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Phosphatidylinositol and Related Phosphoinositides



Although it had long been recognized that phosphatidylinositol was a key membrane constituent, it was initially something of a surprise when the manifold biological activities of this lipid, its phosphorylated metabolites and the hydrolysis products were discovered in animals, plants and microorganisms. Many years later, these continue to be a major focus for research efforts around the world with considerable relevance to human health. Glycosyl-phosphatidylinositol (GPI) as an anchor for proteins is considered to be a sufficiently important topic for its own web page.


1.  Phosphatidylinositol

Structure and Occurrence: Phosphatidylinositol is an important lipid, both as a key membrane constituent and as a participant in essential metabolic processes in all plants and animals, both directly and via a number of metabolites. It is an acidic (anionic) phospholipid that in essence consists of a phosphatidic acid backbone, linked via the phosphate group to inositol (hexahydroxycyclohexane). In most organisms, the stereochemical form of the last is myo-D-inositol (with one axial hydroxyl in position 2 with the remainder equatorial, i.e. a chair-like structure), although other forms (scyllo- and chiro-) have been found on occasion in plants. The 1‑stearoyl,2-arachidonoyl molecular species, which is of considerable biological importance in animals, is illustrated.

Formula of phosphatidylinositol

Phosphatidylinositol is especially abundant in brain tissue, where it can amount to 10% of the phospholipids, but it is present in all tissues, cell types and membranes. In rat liver, it amounts to 1.7 micromoles/g., i.e. less than phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine.

The fatty acid composition of phosphatidylinositol is rather distinctive as shown in Table 1. Thus, in animal tissues, the characteristic feature is a high content of stearic and arachidonic acids. All the stearic acid is linked to position sn-1 and all the arachidonic acid to position sn-2, and as much as 78% of the total lipid may consist of the single molecular species sn-1-stearoyl-sn-2-arachidonoyl-glycerophosphorylinositol (see Table 2 below). Although 1-alkyl- and alkenyl- forms of phosphatidylinositol are known, they tend to be much less abundant than the diacyl form. In plant phosphatidylinositol, e.g. Arabidopsis thaliana as listed, palmitic acid is the main saturated fatty acid in position sn-1, while linoleic and linolenic acids are the main unsaturated components in position sn-2. Similarly in yeast, palmitic acid is in position sn-1 with oleic and palmitoleic acids in position sn-2 predominantly; the Amoebozoa have a C16 alkyl group in position sn-1 and cis-vaccenic acid in position sn-2.

Table 1. Fatty acid composition of phosphatidylinositol (wt % of the total) in animal and plant tissues.
Tissue Fatty acids
16:0 18:0 18:1 18:2 18:3 20:3 20:4 22:3 22:5 22:6
 
Bovine heart [1] 8 40 14 1 1 1 31 1 1 2
Bovine liver [2] 5 32 12 6 1 7 23 4 3 5
Rat liver [3] 5 49 2 2 4 35 1
 
A. thaliana [4] 48 3 2 24 24
[1] = Thompson, W. and MacDonald, G., Eur. J. Biochem., 65, 107-111 (1976). [2] = Thompson, W. and MacDonald, G., J. Biol. Chem., 250, 6779-6785 (1975). [3] = Wood, R. and Harlow, R.D. Arch. Biochem. Biophys., 135, 272-281 (1969). [4] = Browse, J., Warwick, N., Somerville, C.R. and Slack, C.R. Biochem. J., 235, 25-31 (1986).


Biosynthesis: The basic mechanism for biosynthesis of phosphatidylinositol and phosphatidylglycerol is sometimes termed a branch point in phospholipid synthesis, as phosphatidylcholine and phosphatidylethanolamine are produced by a somewhat different route.

As with phosphatidylglycerol (and thence cardiolipin), phosphatidylinositol is formed biosynthetically from phosphatidic acid via the intermediate cytidine diphosphate diacylglycerol in a reaction with inositol catalysed by the enzyme CDP-diacylglycerol inositol phosphatidyltransferase ('phosphatidylinositol synthase') of which only one isoform exists in mammals; the other product of the reaction is cytidine monophosphate (CMP). The enzyme is located in the endoplasmic reticulum, in part in a subcompartment of this associated with mitochondria (mitochondria-associated membranes) and in mitochondria, although it may also occur in the plasma membrane in yeasts, and almost entirely on the cytosolic side of the bilayer. Phosphatidylinositol is then delivered to other membranes either by vesicular transport or via the agency of specific transfer proteins.

