Tyrosine-protein kinase inhibition in human erythrocytes by polyphosphoinositides (PIP and PIP2).

In human erythrocytes Ser/Thr- and Tyr-phosphorylations of cytoplasmic domain of band 3 are catalyzed by casein kinase I and Tyr-protein kinase respectively, both distributed between cytosol and membrane structures. The results reported here show that purified cytosolic Tyr-protein kinase activity, assayed on added substrates such as poly(Glu,Tyr)4:1 and isolated chymotryptic fragments of band 3 cytoplasmic domain (cdb3), is potently inhibited by PIP and even more by PIP2. Similar inhibitory effects are displayed by these polyphosphoinositides also on the endogenous Tyr-phosphorylation of band 3, when they are added to the isolated native membranes, thus suggesting their involvement in regulating in-vivo Tyr-phosphorylation of membrane proteins.     

PIP3, PIP2, and cell movement--similar messages, different meanings?

The inositol lipids PI(4,5)P(2) and PI(3,4,5)P(3) are important regulators of actin polymerization, but their different temporal and spatial dynamics suggest that they perform separate roles. PI(3,4,5)P(3) seems to act as an instructive second messenger, inducing local actin polymerization. PI(4,5)P(2) appears to be present at too high a concentration and homogeneous a distribution to fulfil a similar role. Instead, we suggest that PI(4,5)P(2) acts permissively, restricting new actin polymerization to the region of the plasma membrane.     

PIP2--the master key

The function of inwardly rectifying K+ (Kir) channels is highly diverse and therefore is tightly regulated by various environmental factors. In their article in this issue of Neuron, Rapedius et al. recognize a conserved structural mechanism for Kir channels gating by both pH and PIP2. In light of these findings and accumulated knowledge, PIP2 is suggested to have a common coregulatory role in the gating of Kir channels by all their soluble modulators.     

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。     

PIP1 and PIP2 aquaporins are differentially expressed during tobacco anther and stigma development

Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues, such as occurs during dehiscence of the anther and hydration of the pollen grain after it is deposited on a stigma. To get more insight in these processes, a set of putative aquaporins was cloned and it was found that at least 15 are expressed in reproductive organs, which indicates that the control of water flow is important for reproduction. Functional studies in Xenopus laevis oocytes using two of the cDNAs showed that NtPIP2;1 is an efficient aquaporin, whereas NtPIP1;1 is not. Expression studies on RNA and protein levels showed that PIP1 and PIP2 genes are differently expressed in reproductive organs: PIP1 RNA accumulates in the stigma, and PIP1 and PIP2 RNA can be detected in most tissues of the anther.     

Maize plasma membrane aquaporins belonging to the PIP1 and PIP2 subgroups are in vivo phosphorylated

Aquaporins are channel proteins that facilitate transmembrane water movement. In this study, we showed that plasma membrane intrinsic proteins (PIPs) from maize shoots are in vitro and in vivo phosphorylated on serine residues by a calcium-dependent kinase associated with the membrane fraction. Mass spectrometry identified phosphorylated peptides corresponding to the C-terminal region of (i) ZmPIP2;1, ZmPIP2;2 and/or ZmPIP2;7; (ii) ZmPIP2;3 and/or ZmPIP2;4; (iii) ZmPIP2;6; together with (iv) a phosphorylated peptide located in the N-terminal region of ZmPIP1;1, ZmPIP1;2, ZmPIP1;3 and/or ZmPIP1;4. The role of phosphorylation in the water channel activity of wild-type and mutant ZmPIP2;1 was studied in Xenopus laevis oocytes. Activation of endogenous protein kinase A increased the osmotic water permeability coefficient of ZmPIP2;1-expressing oocytes, suggesting that phosphorylation activates its channel activity. Mutation of S126 or S203, putative phosphorylated serine residues conserved in all plant PIPs, to alanine decreased ZmPIP2;1 activity by 30-50%, without affecting its targeting to the plasma membrane. Mutation of S285, which is phosphorylated in planta, to alanine or glutamate did not affect the water channel activity. These results indicate that, in oocytes, S126 and S203 play an important role in ZmPIP2;1 activity and that phosphorylation of S285 is not required for its activity.     

PIP5K-dependent production of PIP2 sustains microtubule organization to establish polarized transport in the Drosophila oocyte

The attachment of the cytoskeleton to the plasma membrane is crucial in controlling the polarized transport of cell-fate-determining molecules. Attachment involves adaptor molecules, which have the capacity to bind to both the plasma membrane and elements of the cytoskeleton, such as microtubules and actin filaments. Using the Drosophila oocyte as a model system, we show that the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), Skittles, is necessary to sustain the organization of microtubules and actin cytoskeleton required for the asymmetric transport of oskar, bicoid and gurken mRNAs and thereby controls the establishment of cell polarity. We show that Skittles function is crucial to synthesize and maintain phosphatidylinositol 4,5 bisphosphate (PIP2) at the plasma membrane in the oocyte. Reduction of Skittles activity impairs activation at the plasma membrane of Moesin, a member of the ERM family known to link the plasma membrane to the actin-based cytoskeleton. Furthermore, we provide evidence that Skittles, by controlling the localization of Bazooka, Par-1 and Lgl, but not Lkb1, to the cell membrane, regulates PAR polarity proteins and the maintenance of specific cortical domains along the anteroposterior axis.     

