Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease.
It is hard to define the research interest of people who study polyphosphoinositides (PPIs). Naturally, PPIs are lipid molecules, yet many researchers who study PPIs did not initially have a primary interest in lipids. Many of us have gotten interested in PPIs when these lipids became known as the source of second messengers in transducing signals from cell surface receptors. The spectacular progress in the 1980s in defining the pathways by which G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) activated phospholipase C (PLC) enzymes had a major impact on many scientists who showed interest in transmembrane signaling. However, cell biologists also developed immense interest in PPIs because of the importance of PPIs in shaping the membranes and controlling vesicular trafficking and organelle physiology. The attention of scientists who study ion channels also turned toward PPIs as it became obvious that many channels or transporters require PPIs for their activity or control. The discovery of phosphatidylinositol 3-kinases (PI3Ks) has set the stage to widen research interest in PPIs: association of PI3K with oncogenic as well as RTKs and their strong ties with cancer biology has won over cancer researchers, while the importance of PPIs in immune cell functions, chemotaxis, and secretion brought immunologists to the field. If this had not been enough, researchers working with infectious diseases noted that many pathogenic organisms possess enzymes essential for their pathogenic nature that act upon PPIs to invade cells or use the host cells' PPI machinery to evade natural defense mechanisms or reprogram cells to produce the pathogen. Neuroscientists also discovered that synaptic vesicle exocytosis and recycling requires phosphoinositides at multiple steps and that brain development, including neurite outgrowth and axon guidance, is highly dependent on PPIs. Even the invertebrate photo-sensing and signal transduction is dependent on PPIs, further extending the group of scientists showing interest in PPIs. This selected and probably incomplete list increases every day as more and more cellular processes are linked to these universal lipid regulators.
Such an ever-expanding list of processes regulated by PPIs begs an answer to the fundamental question of how and why these lipids gained such a pivotal role in eukaryotic cell regulation during evolution? What structural and functional features make these molecules so widely used and so adaptable to support the functions of a variety of signaling complexes? We have only begun to ask, let alone answer these questions for which evolution may give us some clues. Although PIs have been detected in mycobacteria, their appearance in evolution coincides with the development of internal membranes and organelles. Remarkably, PI kinases surfaced earlier in evolution than tyrosine kinases (190, 986) with common ancestors being a group of serine-threonine kinases, called the PI-kinase related kinases (190, 669). The latter enzymes are all functionally linked to DNA damage control and repair (190, 1350, 1422). PtdIns is unique among phospholipids in that it is a rich phosphorylation target at the cytoplasmic surface of any cellular membrane. In their phosphorylated forms, PPIs can serve as critical reference points for a great variety of proteins to find their docking destinations and/or change their conformation. This is true for cytosolic proteins that are recruited to the membrane by PPIs, as well as for peripheral or integral membrane proteins whose membrane adjacent regions or cytoplasmic “tails” show interaction with PPIs.
With the spectacular expansion of the PI field, it has become impossible to cover all aspects of PPI regulation at great depth in a comprehensive review. In the following overview I will attempt to describe the most basic features of the enzymes that synthesize and degrade PPIs and focus on aspects of this diverse research field that highlight general principles that govern PI-mediated regulation of the many different processes. For a more comprehensive analysis and deeper understanding of the details of the individual processes and their PPI regulation, the reader is referred to the many excellent reviews that have been published over the years and ones that are likely still in preparation. Soluble inositol phosphates partly liberated from inositol lipids and further phosphorylated to highly charged inositol polyphosphates also represent a research topic of great interest but will not be discussed here. The interested reader will find excellent reviews on that topic (29, 1079, 1390). Another aspect of PtdIns function not covered here is related to PtdIns serving as an anchor for a selected group of proteins found on the cell surface linked to the 6-position of the inositol ring through glycan linkages. More about the biology of these GPI-anchored proteins can be found in several comprehensive reviews (473, 1200).