Biosynthesis of phosphatidylinositol in eukaryotes

The phosphatidylinositol synthase per se does not exhibit the fatty acyl specificity observed in the final product, but earlier in the biosynthetic process 1-stearoyl-2-arachidonoyl species of diacyl-sn-glycerols are converted preferentially into phosphatidic acid by the epsilon isoform of diacylglycerol kinase (DGKε) and one of the CDP-diacylglycerol synthases (CDS2) has similar specificity in the generation of the immediate precursor CDP-diacylglycerols from phosphatidic acid. In addition, some specificity may be introduced via lysophosphatidylinositol, formed as a by-product of eicosanoid formation (see below) or as an intermediate as part of the normal cycle of deacylation-acylation of phosphatidylinositol in tissues in which the fatty acid composition is remodelled to give final distinctive composition (the Lands’ cycle - see our web page on phosphatidylcholine). A membrane-bound O-acyltransferase (MBOAT7 or LPIAT1) specific for position sn-2 of lysophosphatidylinositol with a marked preference for arachidonoyl-CoA is ubiquitously expressed in animal tissues, and this may be one means by which free arachidonic acid and eicosanoid levels are regulated. In macrophages subjected to inflammatory stimuli, phosphatidylinositol containing two molecules of arachidonate is produced by remodelling reactions, and there is evidence that it is a novel bioactive phospholipid regulating innate immune responses in these cells. Further specificity may be introduced by lysocardiolipin acyltransferase (LYCAT; also known as LCLAT1 or ALCAT1), which exhibits a preference for lysophosphatidylinositol and lysophosphatidylglycerol over other phospholipids in vitro, and incorporates 18:0 rather than shorter chain fatty acids into position sn-1 of phosphatidylinositol and other phosphoinositides, especially phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3-phosphate.

In contrast, plants have two phosphatidylinositol synthase isoforms, PIS1 and PIS2, which display specificities for particular species of the CDP-diacylglycerol substrate. PIS1 generates phosphatidylinositol with saturated or monounsaturated fatty acids preferentially, while PIS2 generates polyunsaturated species, the two forms possibly having different functions. In protozoan parasites, such as Trypanosoma brucei, the active site of phosphatidylinositol synthase may be the lumen of the endoplasmic reticulum and Golgi. There is evidence for two distinct pools of product in this organism, the bulk membrane form derived from inositol imported from the environment, and a second used for the synthesis of GPI anchors, which uses myo-inositol synthesised de novo.

Phosphatidylinositol is found in all eukaryotes, which are in general able to synthesise inositol de novo via glucose-6-phosphate. In contrast, few bacteria appear to contain phosphatidylinositol, although it is essential for Mycobacteria and phosphatidylinositol-containing lipids are found in the actinomycetes (lipophosphoglycans) and some proteobacteria. Archaeal ether lipids include analogues of phosphatidylinositol, and these are synthesised by a quite different mechanism from that in eukaryotes, i.e. by reaction of inositol 1-phosphate with CDP-archaeol to form archaetidylinositol 3-phosphate and thence archaetidylinositol (see our web page on Archaeal lipids for a more detailed discussion). This mechanism is now believed to be more widespread in bacterial species. In the thermophilic bacterium Rhodothermus marinum, L-myo-inositol-1-phosphate cytidylyltransferase catalyses the formation of CDP-inositol from inositol-1-phosphate and CTP, and this is reacted with dialkylether glycerols to produce phosphoinositol ether lipids (all other pathways involve activation of the lipid group).

Biosynthesis of phosphatidylinositol in R. marinum

In animal tissues, phosphatidylinositol is the primary source of the arachidonic acid required for biosynthesis of eicosanoids, including prostaglandins, via the action of the enzyme phospholipase A2, which releases the fatty acids from position sn-2. The reverse reaction also occurs.

Release of arachidonic acid from phosphatidylinositol

Phosphatidylinositol and the phosphatidylinositol phosphates are also the main source of diacylglycerols that serve as signalling molecules in animal and plant cells, via the action of a family of highly specific enzymes collectively known as phospholipase C (see our web pages on diacylglycerols for further discussion). Diacylglycerols regulate the activity of a group of at least a dozen related enzymes known as protein kinase C, which in turn control many key cellular functions, including differentiation, proliferation, metabolism and apoptosis. Indeed, the biological actions of the various components released have been the subject of intensive study over many years. 2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, may also be a product of phosphatidylinositol catabolism.

In addition to functioning as negatively charged building blocks of membranes, the inositol phospholipids (including the phosphatidylinositol phosphates or 'polyphosphoinositides' discussed below) have crucial roles in interfacial binding of proteins and in the regulation of proteins at the cell interface. As phosphoinositides are polyanionic, they can be very effective in non-specific electrostatic interactions with proteins. However, they are especially efficient in specific binding to so-called ‘PH’ domains of cellular proteins. At least three phosphatidylinositol molecules are present in the crystal structure of human erythrocyte glycophorin, and they are believed to influence binding to other proteins via their head groups. The lipid is also a structural component of yeast cytochrome bc1.


