Probing phosphoinositide functions in signaling and membrane trafficking

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The inositol phospholipids (PIs) comprise a family of eight species with different combinations of phosphate groups arranged around the inositol ring. PIs are among the most versatile signaling molecules known, with key roles in receptor-mediated signal transduction, actin remodeling and membrane trafficking. Recent studies have identified effector proteins and specific lipid-binding domains through which PIs signal. These lipid-binding domains can be used as probes to further our understanding of the spatial and temporal control of individual PI species. New layers of complexity revealed by the use of such probes include the occurrence of PIs at intracellular locations, the identification of phosphatidylinositol signaling hotspots and the presence of non-membrane pools of PIs in cell nuclei.

Introduction

The inositol-containing glycerophospholipids, collectively known as phosphoinositides (inositol phospholipids; PIs), are among the most versatile of regulatory molecules, with strikingly diverse roles in cell signaling and vesicle-based transport mechanisms. This versatility arises from the chemistry of the myo-inositol moiety, which is attached to diacylglycerol (DAG) via a di-ester phosphate at the D-1 position, leaving five free hydroxyls, three of which are phosphorylated in different combinations by lipid kinases. PIs that have been identified in eukaryotic cells, their biosynthesis and metabolic interconversions are illustrated in Figure 1. The enzymes involved in these pathways have been reviewed extensively elsewhere 1, 2, 3, 4, 5, 6. With the recent discovery of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] in fission yeast [7], it now appears that most of the PIs in Figure 1 are conserved from yeast to man. Here, we focus on PI-binding proteins and, in particular, on the use of specific lipid-binding domains as probes for the quantitative temporal and spatial analysis of PIs in cells (Box 1).

The DAG moiety accounts for the fact that PIs are predominantly if not exclusively associated with cell membranes. By contrast, it is the exposed headgroups of PIs that bind to effector proteins and through which their signaling functions are realized. The structural diversity of these headgroups, the existence of effector domains with high affinity and selectivity for particular PI species and the non-uniform distribution of PIs among subcellular membranes are crucial for the fidelity of PI-dependent signaling mechanisms. If the number of PI species seems complex and confusing, the range of effector domains is even more dramatically diverse. They include pleckstrin homology (PH), phagocyte oxidase (PX), epsin N-terminal homology (ENTH) and Fab1p, YOTB, Vac1p and EEA1 (FYVE) domains, as well as an assortment of proteins that bind to PIs with varying degrees of specificity through small patches of basic amino acids 4, 8. These domains serve primarily to target their host proteins to specific cellular locations and, in some cases, perhaps, might more directly regulate protein function. Their use as probes depends upon a high degree of specificity for the target lipid, affinities that allow detection of the target at the levels that occur naturally in cell membranes and an understanding of secondary interactions that might restrict the distribution of the probe to a particular compartment, limiting its use for detection of the target at other sites. With the possible exception of PtdIns5P and PtdIns itself, suitable probes now exist for all of the PIs in Figure 1, implying that each of these lipids has one or more signaling roles. What follows considers how we can use and avoid abusing these probes to study the functions of individual PI species.

Section snippets

PtdIns(4,5)P2: multiple functions and locations

The lipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] undoubtedly represents a focal point in PI-dependent signaling in both metabolic and functional terms. It is synthesized mainly by a diverse family of PtdIns4P 5-kinases and serves as the substrate for two powerful receptor-regulated signal-generating enzymes. PI-phospholipase C (PI-PLC) cleaves PtdIns(4,5)P2, simultaneously producing two second messengers, DAG and inositol (1,4,5)-trisphosphate [15]. Type I PI 3-kinases [1], on

PtdIns(3,4,5)P3 and PtdIns(3,4)P2: lipids mediating PI 3-kinase-dependent signaling pathways

PtdIns(3,4,5)P3 is synthesized by Type I PI 3-kinases (PI3Ks), a reaction that is reversed by the tumor-suppressor lipid phosphatase, PTEN (for ‘phosphatase and tensin homolog deleted on chromosome ten’; see Figure 2). In quiescent cells, PtdIns(3,4,5)P3 is present at low levels [∼0.1% of the level of its precursor, PtdIns(4,5)P2], but, upon stimulation of tyrosine kinase and some G-protein-coupled receptors, its concentration can increase by factors ranging from 2- to 100-fold. The sources of

PtdIns4P, PtdIns3P and PtdIns(3,5)P2: phosphoinositides associated predominantly with intracellular membranes

Research into the roles of PIs on intracellular membranes has lagged well behind that of PIs found at the cell surface. PtdIns(4,5)P2, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are rarely found intracellularly, but a large body of evidence now implicates monophosphorylated PIs in the functions of intracellular organelles, in particular, membrane traffic 3, 56. The two principal lipids of interest are phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 4-phosphate (PtdIns4P), of which

Concluding remarks

The unique versatility of phosphoinositides as intracellular signals arises from four distinct aspects:

•The combinatorial actions of lipid kinases and phosphatases generates the range of molecules depicted in Figure 1, where many of the components are both precursors and products of signaling enzymes.

•A temporal aspect in which individual signals can be produced transiently and metabolized rapidly.

•A spatial aspect in which phosphoinositides are produced and maintained in distinct cellular

Acknowledgements

We thank Yvonne Lyndsay and Terry Smith for help with Figure 1. Research in the C.P.D. laboratory is supported by the Medical Research Council. J.M.L. is supported by the Wellcome Trust and the Lowe Trust.

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