II. THE BASICS: PHOSPHOINOSITIDE STRUCTURES AND ENZYMOLOGY
Polyphosphoinositides are phosphorylated derivatives of PtdIns generated by a number of kinases and phosphatases that act upon their membrane-bound lipid substrates (Figure 1). Phosphorylation occurs in one of the -OH groups of the inositol ring of PtdIns that is linked to the diacylglycerol (DG) backbone via a phosphodiester linkage utilizing the -OH group of the ring at the D1 position. PtdIns contains myo-inositol that assumes a “chair” conformation with five of its six -OH groups being equatorial and the one at position 2 being axial. The best visual representation of the myo-inositol structure and ring numbering was introduced by Bernard William Agranoff in 1978, who likened the “chair” structure of myo-inositol to a turtle whose body represents the inositol ring and the numbering starts at the right front flipper and proceeds counterclockwise through the head and the other appendages (9) (Figure 1A). Since the turtle is taken through its right flipper by the DG backbone, it leaves five hydroxyls for phosphorylation, but only three of these (positions -3, -4, and -5) are actually phosphorylated in naturally occurring PPIs according to current knowledge. The combination of these phosphorylations gives rise to the seven known PPIs (Figure 1B). A distinctive feature of PPIs is their enrichment in arachidonic acid at the sn-2 position of their glycerol backbone. The majority of the PPIs is the 1-stearoyl-2 arachidonyl form, and it has been a puzzling question where this enrichment takes place and what role deacylation-reacylation cycles play in determining this composition. It has been suggested that enzymes responsible for PtdIns synthesis and phosphorylation may show preference to the form(s) of lipids esterified with arachidonic acid at the sn-2 position (405). Arachidonate-rich phosphoinositides are also believed to be the source of PLA2-mediated arachidonate release for the synthesis of prostaglandins and leukotrienes.
The amounts of PPIs within cells have been estimated in different cells and tissues (1121, 1703). These estimates and measurements show significant variations. PtdIns represents ∼10–20% (mol%) of total cellular phospholipids, whereas PtdIns4P and PtdIns(4,5)P2 constitute ∼0.2–1%. Based on long-term [3H]inositol labeling, PtdIns4P and PtdIns(4,5)P2 have about 2–5% of the labeling relative to PtdIns (e.g., Ref. 87). Recent estimates of PtdIns(4,5)P2 density in the plasma membrane (PM) ranged between 5,000–20,000 molecules/μm2 (421). The other PPIs contribute even smaller amounts, PtdIns(3,4,5)P3 being about 2–5% of PtdIns(4,5)P2 and PtdIns3P about 20–30% of PtdIns4P. It is noteworthy that these ratios show great tissue variations and they are also very different in yeast and plants. Yeast do not have detectable amounts of PtdIns(3,4,5)P3 and some plants have a lot more PtdIns4P relative to a smaller pool of PtdIns(4,5)P2, and it is not clear if PtdIns(3,4,5)P3 is at all present in plants (1093, 1469). Remarkably, yeast and plant orthologs of the mammalian PTEN enzyme that has a critical role in PtdIns(3,4,5)P3 dephosphorylation have been found in spite of the apparent lack of detectable PtdIns(3,4,5)P3 in these organisms (611).
PtdIns is synthesized in the endoplasmic reticulum from CDP-DAG and myo-inositol by a PtdIns synthase (PIS) enzyme (11) (see sect. IV) and is then distributed throughout the cell presumably by several PI transfer proteins (PITPs) (277, 689) and possibly via vesicular trafficking. Our recent studies identified the PIS enzyme in an ER-derived highly mobile “organelle” that may serve as a dynamic PtdIns distribution device (796). Early studies already detected the phosphorylation reaction generating two “polyphosphoinositides” that had been previously described in the brain and determined to be PtdIns4P and PtdIns(4,5)P2 (186), thereby postulating PtdIns kinase (PI-kinase) and PtdInsP kinase (PIP kinase) activities associated with membranes (1044). Although these enzymatic activities were associated with various membrane fractions in fractionated tissues, and they showed even some unique features (like sensitivity to different detergents), the general consensus that emerged from these studies was that PI- and PIP-kinase activities were primarily present in the PM, serving what has become known as the signaling pool of PPIs (see sect. III). However, by now it is apparent that multiple isoforms of almost all of the kinase and phosphatase enzymes act upon PPIs, and they do so in different cellular compartments. This explains why in most cases these enzymes are not functionally redundant even if they catalyze the same biochemical reaction. The mechanism(s) that determine the intracellular localization and regulation of the PI kinase and phosphatase enzymes became a central question for each family of these proteins. Generally speaking, most PtdIns mono-phosphorylations (by the PI4Ks and Class III PI3K) occur in endomembranes, such as the endosomes and the Golgi/trans-Golgi network, whereas the phosphorylation of PtdIns4P to PtdIns(4,5)P2 by PIP 5-kinases and further to PtdIns(3,4,5)P3 by class I PI3Ks occurs primarily at the PM.