2.  Phosphatidylinositol Phosphates (Polyphosphoinositides) in Animals


Structure and Occurrence:  The pioneering work of Mable and Lowell Hokin in the 1950s lead to the discovery that phosphatidylinositol was converted to polyphosphoinositides with important signalling and other functional activities in animal cells. This lipid is now known to be phosphorylated by a number of different kinases that place the phosphate moiety on positions 3, 4 and 5 of the inositol ring. Seven different isomers are known, all of which have distinct biological activities. Although the most significant in quantitative and possibly biological terms were long thought to be phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, it is now recognized that 3-phosphorylated forms are also extremely important.

Formulae of phosphatidylinositol phosphates

These lipids are usually present at low levels only in tissues, typically at about 0.5 to 1% of the total lipids of the inner leaflet of the plasma membrane, so they are unlikely to have an appreciable structural role. The positional distributions of fatty acids in the phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate of ox brain are listed in Table 2. In each the saturated fatty acids are concentrated in position sn-1 and polyunsaturated, especially arachidonate, in position sn-2; there are few differences among the three lipids.

Table 2. Distribution of fatty acids (mol % of the total) in positions sn‑1 and sn‑2 in phosphatidylinositol (PI) and the phosphatidylinositol mono- and diphosphates of ox brain.
Fatty acids PI PI monophosphate PI diphosphate
sn-1 sn-2 sn-1 sn-2 sn-1 sn-2
 
16:0 15 9 7
18:0 74 69 69
18:1 10 10 20 13 21 10
18:2 1 2 trace 1 1 1
20:3(n-9) 5 10 10
20:3(n-6) 5 11 12
20:4(n-6) 67 49 52
22:3 7 10 7
22:6(n-3) trace trace trace
Data from Holub, B.J. et al., J. Lipid Res., 11, 558-564 (1970).
Molecular species data, see Traynor-Kaplan, A. et al., Biochim. Biophys. Acta, 1862, 513-522 (2017).

Biosynthesis:  Phosphatidylinositol per se is of course the ultimate precursor of all phosphoinositides, the head groups of which have different charges and structures that impact directly on membrane properties and via the metabolic interactions can function as chemical switches. The individual phosphoinositides are maintained at steady state levels in the inner leaflet of the plasma membrane by a continuous and sequential series of phosphorylation and dephosphorylation reactions by specific kinases, phosphatases and phospholipase C enzymes, which are regulated and/or relocated through cell surface receptors for extracellular ligands. This has been termed a ‘futile cycle’, and can consume a significant proportion of cellular ATP production. Controlled synthesis of these different phosphoinositides occurs in different intracellular compartments for distinct and independently regulated functions with differing target enzymes. In mammals, the complexity is such that 18 phosphoinositide inter-conversion reactions have been identified to date, and these are mediated by 19 phosphoinositide kinases and 28 phosphoinositide phosphatases. As a generality, most mono-phosphorylations occur in endomembranes, such as the endosomes and the Golgi network, while second and third phosphorylations occur primarily at the plasma membrane, and this is reflected in the lipid composition of each membrane. It should be noted that there are links to the metabolism of phosphatidylcholine, which can be hydrolysed by phospholipase D to phosphatidic acid, an important activator of key kinases.

Polyphosphoinositide metabolism

Thus, phosphatidylinositol 4-phosphate is produced by the action of a phosphatidylinositol 4-kinase (PI4K), and is in turn phosphorylated by a phosphatidylinositol phosphate 5-kinase (PIPK I) to form phosphatidylinositol 4,5-bisphosphate, although this can also be formed by phosphorylation of phosphatidylinositol 5-phosphate by a specific 4-kinase (PIPK II). Some selectivity in the formation of molecular species or remodelling may occur to further enrich the arachidonic acid content.

Subsequently, it was discovered that phosphatidylinositol is also phosphorylated by a 3-kinase (PI3K III) to produce phosphatidylinositol 3-phosphate. In fact, three phosphatidylinositol 3-kinases families (eight isoforms) have been described, each with distinct substrate specificities. A second phosphoinositide signalling pathway involves activation of two of these 3-kinases, stimulated by growth factors and hormones, which phosphorylate phosphatidylinositol 4,5-bisphosphate (by PI3K I) and phosphatidylinositol 4-phosphate (by PI3K II) to produce phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, respectively. In addition to the specific kinase, a complex of other binding proteins are required for the reaction. In turn, these lipids stimulate signalling pathways involved in cell growth, survival, proliferation, and motility, often aided by a protein designated AKT, which is recognized as a direct effector of the PI3K signaling cascade. In particular, they mediate insulin-independent glucose transport and many of the physiological actions of insulin. As the class I PI3K isoforms especially have been implicated in the aetiology and maintenance of various diseases and metabolic disorders, including cancer, inflammation and autoimmunity, drug companies are actively pursuing the development of inhibitors. While phosphatidylinositol 3 phosphate and other 3-phosphorylated metabolites amount to only about 0.5% of the total phosphoinositides in resting mammalian cells, they are now recognized to be of profound importance for cellular metabolism.

