PIP2信号与PIP2的再合成

磷脂酰肌醇-4,5-二磷酸（phosphatidylinositol-4,5-bisphosphate,PIP2）是一种分布在细胞膜内侧面的微量磷脂。虽然含量很低,但PIP2在细胞信号转导以及膜蛋白功能调节等方面却起着十分重要的作用。细胞膜中PIP2的含量水平呈动态平衡,在其代谢调节改变时,PIP2局部浓度的变化可影响特定蛋白的功能。该文就近二十年来针对PIP2信号和PIP2代谢调节相关的研究作一综述。     

Suppression of OSCC malignancy by oral glands derived-PIP identified by iTRAQ combined with 2D LC-MS/MS

Oral squamous cell carcinoma (OSCC) is the most common malignancy in head and neck cancer and a global cause of cancer-related death. Due to the poor survival rates associated with OSCC, there is a growing need to develop novel technologies and predictive biomarkers to improve disease diagnosis. The identification of new cellular targets in OSCC tumors will benefit such developments. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics analysis combined with 2-dimensional liquid chromatography and tandem mass spectrometry (2D LC-MS/MS) were used to identify differentially expressed proteins (DEPs) between tumor and normal tissues. Of the DEPs detected, the most significantly downregulated protein in OSCC tissue was prolactin-inducible protein (PIP). Clonogenic and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) experiments showed that the proliferation capacity of OSCC cells overexpressing PIP decreased due to cell cycle arrest at the G0/G1 checkpoint. Wound-healing and transwell assay further showed that PIP overexpression also reduced the migration and invasion of OSCC cells. Immunohistochemistry (IHC) was used to analyze the expression in OSCC, indicating that PIP may be secreted by glandular cells and have an inhibitory effect on OSCC cells to produce. In western blot analysis, silencing studies confirmed that PIP mediates these effects through the AKT/mitogen-activated protein kinase (MAPK) signaling axis in OSCC cells. Taken together, this study reveals PIP as a key mediator of OSCC cell growth, migration, and invasion through its effect on AKT/MAPK signaling.     

GABA/progesterone-induced polyphosphoinositide (PPI) breakdown and its role in the acrosome reaction of guinea pig spermatozoa in vitro

To investigate whether GABA/progesterone (P4) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa2+ for 5.5 h and then labeled with [32P]pi for 1 h. Samples were washed through a three-step gradient Percoll, adjusted to 5×107 cells/mL and exposed to 2 mmol/L Ca2+, 5 μmol/L GABA, 10 μmol/L P4 and other agents. Lipids were separated by t.l.c. and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The results showed that (i) when spermatozoa were treated with GABA, 32P-label diminished rapidly in phosphatidylinositol 4, 5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic acid (PA). The loss of label from PPI was almost completed by 10 min. The time-course of the AR was much slower than PPI when spermatozoa reached a maximal response by 15 min; (ii) the pattern of PPI hydrolysis and stimulation of AR was similar for the three agonists     

陆地棉PIP亚家族基因的克隆及表达特征分析

该研究以陆地棉苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)氨基酸序列为探针,利用Blastp从陆地棉基因组数据库中发现了6个同源性较高的基因。根据6个基因序列设计引物,利用RT-PCR技术从陆地棉纤维细胞中克隆出了这6个基因的全长cDNA序列,分别命名为GhPCBER1、GhPCBER2、GhPCBER3、GhIFR、GhPLR1和GhPLR2。多重序列比对和进化树分析发现,6个蛋白均含有PIP类型蛋白的所有保守性基序和活性残基,属于PIP亚家族。实时荧光定量PCR结果显示,除GhPLR1之外其他5个PIP亚家族基因均在纤维细胞中优势或特异表达;在纤维发育过程中,GhPCBER1、GhPCBER2、GhPCBER3和GhIFR的表达均表现为先上升后下降,GhPCBER1和GhPCBER2在花后21d表达量达到最高,GhPCBER3和GhIFR在花后18d达到最高,GhPLR1和GhPLR2在纤维中的表达量呈持续上升趋势。根据基因的表达特征,推测PIP亚家族可能在棉纤维的发育过程中发挥着重要作用。     

PIP3在中性粒细胞信号通路中的作用及生成调节

趋化性是中性粒细胞参与机体对抗病原体的一个基本的细胞反应。中性粒细胞的趋化过程中涉及一系列信号通路来调节其运动性和极性。信号分子磷酸酰肌醇三磷酸及其参与的信号通路在中性粒细胞趋化过程中起着重要的作用,其自身的生成也受到一系列复杂因素的调节。     

