Type I Phosphotidylinosotol 4-Phosphate 5-Kinase γ Regulates Osteoclasts in a Bifunctional Manner

Type 1 phosphotidylinosotol-4 phosphate 5 kinase γ (PIP5KIγ) is central to generation of phosphotidylinosotol (4,5)P₂ (PI(4,5)P₂). PIP5KIγ also participates in cytoskeletal organization by delivering talin to integrins, thereby enhancing their ligand binding capacity. As the cytoskeleton is pivotal to osteoclast function, we hypothesized that absence of PIP5KIγ would compromise their resorptive capacity. Absence of the kinase diminishes PI(4,5) abundance and desensitizes precursors to RANK ligand-stimulated differentiation. Thus, PIP5KIγ^−/− osteoclasts are reduced in number in vitro and confirm physiological relevance in vivo. Despite reduced numbers, PIP5KIγ^−/− osteoclasts surprisingly have normal cytoskeletons and effectively resorb bone. PIP5KIγ overexpression, which increases PI(4,5)P₂, also delays osteoclast differentiation and reduces cell number but in contrast to cells lacking the kinase, its excess disrupts the cytoskeleton. The cytoskeleton-disruptive effects of excess PIP5KIγ reflect its kinase activity and are independent of talin recognition. The combined arrested differentiation and disorganized cytoskeleton of PIP5KIγ-transduced osteoclasts compromises bone resorption. Thus, optimal PIP5KIγ and PI(4,5)P₂ expression, by osteoclasts, are essential for skeletal homeostasis.     

Phosphatidic Acid and Phosphoinositide Turnover in Myelin and Its Stimulation by Acetylcholine

Brain slices obtained from the forebrains of adult female rats were incubated with [32P]phosphate and [3H]glycerol for 60 min, and lipids extracted and analyzed by TLC. The 32P in brain slice lipids was primarily in polyphosphoinositides, phosphatidylinositol (PI), and phosphatidate (PA). Distribution of the 32P-labeled lipids in isolated myelin was biased toward PA, 38%, relative to 16% in whole tissue slice lipids. About 33% of the total labeled PA in brain slices was accounted for by that in myelin. On a per milligram protein basis, PA labeling in myelin is about 2.5-fold greater than that of whole brain slice. Since incorporation of [3H]glycerol (indicative of synthesis by the de novo synthetic pathway) was at very low levels, we conclude that [32P]phosphate entered into myelin PA primarily through a pathway involving phospholipase C activity. Much of the production of PA relates to hydrolysis of phosphoinositides, yielding diacylglycerol which is then phosphorylated within myelin. The distribution of label among the inositol-containing lipids suggests that only a fraction of the myelin polyphosphoinositides serve as substrate for rapid diglyceride production. In the presence of 10 mM acetylcholine (ACh) there was a 20-60% stimulation of [32P]phosphate incorporation into PA and PI of brain slice lipids and purified myelin. Stimulation by ACh was blocked by atropine. The observed increase in the 32P/3H ratio, relative to controls, indicated that for both total lipids and myelin lipids there was selective stimulation of a phospholipase C-dependent cycle relative to de novo biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronically Administered Lithium Alters Neither myo-Inositol Monophosphatase Activity nor Phosphoinositide Levels in Rat Brain

Rats were exposed to either 29 consecutive days of LiCl injections or 27 and 39 days of dietary Li2CO3, followed by injected LiCl at the end of the diet to insure a constant level of exposure to the drug. At the end of the period of chronic exposure to lithium, the rats were sacrificed and brain myo-inositol-1-phosphate phosphohydrolase (myo-inositol monophosphatase) activity was measured. In none of the experiments was there any difference in the lithium-sensitive activity toward myo-inositol monophosphatase when comparing the control and chronic groups. These brains and those from another group of rats that had been given Li2CO3 in their diet for 41 days, followed by 7 additional days of LiCl injections, were also examined for changes in the levels of the phosphoinositides. No reproducible differences in the absolute tissue levels of those lipids were found when control and chronic lithium groups were compared. These results are contrary to published reports which suggest that myo-inositol monophosphatase activity increases and that the phosphatidylinositol level decreases in rat brain as a result of chronic administration of lithium.     

Cerebral Phosphoinositide and Energy Metabolism During and After Insulin-Induced Hypoglycemia

During and after insulin-induced hypoglycemia, changes in levels of cerebral phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA), triacylglycerol (TAG), diacylglycerol (DAG), and free fatty acids (FFAs) as well as the cerebral energy state were studied in relation to the EEG. In hypoglycemic rats with an EEG pattern of quasiperiodic sharp or slow sharp waves, which preceded the development of an isoelectric EEG, PIP2 levels increased significantly, together with a slight decrease in PI content. Levels of the other lipids did not change during this period. The cerebral energy state was affected only slightly in spite of profound decreases in plasma and tissue glucose levels. With 30 min of an isoelectric EEG, levels of all phosphoinositides and PA decreased significantly; total FFA and DAG contents increased seven- and twofold, respectively; the TAG-palmitate level decreased, and that of TAG-arachidonate increased. Plasma and tissue glucose were nearly depleted, and the cerebral energy state deteriorated severely. The increment in fatty acids in the DAG and FFA pools was less than their loss from phosphoinositides and PA, an observation suggesting vascular washout or oxidation of a portion of the FFAs produced. Following 90 min of glucose infusion, PIP and PA levels recovered to control values; however, the PIP2 content exceeded control levels, and that of PI remained below control levels. DAG and FFA contents returned to normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