In addition to the activity of kinases, the amounts of these various metabolites are regulated by the activities of specific phosphoinositide phosphatases, which are highly conserved in eukaryotes and dephosphorylate phosphoinositides at the 3, 4 and 5 positions of the inositol ring. For example, so-called ‘SHIP’ phosphatases convert phosphatidylinositol 4,5-bisphosphate back to phosphatidylinositol 4-phosphate by hydrolysis of the 5-phosphate group. 3-Phosphorylated phosphoinositides are only degraded by phosphatases, especially those of the PTEN family, and not by phospholipase C (see below).

Scottish thistleThe various organelles in cells have membranes with distinct functions and molecular compositions. Yet, they are all formed primarily at the endoplasmic reticulum, and the different membrane lipids and proteins must be transported to each site via specific membrane trafficking processes. A concept has emerged in which each phosphoinositide has its own role – the ‘lipid code’ hypothesis, in which defined lipids act as labels for each cellular membrane to maintain the orderly flow required for the complexities of membrane trafficking and spatio-temporal signalling reactions. Thus, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate are found mainly on the Golgi, plasma membrane, early endosomes and late endocytic organelles, respectively, where they are sometimes regarded as landmarks for these compartments. In consequence, there is selective recruitment of effector proteins to particular membranes by binding specifically to each type of phosphoinositide, and this is followed by interactions between the phosphoinositide-binding proteins and various enzymes. Further phosphorylation via kinases or removal of phosphates by phosphatases continues in the various organelles.

Function: The distinctive subcellular location of the different phosphoinositide species, together with the rapid and reversible nature of phosphorylation, gives them a central and general position in the fields of cell signalling cascades and intracellular membrane trafficking. They are able to achieve signalling effects directly by binding to specific cytosolic domains of membrane proteins via their polar head groups, thereby triggering downstream signalling cascades. In this way, they can regulate the function of innumerable proteins integral to membranes, for example by relocating a protein from one area of the cell to another, usually from the cytosol to the inner leaflet of the plasma membrane, or they can attract cytoskeletal and signalling components to the membrane. Amongst the proteins that bind to phosphoinositides in this way are phospholipases, protein kinases, regulators of membrane trafficking, and cytoskeletal, scaffold and ion channel proteins.

Binding usually involves electrostatic interactions with the negative charges of the phosphate groups on the inositol ring with characteristic clusters of basic amino acid residues in proteins, and it can lead to folding and thence increased activity of unstructured peptides. In particular, a binding region termed the pleckstrin homology (PH) domain, consisting of ~100 amino acids, is the most abundant lipid-binding domain with more than 225 examples identified, and this can exhibit great specificity for particular polyphosphoinositides, often binding simultaneously with other proteins. While the interaction is driven by non-specific electrostatic interactions initially, it is followed by specific binding to increase the membrane residence time. The distinctive phosphoinositide composition of membranes in different organelles adds strength and specificity to the interactions by cooperative binding with other membrane proteins.

The phosphatidylinositol monophosphates are present in cells at low levels only, although their levels do not appear to fluctuate greatly. Phosphatidylinositol 3-phosphate (PI(3)P) has been implicated in membrane trafficking through its interactions with certain proteins in endosomes. Indeed, it is a major determinant of the identity of the membrane of early endosomes, and it participates in most aspects of endosomal function, i.e. the controlled internal degradation and turnover of cellular constituents (autophagy). In general, it controls cellular processes by recruiting effector proteins through low to moderate affinity interaction with specific PI(3)P binding domains. After sorting of the lysosomal contents, components of the internalized cargo are recycled to the plasma membrane and PI(3)P is dephosphorylated to phosphatidylinositol by a specific phosphatase. Thus the processes of internalization, sorting, and trafficking of membrane proteins depends on the interconversion of phosphoinositide species by coordinated phosphorylation-dephosphorylation reactions. A further function of PI(3)P is in the regulation of the final stage of cell division (cytokinesis), and the lipid is known to accumulate where cells divide. It may also have a role as a second messenger with an involvement in a number of physiological and pathological processes, including cancer as mutations in phosphatidylinositol 3-kinase are found in a high proportion of human cancers.