Repair of potentially lethal and sublethal damage induced by neocarzinostatin in normal and ataxia-telangiectasia skin fibroblasts

Changes in 32P labeling of phosphatidylinositol-4,5-bisphosphate (PIP2) were examined during ADP-induced aggregation of washed rabbit platelets prelabeled with [32P]phosphate. ADP caused a significant decrease in the amount and 32P labeling of PIP2 at 10 and 60 sec. The decrease in labeling persisted at 2.5 min when the platelets were still aggregated, but disappeared by 10 min. Platelets refractory to ADP showed no further significant change in 32P in PIP2 when exposed to ADP; a decrease in PIP2 labeling could be induced, however, after platelets had recovered their disc shape and sensitivity to ADP. These data indicate that PIP2 may play a role in the response of platelets to ADP.     

PIP5KIC在自噬体形成过程中的重要作用

自噬（autophagy）是一种在真核生物中十分保守的溶酶体依赖性降解途径,它通过形成双层膜结构包裹胞内堆积的蛋白质和受损细胞器并将其运送到溶酶体中进行降解。在实验中发现,一型磷脂酰肌醇4-磷酸5-激酶C亚型（type I phosphatidylinositol 4-phosphate 5-kinase isoform C,PIP5KIC）会参与到自噬过程中。在哺乳动物细胞中,敲低一型磷脂酰肌醇4-磷酸5-激酶C亚型会造成欧米茄体（omegasome）的形状异常,进而造成自噬水平的降低。同样,在酵母中敲掉其同源物磷脂酰肌醇5-激酶Mss4后也会导致类似的现象。因此,推测一型磷脂酰肌醇4-磷酸5-激酶C亚型在自噬体的生成中起着很重要的作用。     

腺苷及其类似物对猪红细胞膜上磷脂酰肌醇磷酸化的抑制作用

本文研究了腺苷及其类似物对猪红细胞膜上磷脂酰肌醇磷酸化的影响。研究结果表明:1、腺苷对磷脂酰肌醇磷酸化有明显的抑制作用,IC_(50)=15μmol/L;动力学分析表明,这种抑制作用机理是与ATP竞争性的;2、腺嘌呤、AMP、ADP、5＇-氯-5＇-脱氧腺苷、阿糖腺苷、2＇-脱氧腺苷对磷脂酰肌醇磷酸化有不同程度的抑制作用;3、cAMP对磷脂酰肌醇磷酸化也有抑制作用,这提示了cAMP与肌醇脂质信使系统有联系;5、6-氯-嘌呤核苷(100μmol/L)对该磷酸化无显著抑制作用。     

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

Upland rice and lowland rice exhibited different P/P expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations.To further understand the aquaporin function in plants underwater stress,the expression of a subgroup of aquaporins,plasma membrane intrinsic proteins(PIPs),was studied at boththe protein and mRNA level in upland rice(Oryza sativa L.cv.Zhonghan 3)and lowland rice(Oryza sativa L.cv.Xiushui63)when they were water stressed by treatment with 20% polyethylene glycol(PEG).Plants responded differently to20% PEG treatment.Leaf water content of upland rice leaves was reduced rapidly.PIP protein level increased markedlyin roots of both types,but only in leaves of upland rice after 10h of PEG treatment.At the mRNA level,OsPIP1;2,Os-PIP1;3,OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1;2 and OsPIP1;3 in leaves were significantly up-regulatedin upland rice,whereas the corresponding genes remained unchanged or down-regulated in lowland rice.Meanwhile,weobserved a significant increase in the endogenous abscisic acid(ABA)level in upland rice but not in lowland rice underwater deficit.Treatment with 60μM ABA enhanced the expression of OsPIP1;2,OsPIP2;5 and OsPIP2;6 in roots andOsPIP1;2,OsPIP2;4 and OsPIP2;6 in leaves of upland rice.The responsiveness of PIP genes to water stress and ABAwere different,implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signalingpathways during water deficit.     