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.     

PIP3-dependent macropinocytosis is incompatible with chemotaxis

In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP₃) has been particularly controversial. PIP₃ has many cellular roles, notably in growth control and macropinocytosis as well as cell motility. Here we show that PIP₃ is not only unnecessary for Dictyostelium discoideum to migrate toward folate, but actively inhibits chemotaxis. We find that macropinosomes, but not pseudopods, in growing cells are dependent on PIP₃. PIP₃ patches in these cells show no directional bias, and overall only PIP₃-free pseudopods orient up-gradient. The pseudopod driver suppressor of cAR mutations (SCAR)/WASP and verprolin homologue (WAVE) is not recruited to the center of PIP₃ patches, just the edges, where it causes macropinosome formation. Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP₃ patches, but migrate more efficiently toward folate. Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases). Thus PIP₃ promotes macropinocytosis and interferes with pseudopod orientation during chemotaxis of growing cells.     

Chemotaxis in the absence of PIP3 gradients

Chemotaxing neutrophils and Dictyostelium amoebae produce in their plasma membranes the signaling lipid PI(3,4,5)P3 (PIP3) in gradients, which are orientated with the external chemotactic gradient and have been proposed to act as an internal compass, guiding movement of the cell. Evidence for and against this idea exists, but in all cases it depends on the use of inhibitors or gene knockouts, which may only incompletely abolish the PIP3 gradient. We have created a multiple gene-knockout strain in Dictyostelium lacking all five type-1 phosphoinositide 3-kinases encoded in the genome and the PTEN phosphatase and have thus removed all known ways for chemoattractant to produce PIP3 gradients in the plasma membrane. The resulting sextuple mutant is able to chemotax to cyclic-AMP with near wild-type efficiency and to trigger actin polymerization without apparent defect. There is, however, a consistent defect in movement speed in chemotaxis and especially in random movement. This work shows that polarization of membrane PIP3 is not necessary for accurate chemotaxis, but it can affect cell speed. A signaling pathway from receptor to cytoskeleton able to guide cells independently of polarized PIP3 and type-1 phosphoinositide 3-kinases must exist.     

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。     

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。     

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP₂) levels, consistent with prior evidence that MARCKS can regulate PIP₂ levels. We also found increased staining for PIP₂ in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP₂ co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP₂ levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.     

Studies on PIP2-phosphomonoesterase activity in human platelets

An occurrence of phosphatidylinositol 4,5-bisphosphate (PIP2) phosphomonoesterase in human platelets was demonstrated by analyzing phosphoinositides metabolism. The activity of the enzyme was maximum at pH 7.0. It was active even in the absence of Ca2+ or Mg2+ but it was enhanced in the presence of Mg2+ or NaF. The activity was inhibited by pyrophosphate. The activity was not altered in the presence of Ca2+. Thereby, besides phosphodiesteric cleavage by phospholipase C, the amount of PIP2 in activated platelets may be reduced by the combined effect of PIP2-phosphomonoesterase and suppressed activity of PI-kinase by Ca2+.     

Cooperative adsorption of ezrin on PIP2-containing membranes

By means of the quartz crystal microbalance (QCM) and scanning force microscopy (SFM), the adsorption of ezrin, a member of the ezrin/radixin/moesin protein family, on l-alpha-phosphatidylinositol-4,5-bisphosphate (PIP(2)) containing solid-supported membranes was investigated. An increase in the PIP(2) content in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes resulted in an increased amount of bound ezrin strongly supporting the crucial role of PIP(2) for ezrin recruitment to membranes. No ezrin adsorption to membranes composed of pure POPC was detected. To characterize the binding process in more detail, the kinetics and reversibility of ezrin adsorption were investigated by the QCM technique, showing that the protein remains partly bound after rinsing with pure buffer, which we suspected to be a result of lateral interactions between the proteins. SFM images revealed the formation of two-dimensional ezrin clusters on PIP(2)-doped POPC membranes. Time-elapsed SFM images show that the growth of protein domains occurs from a few nucleation sites. The QCM data in conjunction with the results obtained by SFM led us to propose that the binding process of ezrin occurs in a positive cooperative manner. When lateral interactions of the proteins on the membrane were taken into account, we were able to simulate the kinetics obtained from time-resolved QCM readouts by employing a model developed by Minton. On the basis of the kinetic analysis, we were also able to reconstruct the adsorption isotherm.