Phosphatidylinositol 4-phosphate is the precursor for the 4,5-bisphosphate, but it binds to a protein on the cytoskeleton of the cell and has its own characteristic functions. In particular, it is essential for the structure and function of the Golgi complex, where it is required for the recruitment of specific proteins. Some of these participate in vesicle formation, while others like the oxysterol binding protein are involved in lipid transfer. It has been called the 'fuel' that drives sterol transport and allows the establishment of active sterol concentration gradients across membrane-bound compartments. In the plasma membrane, it can support the functions of ion channels, and it also contributes to the anchoring of proteins with polybasic domains.

While the biological properties of phosphatidylinositol 5-phosphate have taken longer to unravel, because of the difficulties of separation of this isomer, it is now apparent that it is involved in osmoregulation both in plants and animals. It also has signalling functions, and although it is the least abundant phosphatidylinositol monophosphate, it is involved in signalling at the nucleus and in the cytoplasm, modulating cellular responses to various stresses, hormones and growth factors.

Phosphatidylinositol 4,5-bisphosphate is an essential lipid messenger with vital signalling functions as well as serving as a precursor of key metabolites, especially diacylglycerols (see below). In the plasma membrane, it complexes with and regulates many cytoplasmic and membrane proteins, including those concerned with ion channels for potassium, calcium, sodium and other ions. In most instances, it increases channel activity, while its hydrolysis by phospholipase C reduces such activity. In particular, it appears to interact with cationic residues of a large array of proteins in concert with cholesterol to form localized membrane domains that are distinct from the sphingolipid-enriched rafts. Indeed, it has a much higher concentration than other phosphoinositide species in cells, but most of this is in effect sequestered by binding proteins. Also, phosphatidylinositol 4,5-bisphosphate and its diacylglycerol metabolites are important for vesicle formation in membranes. For example, a major pathway in cells for internalization of cell surface proteins such as transferrin is the clathrin-coated vesicle pathway. Phosphatidylinositol 4,5-bisphosphate is essential to this process in that it binds to the machinery involved in the membrane, increasing the number of clathrin-coated pits and permitting internalization of proteins. It also has a function in caveolae, where it is concentrated at the rim.

Scottish thistlePhosphatidylinositol 4,5-bisphosphate is intimately involved in the development of the actin cytoskeleton and its attachment to the plasma membrane, thereby controlling cell shape, motility, and many other processes. In particular it binds with high specificity to vinculin, a membrane-cytoskeletal protein that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton. In the cell nucleus, this lipid is believed to be involved in maintaining chromatin, the complex combination of DNA, RNA, and protein that makes up chromosomes in a transcriptionally active conformation, as well as being a precursor for further signalling molecules.

It is the primary precursor of the endocannabinoid 2-arachidonoylglycerol in neurons, and it is also an essential cofactor for phospholipase D and so affects the cellular production of phosphatidic acid with its specific signalling functions. By binding specifically to ceramide kinase, the enzyme responsible for the synthesis of ceramide-1-phosphate, it has an influence on sphingolipid metabolism. Like ceramide-1-phosphate, it binds to and activates the Ca2+-dependent phospholipase A2, which generates the arachidonate for eicosanoid production. One molecule of phosphatidylinositol 4,5-bisphosphate is bound to each subunit of the protein in the X-ray crystal structures of mammalian GIRK2 potassium channel, where it enables a conformational change that assists the transport function of the protein.

Perhaps, the best characterized of the phosphoinositide signalling functions results from the hydrolysis of phosphatidylinositol phosphates by phospholipase C isoforms in this instance to produce sn-1,2-diacylglycerols and inositol 3,4,5-trisphosphate (see below), which act as second messengers. Only those polyunsaturated diacylglycerol species derived from phosphatidylinositol 4,5-bisphosphate are able to bind and activate protein kinase C (α, ε, δ) isoforms both in vitro and in vivo. This lipid is doubly important as it also binds strongly to these enzymes via a basic patch distal to a Ca2+ binding site, and this targets them selectively to the plasma membrane.

Phosphatidylinositol 3,5-bisphosphate is present at low levels only in cells (0.04-0.1% of the total phosphatidylinositides), but it is important in membrane and protein trafficking, especially in the late endosomes in eukaryotes and in yeast vacuoles. It is also involved in the mediation of signalling in response to stress and hormonal cues and in the control of ion transport in membranes, while genetic studies confirm that it is essential for healthy embryonic development Phosphatidylinositol 3,4-bisphosphate regulates a variety of cellular processes with relevance to health and disease that include B cell activation and autoantibody production, insulin sensitivity, neuronal dynamics, endocytosis and cell migration; it is known to bind selectively to a number of proteins and especially one of the protein kinases.