The two main routes of PPI elimination are through dephosphorylation by PPI phosphatases and hydrolysis by phosphoinositide-specific phospholipase C enzymes (PLCs). Some of the PPI phosphatases are quite specific for the position of the phosphate group that they remove, while others, mainly the ones that dephosphorylate monophosphorylated PPIs, are more promiscuous and their functional specificity lies in their localization. The diversity of the PPI phosphatases parallels, in fact, exceeds that of the kinases (Figure 1B), and several human diseases have been traced to the dysfunction of PPI phosphatases (see sect. VI). The diversity of phosphoinositide-specific PLCs is also remarkable (see sect. VII). The preferred in vivo substrate of the mammalian PLC enzymes is believed to be PtdIns(4,5)P2, although this question has not been satisfactorily answered in whole cell studies and purified PLC enzymes can also hydrolyze PtdIns4P and PtdIns in vitro.
Although individual groups of PPI metabolizing enzymes will be featured in more details below, a few important general questions are worth highlighting here. The first is related to their substrate recognition. Most, if not all, of the PPI kinase and phosphatase enzymes are loosely membrane-associated peripheral membrane proteins that utilize a substrate that is part of the membrane with the inositol headgroup facing the cytoplasmic leaflet of the membrane. When enzyme activities of these proteins are measured in vitro, the assay usually contains the lipid substrate in some form of lipid micelle, and the type and amount of detergent yielding optimal activity greatly differ for each of these enzymes. For example, the in vitro activity of PI 4-kinases depends on the presence of detergents such as Triton X-100, while those of the class I PI 3-kinases are inhibited by detergents and the activity of class III PI 3-kinases are usually assayed in the presence of Mn2+ instead of the usual Mg2+. Overexpression in cells of some of the PI kinases (such as the type III PI 4-kinases) hardly yields an increase in the phosphorylation of their endogenous lipid substrates. This indicates that the mechanism(s) that ensure recruitment of the enzymes to the membrane and their access to the membrane-bound PtdIns substrate are major determinants of their in situ activities. Few studies have been designed to understand the exact nature of the lipid substrate PI kinase interactions, and most of the solved structures of the kinase enzymes do not resolve the activation loop (a mobile part of the molecule that is critical for substrate presentation) within their catalytic center. Similarly, the lipid substrate in these structures is either missing or, if present, was provided in the form of a soluble inositol-phosphate headgroup. Therefore, there is a major gap in our understanding of how these enzymes work in their natural membrane environment.
Phosphoinositides affect cellular functions by interacting with molecules that either reside in the membrane, such as ion channels and transporters, or get recruited to the membrane by reversible mechanisms. Several signaling molecules are recruited to the membrane through interaction with PPIs via inositide-binding protein modules (see Table 1). The first such protein module was identified in pleckstrin (574), and ever since, the homologous modules have been termed pleckstrin homology (PH) domains. PH domains are present in a large number of regulatory molecules (296). It is important to note, however, that not all PH domains bind lipids and probably all PH domains also bind proteins (880). Often simultaneous protein and lipid binding are required for the membrane recruitment or conformational change of PH domains; hence, these (and other PI binding modules) are called coincidence detectors (214). Most frequently the protein input for PH domains come from interaction with small GTP binding proteins. Other domains, such as the FERM domains link the actin cytoskeleton to the PM (255). Both FERM and GLUE-domains contain a structural fold similar to that of PTB (phosphotyrosine binding) or PH domains (1062, 1416). EHD domains (1120) and BAR domains also bind anionic lipids including inositol lipids and also sense and/or generate membrane curvatures (466). This list is ever expanding and now also includes PDZ domains (1825) and the KA1 domain, a novel fold found at the COOH terminus of a range of proteins and which binds PtdIns(4,5)P2 but also other anionic phospholipids such as PS (1083). A recently described PtdIns(3,4,5)P3 binding domain found at the COOH termini of some IQ domain containing GAP proteins has a structure reminiscent of the integral fold of C2 domains (363). In addition, several proteins contain polybasic stretches that do not amount to a characteristic domain, but also interact with acidic phospholipids with electrostatic interaction. Good examples are the MARCKS proteins (1670) and the K-Ras COOH terminus (601), but many other proteins show membrane association using this mechanism. Importantly, many of these targeting sequences can use PtdIns4P as well as PtdIns(4,5)P2 as their membrane anchor lipid (559).