磷酸肌醇家族与细胞功能

磷酸肌醇具有信号前体的作用,在激动剂作用下可产生第二信使,近年研究表明磷酸肌醇家族成员要 也可作为脂质第二信使将效应器蛋白聚集于特定膜区以调控它们的组装或活性。磷酸肌醇与蛋白的结合主要依赖于ＰＨ、ＦＹＶＥ及ＰＴＢ结构域。磷酸肌醇家族成员参与许多细胞活动诸如离子转运,膜泡运输,信号转导,细胞骨架再组装和核基因调控。     

1,2-Diacylglycerol Content and Its Arachidonyl-Containing Molecular Species Are Reduced in Sciatic Nerve from Streptozotocin-induced Diabetic Rats

The content of 1,2-diacylglycerol (DAG) was determined in sciatic nerves from normal and streptozotocin-induced diabetic rats. In nerves frozen in situ, DAG content was reduced 22% in the proximal region and 77% in the distal region of diabetic nerve, principally because of the loss of associated fat. DAG levels in freshly dissected and desheathed diabetic nerve were decreased from 23 to 30% as compared with normal nerve. Determination of DAG molecular species distribution in desheathed normal nerve indicated that 18:0/20:4 accounted for 34%, 16:0/18:1 for 17%, and several other polyunsaturated fatty acid-containing species for 17% of the total. In diabetic nerve, the quantity of the 18:0/20:4 DAG, species was reduced by 37%, and this drop was 62% of the reduction in all molecular species. The content of the minor species, 16:0/20:4 DAG, was decreased by 48%. Our results suggest that nerve DAG arises in large part from phosphoinositide degradation. Moreover, these results provide support for the hypothesis that reduced Na+,K(+)-ATPase activity in diabetic nerve is a consequence of decreased phosphoinositide turnover, which thereby generates insufficient DAG to maintain a protein kinase C-mediated step necessary for activation of Na+,K(+)-ATPase.     

Endosomal Sorting Complex Required for Transport (ESCRT) Complexes Induce Phase-separated Microdomains in Supported Lipid Bilayers

The endosomal sorting complex required for transport (ESCRT) system traffics ubiquitinated cargo to lysosomes via an unusual membrane budding reaction that is directed away from the cytosol. Here, we show that human ESCRT-II self-assembles into clusters of 10-100 molecules on supported lipid bilayers. The ESCRT-II clusters are functional in that they bind to ubiquitin and the ESCRT-III subunit VPS20 at nanomolar concentrations on membranes with the same stoichiometries observed in solution and in crystals. The clusters only form when cholesterol is included in the lipid mixture at >10 mol %. The clusters induce the formation of ordered membrane domains that exclude the dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbo-cyanine perchlorate. These results show that ESCRT complexes are capable of inducing lateral lipid phase separation under conditions where the lipids themselves do not spontaneously phase-separate. This property could facilitate ESCRT-mediated membrane budding.     

First came the link between phosphoinositides and Ca signalling, and then a deluge of other phosphoinositide functions

The link between phosphoinositide turnover and Ca²⁺-regulated cell processes goes back half a century, but only began to be understood in the 1970s. This article briefly outlines how the roles of these minority membrane lipids in Ca²⁺ signalling and then in multifarious other biological processes were recognised during the latter half of the 20th century.     

Biochemistry and structure of phosphoinositide phosphatases

Phosphoinositides are the phosphorylated derivatives of phosphatidylinositol, and play a very significant role in a diverse range of signaling processes in eukaryotic cells. A number of phosphoinositide-metabolizing enzymes, including phosphoinositide-kinases and phosphatases are involved in the synthesis and degradation of these phospholipids. Recently, the function of various phosphatases in the phosphatidylinositol signaling pathway has been of great interest. In the present review we summarize the structural insights and biochemistry of various phosphatases in regulating phosphoinositide metabolism. [BMB Reports 2013; 46(1): 1-8]     

Phosphatidylserine stimulation of Drs2p·Cdc50p lipid translocase dephosphorylation is controlled by phosphatidylinositol-4-phosphate

Here, Drs2p, a yeast lipid translocase that belongs to the family of P(4)-type ATPases, was overexpressed in the yeast Saccharomyces cerevisiae together with Cdc50p, its glycosylated partner, as a result of the design of a novel co-expression vector. The resulting high yield allowed us, using crude membranes or detergent-solubilized membranes, to measure the formation from [γ-(32)P]ATP of a (32)P-labeled transient phosphoenzyme at the catalytic site of Drs2p. Formation of this phosphoenzyme could be detected only if Cdc50p was co-expressed with Drs2p but was not dependent on full glycosylation of Cdc50p. It was inhibited by orthovanadate and fluoride compounds. In crude membranes, the phosphoenzyme formed at steady state at 4 °C displayed ADP-insensitive but temperature-sensitive decay. Solubilizing concentrations of dodecyl maltoside left this decay rate almost unaltered, whereas several other detergents accelerated it. Unexpectedly, the dephosphorylation rate for the solubilized Drs2p·Cdc50p complex was inhibited by the addition of phosphatidylserine. Phosphatidylserine exerted its anticipated accelerating effect on the dephosphorylation of Drs2p·Cdc50p complex only in the additional presence of phosphatidylinositol-4-phosphate. These results explain why phosphatidylinositol-4-phosphate tightly controls Drs2p-catalyzed lipid transport and establish the functional relevance of the Drs2p·Cdc50p complex overexpressed here.