Phosphatidylinositol 3,4,5-trisphosphate has been implicated in a variety of cellular functions, such as growth, cell survival, proliferation, cytoskeletal rearrangement, intracellular vesicle trafficking, and cell metabolism. In particular, it is an important component of a signalling pathway in the cell nucleus. In contrast to phosphatidylinositol 3-phosphate, it opposes autophagy. During feeding, various physiological responses lead to the secretion of insulin, which stimulates the phosphorylation of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate and triggers a signalling cascade that leads to the suppression of autophagy. When this pathway is impaired it has deleterious effects upon the insulin resistance associated with various metabolic diseases including obesity and diabetes.

The human immune system utilizes neutrophils, which are highly mobile cells, to eliminate pathogens from infected tissue. The first step is to track and then pursue molecular signals, such as cytokines, emitted by pathogens. It has been established that two phospholipids operate in sequence to point the neutrophils in the correct direction. The first of these is phosphatidylinositol 3,4,5-trisphosphate, which binds to a specific protein DOCK2 and enables it to translocate to the plasma membrane. Then phosphatidic acid, generated by the action of phospholipase D on phosphatidylcholine, takes over and directs the DOCK2 to the leading edge of the plasma membrane. This causes polymerization of actin within the cell and in effect reshapes the neutrophil and points it in the direction from which the pathogens signals are coming.


3.  Water-Soluble Inositol Phosphates

As mentioned briefly above, hydrolysis of phosphatidylinositol phosphates by calcium-dependent phospholipase C (or 'phosphoinositidase C') leads to generation of sn-1,2-diacylglycerols, which act as second messengers in animal cells and are of enormous metabolic importance. There are many different enzymes of this type, but the activity of the phosphoinositide-specific phospholipase C constitutes an essential step in the inositide signalling pathways. The enzyme exists in six families consisting of at least 13 isoenzymes, each of which has a distinctive role and can have a characteristic cell distribution that is linked to a specific function. Activity of these enzymes is stimulated by signalling molecules such as G-protein coupled receptors, receptor tyrosine kinases, Ras-like GTPases and calcium ions, thus linking the hydrolysis of phosphatidylinositol phosphates to a wide range of other cellular signals. As phospholipase C is a soluble protein located mainly in the cytosol, translocation to the plasma membrane is a crucial step in signal transduction. Regulation of these isoenzymes is vital for health as they are associated with the activation or inhibition of important pathophysiological processes.

The other products of this reaction that are of special relevance because of their many essential functions are water-soluble inositol phosphates. Up to 60 different compounds of this type are possible, and at least 37 of these have been found in nature at the last count, all of which are also extremely important biologically. However, polyphosphoinositides with a phosphate in position 3 are not substrates for phospholipase C.

Generation of water-soluble inositol phosphates

For example, under the action of various physiological stimuli in animals and acting via various G-protein-coupled receptors, phosphatidylinositol 4,5-bisphosphate releases inositol 1,4,5-trisphosphate, an important cellular messenger that diffuses into the cytosol and stimulates calcium release from an ATP-loaded store in the endoplasmic reticulum via ligand-gated calcium channels (the diacylglycerols remain in the membrane to recruit and activate members of the protein kinase C family). The increase in calcium concentration, together with the altered phosphorylation status, activates or de-activates many different protein targets, enabling cells to respond in an appropriate manner to the extracellular stimulus.

All of the various inositol phosphates appear to be involved in the control of cellular events in very specific ways, but especially in the organization of key signalling pathways, the rearrangement of the actin cytoskeleton or intracellular vesicle trafficking. They have also been implicated in gene transcription, RNA editing, nuclear export and protein phosphorylation. As these remarkable compounds can be rapidly synthesised and degraded in discrete membrane domains or even sub-nuclear structures, they are considered to be ideal regulators of dynamic cellular mechanisms. From structural studies of inositol polyphosphate-binding proteins, it is believed that the inositides may act in part at least by modifying protein function by acting as structural cofactors, ensuring that proteins adopt their optimum conformations. In addition, phosphoinositides and the inositol polyphosphates are key components of the nucleus of the cell, where they have many essential functions, including DNA repair, transcription regulation and RNA dynamics. It is believed that they may be activity switches for the nuclear complexes responsible for such processes, with the phosphorylation state of the inositol ring being of primary importance. As different isomers appear to have specific functions at each level of gene expression, extracellular events must coordinate the production of these compounds in a highly synchronous manner.