III. HISTORICAL OVERVIEW
Although one can argue that a newcomer to a field can benefit from not being biased by existing dogmas, ignorance of the history of a research field and lack of understanding of the milestones and breakthroughs are making it difficult, if not impossible, to put any new research findings into perspective. One also has to know and respect the contribution of the scientists whose findings serve as the foundation upon which new knowledge can be built. Therefore, I give a short overview of the field that is certainly biased by my experiences, but can still give some ideas about the major milestones as this research field has evolved. Several reviews and recollections have been published on the historical aspects of this huge research field, and they are highly recommended for interested readers (10, 634, 701, 1039).
Inositol had already been discovered as a component of muscle by the end of the 19th century and its structure established as a hexa-hydroxyl-cyclohexane (990). Nine different stereoisomers of inositol were described in the 1940s, and myo-inositol was found to be the main eukaryotic isomer (1234). The importance of inositol was recognized in the era of vitamins when it was realized that inositol was an important dietary ingredient for rodents, especially when animals were kept in germ-free conditions. Animals kept on inositol-free diets developed alopecia and “fatty liver” (reviewed in Ref. 642). Subsequently, it was found that mammalian cells required myo-inositol to grow properly in culture (394). In the fungus neurospora, lack of inositol caused a defect in lysosomal membrane integrity and autolysis (1006). The notion that inositol was a component of lipid membranes was first recognized in mycobacteria [the lipid was phosphatidylinositol mannoside (96)] and a phosphoinositide was also described in soybean (803).
A. Identification of Phosphoinositides and Their Metabolic Fate
The real beginning of the “modern” era of PI research was marked by a series of ground-breaking studies in the 1940s by Jordi Folch who identified inositol in the ethanol-insoluble phospholipid fraction of bovine brain and determined that it contained phosphates and inositol in a molar ratio of 2:1 (450). This lipid, termed diphosphoinositide or DPI, was found primarily in myelin in tight association with proteins (“neurokeratin”) (449) and showed rapid metabolic labeling when guinea pigs were injected with [32P-\]phosphate (328, 329). Subsequent work mainly by three groups (led by Clinton Ballou, Rex Dawson, and Tim Hawthorne) identified the structures of mono-, di-, and tri-phosphoinositides (abbreviated at the time as MPI, DPI, and TPI, respectively) as glycerophospholipids with an inositol ring linked to an sn-1,2-DG backbone via the D1-OH group of myo-inositol, and containing a phosphate at the 4- and both the 4- and 5-positions, in DPI and TPI, respectively (359, 581, 1563). Although these lipids had been isolated and identified primarily from brain, where they are most abundant, it had become evident by the early 1960s that they were present in small amounts in all eukaryotic tissues (1651).
To understand the meaning of their 32P labeling, it was essential to understand the synthetic and degradation pathways of PPIs. From the pioneering work of Eugene Kennedy and his colleagues on the synthesis of glycerolipids, it had been understood that cytidine nucleotide intermediates (such as CDP-choline and CDP-ethanolamine) donate the headgroups to the sn-1,2-DG backbone during phosphatidylcholine and -ethanolamine synthesis. However, Bernard Agranoff and his colleagues found that this was not the case for PtdIns. Here, the lipid backbone itself was “activated” by the cytidine nucleotide in the form of CDP-DG (11). Since the precursor of this intermediate is phosphatidic acid (PtdOH), which is produced by phosphorylation of sn-1,2-DG by a DG kinase using the terminal phosphate of ATP, described by Hokin and Hokin (640), an alternative name of CMP-PtdOH was suggested, to indicate that the phosphate was carried over to PtdIns not from CTP but ATP. PtdIns synthesis was found primarily associated with “microsomal” fractions and hence attributed to the ER, but CDP-DG is also a precursor of the mitochondrial lipids phosphatidylglycerol and cardiolipin (324), suggesting an important compartmentalization of these metabolic pathways.