PDZ结构域介导的PIP脂质信号转导

PDZ结构域是调控蛋白质/蛋白质相互作用的一类重要结构域,能特异结合蛋白质C末端一段有规律的氨基酸序列。含有PDZ结构域的支架蛋白能够组装成超大的蛋白质复合体来调控细胞内的信号转导通路。最新研究表明,PDZ结构域还能与PIP脂质直接相互作用,从而参与调控PIP脂质信号通路。将综合最新研究进展,阐明PDZ结构域与PIP脂质的作用方式,以及对相关PIP脂质信号转导的调控过程。     

Characterisation of the PTEN inhibitor VO-OHpic

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a phosphatidylinositol triphosphate 3-phosphatase that counteracts phosphoinositide 3-kinases and has subsequently been implied as a valuable drug target for diabetes and cancer. Recently, we demonstrated that VO-OHpic is an extremely potent inhibitor of PTEN with nanomolar affinity in vitro and in vivo. Given the importance of this inhibitor for future drug design and development, its mode of action needed to be elucidated. It was discovered that inhibition of recombinant PTEN by VO-OHpic is fully reversible. Both K _m and V _max are affected by VO-OHpic, demonstrating a noncompetitive inhibition of PTEN. The inhibition constants K _ic and K _iu were determined to be 27 ± 6 and 45 ± 11 nM, respectively. Using the artificial phosphatase substrate 3-O-methylfluorescein phosphate (OMFP) or the physiological substrate phosphatidylinositol 3,4,5-triphosphate (PIP₃) comparable parameters were obtained suggesting that OMFP is a suitable substrate for PTEN inhibition studies and PTEN drug screening.     

Regulation of the actin cytoskeleton by PIP2 in cytokinesis

Cytokinesis is a sequential process that occurs in three phases: assembly of the cytokinetic apparatus, furrow progression and fission (abscission) of the newly formed daughter cells. The ingression of the cleavage furrow is dependent on the constriction of an equatorial actomyosin ring in many cell types. Recent studies have demonstrated that this structure is highly dynamic and undergoes active polymerization and depolymerization throughout the furrowing process. Despite much progress in the identification of contractile ring components, little is known regarding the mechanism of its assembly and structural rearrangements. PIP2 (phosphatidylinositol 4,5-bisphosphate) is a critical regulator of actin dynamics and plays an essential role in cell motility and adhesion. Recent studies have indicated that an elevation of PIP2 at the cleavage furrow is a critical event for furrow stability. In this review we discuss the role of PIP2-mediated signalling in the structural maintenance of the contractile ring and furrow progression. In addition, we address the role of other phosphoinositides, PI(4)P (phosphatidylinositol 4-phosphate) and PIP3 (phosphatidylinositol 3,4,5-triphosphate) in these processes.     

Plasma Membrane Domain Patterning and Self-Reinforcing Polarity in Arabidopsis

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Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family

Plasma membrane intrinsic proteins （PIPs） are a subfamily ofaquaporins that enable fast and controlled translocation of water across the membrane. In this study, we systematically identified and cloned ten PIP genes from rice. Based on the similarity of the amino acid sequences they encoded, these rice PIP genes were classified into two groups and designated as OsPIP1-1 to OsPIP1-3 and OsPIP2-1 to OsPIP2-7 following the nomenclature of PIP genes in maize. Quantitative RT-PCR analysis identified three root-specific and one leaf-specific OsPIP genes. Furthermore, the expression profile of each OsPIP gene in response to salt, drought and ABA treatment was examined in detail. Analysis on transgenic plants over-expressing of either OsPIP1 （OsPIP1-1） or OsPIP2 （OsPIP2-2） in wild-type Arabidopsis, showed enhanced tolerance to salt （100 mM of NaCl） and drought （200 mM ofmannitol）, but not to salt treatment of higher concentration （150 mM of NaCl）. Taken together, these data suggest a distinct role of each OsPIP gene in response to different stresses, and should add a new layer to the understanding of the physiological function of rice PIP genes.     

Activation of inwardly rectifying potassium (Kir) channels by phosphatidylinosital-4,5-bisphosphate (PIP2): interaction with other regulatory ligands

All members of the inwardly rectifying potassium channels (Kir1-7) are regulated by the membrane phospholipid, phosphatidylinosital-4,5-bisphosphate (PIP₂). Some are also modulated by other regulatory factors or ligands such as ATP and G-proteins, which give them their common names, such as the ATP sensitive potassium (K_ATP) channel and the G-protein gated potassium channel. Other more non-specific regulators include polyamines, kinases, pH and Na⁺ ions. Recent studies have demonstrated that PIP₂ acts cooperatively with other regulatory factors to modulate Kir channels. Here we review how PIP₂ and co-factors modulate channel activities in each subfamily of the Kir channels.     