The extraordinary range of activities of phosphoinositides is relevant to major human diseases, including cancer and diabetes, making them important targets for pharmacological research and intervention. It should also be noted that the phospholipase C isoenzymes regulate the concentration of phosphatidylinositol 4,5-bisphosphate and related lipids and thence their activities in addition to the generation of new biologically active metabolites.


4.  Phosphatidylinositides in Plants

In plants as in animals, phosphatidylinositol and polyphosphoinositides have essential biological functions, exerting their regulatory effects by acting as ligands that bind to protein targets via specific lipid-binding domains and so alter the location of proteins and their enzymatic activities. However, it appears that polyphosphoinositide metabolism developed in different ways after the divergence of the animal and plant kingdoms so the details of the processes in each are very different. Phosphatidylinositol per se is of course the precursor of the phosphorylated forms and determines their fatty acid compositions. It also has a role in inhibiting programmed cell death by acting as the biosynthetic precursor of the sphingolipid ceramide phosphoinositol and so reducing the levels of ceramide. As in animals, the various phosphoinositides are produced by a series of kinases and phosphatases (in many isoforms) in different cellular membranes. For example, phosphatidylinositol is generated mainly in the endoplasmic reticulum, while PI 4-kinases and their product are located in the trans-Golgi network and nucleus, and PI4P 5-kinases and product are present in the plasma membrane. How the various metabolites are transported between membranes has yet to be determined.

Polyphosphoinositide metabolism in plants

Although what might be considered normal levels of phosphatidylinositol 4-phosphate are present, phosphatidylinositol 4,5-bisphosphate levels are extremely low in plants (10 to 20-fold lower than in mammalian cells). Most other metabolites are produced via phosphatidylinositol 3-phosphate, and reports that some phosphatidylinositol 3,4,5-trisphosphate may be produced from phosphatidylinositol 4,5-bisphosphate require confirmation. In contrast to mammalian phosphatidylinositol 3-kinases, which accept both phosphatidylinositol and its monophosphates as substrates, the plant enzyme acts only on the former. Specific functions are now being discovered for each of the plant phosphoinositides, which are produced rapidly in response to osmotic and heat stress, and it has become evident that a continuous turnover is essential for cell growth and development. For example, they have marked effects on the growth of many cell types and on guard cell function.

Highly polarized distributions of phosphoinositides are found within membranes, especially in microdomains at the tip of growing cells such as pollen tubes and root hairs. For example, phosphatidylinositol-4-phosphate is an important constituent of the plasma membrane in plant cells, where it controls the electrostatic state and is involved in cell division; it may control the location and function of many membrane proteins, including those required for development, reproduction, immunity, nutrition and signalling. In addition, phosphatidylinositol 4-phosphate may interact with salicylic acid in the plant immune response.

Although its concentration is low, phosphatidylinositol 4,5-bisphosphate has been shown to have signalling functions by binding to a number of different target proteins, which have characteristic binding domains. For example, together with phosphatidic acid, phosphatidylinositol 4,5-bisphosphate regulates the activity of a number of actin-binding proteins, which in turn control the activity of the actin cytoskeleton. This has a key role in plant growth, the movement of subcellular organelles, cell division and differentiation, and plant defence. In addition, this lipid exerts a control over ion channels, ATPases and phospholipase C-mediated lipid degradation and the production of further second messengers. The specificity of the interactions may be dependent on the fatty acid composition of the lipid and on the activity of phosphatidylinositol 4-phosphate 5-kinase.

Phosphatidylinositol 3,5-bisphosphate is believed to have a role in the central or lytic vacuole in plant cells, both in the structural dynamics of the cellular cytoskeleton and in the function of ion transporters.

A number of different enzymes of the phospholipase C type that are specific for polyphosphoinositides have been isolated from higher plants; they are activated by Ca2+ and unlike their mammalian counterparts, they are not regulated by G proteins. It is not certain whether phosphatidylinositol is itself a substrate for these enzymes in vivo. Less is known of the metabolism of the water-soluble inositol phosphates produced in comparison to animals, and plants appear to lack a receptor for inositol 1,4,5-trisphosphate (IP3), although it is the most abundant metabolite of this type and is reported to induce release of calcium ions to trigger stomatal closure. There is a general if contested belief that inositol hexakisphosphate (phytic acid or IP6), produced at least in part by sequential phosphorylation of inositol 1,4,5-trisphosphate, is a more important cellular messenger in plants and mobilizes an endomembrane store of calcium ions. Inositol-1,2,4,5,6-pentakisphosphate (IP5) is a structural co-factor of the jasmonic acid receptor coronatine insensitive 1, linking phosphoinositide signalling with phytohormone-controlled pathways.