Several studies then indicated that DPI was a phosphorylation product by PtdIns kinase activities associated with various membrane fractions (283, 636, 1044), including the PM (1043) and, importantly, similar activities were also present in red blood cell membranes where it was possible to show generation of PtdIns4P and PtdIns(4,5)P2 by presumed sequential phosphorylations of PtdIns (636). These observations, together with the notion that PPIs were highly enriched in myelin sheets that are essentially rolled up plasma membranes, gave strong support to the idea that PPIs were primarily associated with the PM. The metabolism of DPI and TPI was also explored in early studies. Sloane-Stanley identified a phospholipase C (PLC) activity (although not called it PLC yet) capable of hydrolyzing brain phosphoinositides (1420), and Rodnight found this activity increased by Ca2+ (1276). These early observations were followed by the realization that TPI is metabolized in two different ways: one route with dephosphorylation to DPI and PtdIns and another, via hydrolysis to InsP3 and diacylglycerol (what is now known as PLC) (1554).
The very rapid labeling kinetics of PtdIns(4,5)P2 and PtdIns4P in erythrocyte membrane and in intact cells relative to the much slower labeling kinetics of PtdIns and other phospholipids suggested high turnovers of the phospho-monoester groups due to rapid “futile” phosphorylation-dephosphorylation cycles (reviewed in Ref. 385). This also indicated that dephosphorylation of the monophosphate groups and rephosphorylation can all take place in the PM. PLC activities were then found in several tissues both in soluble and membrane fractions (464, 775), and association of PLC activity with the PM was also described (852, 1047). The distribution of PLC activities between soluble and membrane fractions and the enzymatic characteristics of the activities associated with different fractions showed significant variations between various laboratories (e.g., see Ref. 702). These discrepancies are now better understood knowing how many PLC enzymes and membrane-recruitment mechanisms exist (see sect. VII).
B. Agonists Stimulate Phosphoinositide Metabolism
In 1953 Mabel and Lowell Hokin (638) reported that [32P]phosphate incorporation into a phospholipid fraction was strikingly increased in the exocrine pancreas when the tissue was stimulated with secretagogues that induced protein secretion. In a series of subsequent work (635), the Hokins found that the increased 32P-labeling was limited to the two lipids, “DPI” and PtdOH that were identified by Dawson as acutely 32P-labeled in the brain slices (328). The Hokins also found that this increase was a general phenomenon observed in various cells associated with a stimulated secretion response (635). They suggested that the primary response was an increased phosphoinositide-specific PLC-catalyzed hydrolysis of PtdIns with production of DG, which was then converted to PtdOH (a step where the 32P incorporation took place) and then back to PtdIns to complete a cycle that was dubbed the “PI cycle” (639) (Figure 2A). These studies implicated PIs in secretion, but how increased inositide labeling was linked to any specific biochemical process in secretion remained elusive. In fact, more and more studies indicated that the increased PI turnover could be dissociated from secretion: it was observed in cells and with stimuli that did not evoke secretion, such as in postganglionic neurons (632) or lymphocytes (441). Also, the increased PI turnover was preserved in Ca2+-free medium, whereas secretion was eliminated under those conditions (633). Moreover, increased PI labeling needed higher concentrations of agonists than those for secretion (637), and it was not evoked by some agonists that increased secretion via cAMP (1337). In the meantime, a series of important experiments linked the PI turnover to cell proliferation. Fisher and Muller (442) found that lymphocytes stimulated with the mitogen phytohemagglutinin increased 32P- or [3H]inositol labeling of PtdIns and a rapid appearance of PtdOH, consistent with increased turnover of PI. A close correlation between cell proliferation and specifically inositol lipid turnover was found in cells subjected to various stimuli, including transformation with viruses, such as Rous sarcoma or SV40 (358). A dramatic drop in PtdIns turnover was shown to correlate with the transition from proliferation to differentiation during lens development in chicken embryos (1791).