hydrolysis mediated by a Ca(2+)-dependent phospholipase C. PIP(2) could reactivate channel activity after a brief exposure to Ca(2+), but not after prolonged exposure. However, in both cases, PIP(2) reversed the increase in ATP sensitivity, indicating that PIP(2) lowers the ATP sensitivity by increasing P(o) as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca(2+) facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIP(2) and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP(2) responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP(2) and phosphorylation.     

Extracellular blockade of K(+) channels by TEA: results from molecular dynamics simulations of the KcsA channel

TEA is a classical blocker of K(+) channels. From mutagenesis studies, it has been shown that external blockade by TEA is strongly dependent upon the presence of aromatic residue at Shaker position 449 which is located near the extracellular entrance to the pore (Heginbotham, L., and R. MacKinnon. 1992. Neuron. 8:483-491). The data suggest that TEA interacts simultaneously with the aromatic residues of the four monomers. The determination of the 3-D structure of the KcsA channel using X-ray crystallography (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) has raised some issues that remain currently unresolved concerning the interpretation of these observations. In particular, the center of the Tyr82 side chains in KcsA (corresponding to position 449 in Shaker) forms a square of 11.8-A side, a distance which is too large to allow simultaneous interactions of a TEA molecule with the four aromatic side chains. In this paper, the external blockade by TEA is explored by molecular dynamics simulations of an atomic model of KcsA in an explicit phospholipid bilayer with aqueous salt solution. It is observed, in qualitative accord with the experimental results, that TEA is stable when bound to the external side of the wild-type KcsA channel (with Tyr82), but is unstable when bound to a mutant channel in which the tyrosine residue has been substituted by a threonine. The free energy profile of TEA relative to the pore is calculated using umbrella sampling simulations to characterize quantitatively the extracellular blockade. It is found, in remarkable agreement with the experiment, that the TEA is more stably bound by 2.3 kcal/mol to the channel with four tyrosine residues. In the case of the wild-type KcsA channel, TEA (which has the shape of a flattened oblate spheroid) acts as an ideal plug blocking the pore. In contrast, it is considerably more off-centered and tilted in the case of the mutant channel. The enhanced stability conferred by the tyrosine residues does not arise from Pi-cation interactions, but appears to be due to differences in the hydration structure of the TEA. Finally, it is shown that the experimentally observed voltage dependence of TEA block, which is traditionally interpreted in terms of the physical position of the TEA along the axis of the pore, must arise indirectly via coupling with the ions in the pore.     

Identification of a conserved 8 aa insert in the PIP5K protein in the Saccharomycetaceae family of fungi and the molecular dynamics simulations and structural analysis to investigate its potential functional role

Homologs of the phosphatidylinositol‐4‐phosphate‐5‐kinase (PIP5K), which controls a multitude of essential cellular functions, contain a 8 aa insert in a conserved region that is specific for the Saccharomycetaceae family of fungi. Using structures of human PIP4K proteins as templates, structural models were generated of the Saccharomyces cerevisiae and human PIP5K proteins. In the modeled S. cerevisiae PIP5K, the 8 aa insert forms a surface exposed loop, present on the same face of the protein as the activation loop of the kinase domain. Electrostatic potential analysis indicates that the residues from 8 aa conserved loop form a highly positively charged surface patch, which through electrostatic interaction with the anionic portions of phospholipid head groups, is expected to play a role in the membrane interaction of the yeast PIP5K. To unravel this prediction, molecular dynamics (MD) simulations were carried out to examine the binding interaction of PIP5K, either containing or lacking the conserved signature insert, with two different membrane lipid bilayers. The results from MD studies provide insights concerning the mechanistic of interaction of PIP5K with lipid bilayer, and support the contention that the identified 8 aa conserved insert in fungal PIP5K plays an important role in the binding of this protein with membrane surface. Proteins 2017; 85:1454–1467. © 2017 Wiley Periodicals, Inc.     

Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein

DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf.     

Phosphatidylinositol 4‐kinase IIα function at endosomes is regulated by the ubiquitin ligase Itch

Phosphatidylinositol (PI) 4‐phosphate (PI(4)P) and its metabolizing enzymes serve important functions in cell signalling and membrane traffic. PI 4‐kinase type IIα (PI4KIIα) regulates Wnt signalling, endosomal sorting of signalling receptors, and promotes adaptor protein recruitment to endosomes and the trans‐Golgi network. Here we identify the E3 ubiquitin ligase Itch as binding partner and regulator of PI4KIIα function. Itch directly associates with and ubiquitinates PI4KIIα, and both proteins colocalize on endosomes containing Wnt‐activated frizzled 4 (Fz4) receptor. Depletion of PI4KIIα or Itch regulates Wnt signalling with corresponding changes in Fz4 internalization and degradative sorting. These findings unravel a new molecular link between phosphoinositide‐regulated endosomal membrane traffic, ubiquitin and the modulation of Wnt signalling.