In plants in contrast to animals, diacylglycerols, the other product of phospholipase C hydrolysis of phosphoinositides, are rapidly converted to phosphatidic acid by diacylglycerol kinases and have not been considered important in signal transduction. Plants lack protein kinase C but they do have proteins with related properties that appear to be influenced by diacylglycerols. Via the action of phospholipase D, inositol phospholipids are a source of phosphatidic acid with its well-characterized signalling functions in plants, especially in defence.

Yeasts produce only five phosphoinositides with more phosphatidylinositol 4,5-bisphosphate than plants but no detectable phosphatidylinositol 3,4-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate. They are produced rapidly in response to nitrogen starvation, and phosphatidylinositol 3,5-bisphosphate synthesis in particular is induced by osmotic stress. In contrast, the Amoebozoa, such as Dictyostelium discoideum, possess Class I PI3Ks, which produce phosphatidylinositol 3,4,5-trisphosphate directly at the plasma membrane from 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), i.e with an ether-linked 16:0 chain at the sn-1 position.


5.  Lyso-Phosphoinositides

Formula of lysophosphatidylinositolIt has become apparent relatively recently that like other lysophospholipids, lysophosphatidylinositol, i.e. with a single fatty acid only linked to the glycerol moiety, and the polyphospho-analogues may have messenger functions. For example, it has long been known to stimulate the release of insulin from pancreatic cells, suggesting a role in glucose homeostasis. Lysophosphatidylinositols are formed as intermediates in the remodelling of the fatty acid compositions of the lipids by the action of phospholipase A1 or phospholipase A2 (e.g. cPLA2α), and when arachidonic acid is released for eicosanoid biosynthesis (see above).

sn-2-Arachidonoyl-lysophosphatidylinositol, in particular, is an endogenous ligand for a G protein-coupled receptor GPR55, and thereby can induce rapid phosphorylation of certain enzymes, including a protein kinase, which promote cancer cell proliferation, migration and metastasis. Indeed, lysophosphatidylinositol is a biomarker for poor prognosis in cancer patients, and its concentration is elevated significantly in highly proliferative cancer cells in vitro. GPR55 is expressed in many regions of the brain, the intestines, endocrine pancreas and islets (where it may stimulate insulin release). In addition to its role in cancer, lysophosphatidylinositol has been implicated in a number of metabolic diseases. It is potentially a precursor of the endocannabinoid 2-arachidonoylglycerol.

glycerophosphoinositolSequential removal of both fatty acids from phosphatidylinositol by a specific phospholipase A2 (PLA2IVα) with both phospholipase A2 and lysophospholipase activities releases water-soluble glycerophosphoinositol. While this can be hydrolysed by a glycerophosphodiester phosphodiesterase to inositol 1-phosphate, glycerophosphoinositol per se has distinctive biological activities and functions, as do related compounds derived from the phosphatidylinositol phosphates. In particular, glycerophosphoinositol has anti-inflammatory activity in that it inhibits the inflammatory and thrombotic responses induced by bacterial lipopolysaccharides (endotoxins).


6.  Glycosyl-Phosphatidylinositol Anchors for Proteins, Lipophosphoglycans and Phosphatidylinositol Mannosides

Phosphatidylinositol is known to be the anchor that links a variety of proteins to the external leaflet of the plasma membrane via a glycosyl bridge (glycosyl-phosphatidylinositol(GPI)-anchored proteins). However, this was considered a sufficiently important topic for its own web page. Lipophosphoglycans (lipoarabinomannans and arabinogalactans) and phosphatidylinositol mannosides are important components of the membranes of parasitic protozoa and bacteria, but for convenience they are also discussed with the GPI-anchored proteins.


7.  Analysis

The book by Kuksis cited below is a definitive guide to the analysis of phosphoinositides. Like all acidic phospholipids, phosphatidylinositol is not particularly easy to isolate in a pure state, special care being necessary to ensure that it is fully resolved from phosphatidylserine. However, this can be accomplished by adsorption TLC or HPLC with care. The phosphatidylinositol phosphates are a different matter, however, because of their high polarity and low abundance in tissues. It is necessary to used acidified solvents to extract them efficiently from tissues and to ensure that they are in a single salt form. For isolation of individual components, TLC methods are usually favoured, although detection can be a problem - one approach being to equilibrate with radioactive phosphorus to facilitate detection and quantification by liquid scintillation counting. HPLC with mass spectrometric (electrospray ionization) detection is now showing great promise. Analysis of the lipid-glycoconjugate-protein complexes and of the lipophosphoglycans is a rather specialized task for which modern mass spectrometric and NMR facilities are essential.


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Lipid listings Credits/disclaimer Updated: August 7th, 2017 Author: William W. Christie LipidWeb icon