PIP3 is involved in neuronal polarization and axon formation

Recent experiments in various cell types such as mammalian neutrophils and Dictyostelium discoideum amoebae point to a key role for the lipid product of PI 3-kinase, PIP(3), in determining internal polarity. In neurons, as a consequence of the elongation of one neurite, the axon is specified and the cell acquires its polarity. To test the hypothesis that PI 3-kinase and PIP(3) may play a role in neuronal polarity, and especially in axon specification, we observed localization of PIP(3) visualized by Akt-PH-GFP in developing hippocampal neurons. We found that PIP(3) accumulates in the tip of the growing processes. This accumulation is inhibited by addition of PI 3-kinase inhibitors. Those inhibitors, consistently with a role of PIP(3) in process formation and elongation, delay the transition from stage 1 neurons to stage 3 neurons, and both axon formation and elongation. Moreover, when the immature neurite contacts a bead coated with laminin, a substrate known to induce axon specification, PIP(3) accumulates in its growth cone followed by a rapid elongation of the neurite. In such conditions, the addition of PI 3-kinase inhibitors inhibits both PIP(3) accumulation and future axon elongation. These results suggest that PIP(3) is involved in axon specification, possibly by stimulating neurite outgrowth. In addition, when a second neurite contacted the beads, this neurite rapidly elongates whereas the elongation of the first laminin-contacting neurite stops, consistently with the hypothesis of a negative feedback mechanism from the growing future axon to the other neurites.     

Regulation of Insulin Secretion by Phosphatidylinositol-4,5-Bisphosphate

The role of PIP₂ in pancreatic beta cell function was examined here using the beta cell line MIN6B1. Blocking PIP₂ with PH-PLC-GFP or PIP5KIγ RNAi did not impact on glucose-stimulated secretion although susceptibility to apoptosis was increased. Over-expression of PIP5KIγ improved cell survival and inhibited secretion with accumulation of endocytic vacuoles containing F-actin, PIP₂, transferrin receptor, caveolin 1, Arf6 and the insulin granule membrane protein phogrin but not insulin. Expression of constitutively active Arf6 Q67L also resulted in vacuole formation and inhibition of secretion, which was reversed by PH-PLC-GFP co-expression. PIP₂ co-localized with gelsolin and F-actin, and gelsolin co-expression partially reversed the secretory defect of PIP5KIγ-over-expressing cells. RhoA/ROCK inhibition increased actin depolymerization and secretion, which was prevented by over-expressing PIP5KIγ, while blocking PIP₂ reduced constitutively active RhoA V14-induced F-actin polymerization. In conclusion, although PIP₂ plays a pro-survival role in MIN6B1 cells, excessive PIP₂ production because of PIP5KIγ over-expression inhibits secretion because of both a defective Arf6/PIP5KIγ-dependent endocytic recycling of secretory membrane and secretory membrane components such as phogrin and the RhoA/ROCK/PIP5KIγ-dependent perturbation of F-actin cytoskeleton remodelling.     

Profilin binding to sub-micellar concentrations of phosphatidylinositol (4,5) bisphosphate and phosphatidylinositol (3,4,5) trisphosphate

Profilin is a small (12-15 kDa) actin binding protein which promotes filament turnover. Profilin is also involved in the signaling pathway linking receptors in the cell membrane to the microfilament system within the cell. Profilin is thought to play critical roles in this signaling pathway through its interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] (P.J. Lu, W.R. Shieh, S.G. Rhee, H.L. Yin, C.S. Chen, Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity, Biochemistry 35 (1996) 14027-14034). To date, profilin's interaction with polyphosphoinositides (PPI) has only been studied in micelles or small vesicles. Profilin binds with high affinity to small clusters of PI(4,5)P₂ molecules. In this work, we investigated the interactions of profilin with sub-micellar concentrations of PI(4,5)P₂ and PI(3,4,5)P₃. Fluorescence anisotropy was used to determine the relevant dissociation constants for binding of sub-micellar concentrations of fluorescently labeled PPI lipids to profilin and we show that these are significantly different from those determined for profilin interaction with micelles or small vesicles. We also show that profilin binds more tightly to sub-micellar concentrations of PI(3,4,5)P₃ (K_D = 720 μM) than to sub-micellar concentrations of PI(4,5)P₂ (K_D = 985 μM). Despite the low affinity for sub-micellar concentration of PI(4,5)P₂, profilin was shown to bind to giant unilamellar vesicles in presence of 0.5% mole fraction of PI(4,5)P₂ The implications of these findings are discussed.