Progress in Lipid Research - sorbonne-universite...1.3. Lateral distribution in the plasma membrane Although the overall PI(4,5)P 2 level is relatively constant in mammalian cells,

Progress in Lipid Research 52 (2013) 1–14

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Progress in Lipid Research

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Review

Signal transduction pathways involving phosphatidylinositol 4-phosphateand phosphatidylinositol 4,5-bisphosphate: Convergences and divergencesamong eukaryotic kingdoms

Elise Delage ⇑, Juliette Puyaubert, Alain Zachowski, Eric RuellandUR5 Université Pierre et Marie Curie, EAC7180 CNRS, Physiologie Cellulaire et Moléculaire des Plantes, 4 Place Jussieu, Case Courrier 156, 75252 Paris Cedex 05, Paris, France

a r t i c l e i n f o

Article history:Received 25 June 2012Received in revised form 22 August 2012Accepted 23 August 2012Available online 8 September 2012

Keywords:Phosphatidylinositol 4-phosphatePhosphatidylinositol 4,5-bisphosphateSignal transduction

0163-7827/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.plipres.2012.08.003

Abbreviations: ANTH, AP180 N-terminal homologyDAGK, diacylglycerol kinase; DGPP, diacylglycerol pyhomology; ER, endoplasmic reticulum; ERK, extracelluguanine nucleotide exchange factor; IP3, inositol 3,4,5alanine-rich C kinase substrate; PA, phosphatidic acpleckstrin homology; PI, phosphatidylinositol; PI(3,4,ositol 3-kinase; PI4K, phosphatidylinositol 4-kinase; P5-phosphate; PI5P4K, phosphatidylinositol 5-phosphkinase B; PKC, protein kinase C; PLC, phospholipase C;SH2-containing inositol phosphatase; Star-PAP, nucle⇑ Corresponding author. Tel.: +33 1 44 27 96 47; fa

E-mail addresses: [email protected] (E. DelRuelland).

a b s t r a c t

Phosphoinositides are minor constituents of eukaryotic membranes but participate in a wide range of cel-lular processes. The most abundant and best characterized phosphoinositide species are phosphatidylin-ositol 4,5-bisphosphate (PI(4,5)P2) and its main precursor, phosphatidylinositol 4-phosphate (PI4P). PI4Pand PI(4,5)P2 regulate various structural and developmental functions but are also centrally involved in aplethora of signal transduction pathways in all eukaryotic models. They are not only precursors of secondmessengers but also directly interact with many protein effectors, thus regulating their localisation and/or activity. Furthermore, the discovery of independent PI(4,5)P2 signalling functions in the nucleus ofmammalian cells have open a new perspective in the field. Striking similarities between mammalian,yeast and higher plant phosphoinositide signalling are noticeable, revealing early appearance and evolu-tionary conservation of this intracellular language. However, major differences have also been high-lighted over the years, suggesting that organisms may have evolved different PI4P and PI(4,5)P2

functions over the course of eukaryotic diversification. Comparative studies of the different eukaryoticmodels is thus crucial for a comprehensive view of this fascinating signalling system. The present reviewaims to emphasize convergences and divergences between eukaryotic kingdoms in the mechanismsunderlying PI4P and PI(4,5)P2 roles in signal transduction, in response to extracellular stimuli.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1. Spatio-temporal regulation of PI4P and PI(4,5)P2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2. Distribution over the cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3. Lateral distribution in the plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.4. Distribution in biochemically different pools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.5. Synthesis and degradation of PI4P and PI(4,5)P2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.6. Non-redundant functions of phosphoinositide-modifying enzyme isoforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.7. Spatio-temporal regulation of phosphoinositide-modifying enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
ll rights reserved.

; BAF, brahma-related gene associated factor; C2, PKC homology-2; CKIa, casein kinase Ia; DAG, diacylglycerol kinase;rophosphate; DRM, detergent-resistant membrane; EGFR, epidermal growth factor receptor; ENTH, epsin N-terminallar signal-regulated kinase; FERM, acronym of band four.1 ezrin radixin moesin; GAP, GTPase activating protein; GEF,-trisphosphate; Ipk, inositol polyphosphate kinase; MAPK, mitogen-activated protein kinase; MARCKS, myristoylatedid; PDK1, phosphoinositide-dependent kinase 1; PDZ, postsynaptic density protein disc large zona occludens; PH,5)P3, phosphatidylinositol 3,4,5-trisphophate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI3K, phosphatidylin-I4P, phosphatidylinositol 4-phosphate; PI4P5K, phosphatidylinositol 4-phosphate 5-kinase; PI5P, phosphatidylinositolate 4-kinase; PIPK, phosphatidylinositol phosphate kinase; PITP, phosphatidylinositol transfer protein; PKB, proteinPLD, phospholipase D; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PX, phox homology; SHIP,

ar speckle targeted PI4PK5a regulated poly(A) polymerase.x: +33 1 44 27 61 51.age), [email protected] (J. Puyaubert), [email protected] (A. Zachowski), [email protected] (E.

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2 E. Delage et al. / Progress in Lipid Research 52 (2013) 1–14

2. PI4P and PI(4,5)P2 as precursors of second messengers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1. Biological functions of the PLC pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.2. Second messengers produced by the PLC pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.3. Substrate supply to PLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.4. PI3K signalling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3. Downstream effectors of PI4P and PI(4,5)P2 signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.1. Regulation of transmembrane proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.2. Membrane anchorage of signalling proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.3. Regulation of enzymatic activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.4. Protein sequestration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.5. Phosphoinositide binding domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4. PI4P and PI(4,5)P2 in nuclear signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.1. Nuclear pools of PI(4,5)P2 in mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.2. Nuclear PI(4,5)P2 as a precursor of second messengers in mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.3. Nuclear PI(4,5)P2 downstream effectors in mammalian cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.4. Existence of a nuclear phosphoinositide system in other eukaryotic models? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1. Introduction

Phosphatidylinositol (PI) performs a dual role in eukaryoticcells, since it is both a structural lipid and the precursor of impor-tant signalling molecules, namely phosphoinositides. Phosphoino-sitides are the products of phosphatidylinositol phosphorylation atD3-, D4- or D5-positions of the inositol ring, in all possible combi-nations. They represent a minor percentage of total phospholipids,but regulate a striking number of fundamental cellular processes.The most abundant and best characterized phosphoinositide spe-cies are phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and itsmain precursor, phosphatidylinositol 4-phosphate (PI4P). In alleukaryotic models, they regulate various structural and develop-mental functions, such as membrane biogenesis, lipid homeostasis,vesicular trafficking, and cytoskeleton organization and dynamic[1–4]. Furthermore, PI4P and/or PI(4,5)P2 are centrally involvedin a plethora of signal transduction pathways, not only as precur-sors of second messengers but also as membrane-bound regulatorsof signalling proteins. Numerous protein effectors of these lipidshave been identified over the last decade. Besides, recent method-ological advances have allowed the visualization of phosphoinosi-tide dynamics in living cell and their restricted manipulation inspecific membranes (for instance via organelle-specific recruit-ment of phosphatases), sheding new light on their functional com-partmentalization at the cellular level. Stunning similaritiesbetween mammalian, yeast and higher plant ‘‘phosphoinositidesystems’’ are noticeable, revealing early appearance and evolution-ary conservation of this intracellular language. However, major dif-ferences between organisms have also been highlighted over theyears. Recently, the discovery of an independent phosphoinositidesignalling occurring in the nucleus of mammalian cells have open anew perspective in the field. This review will focus on the mecha-nisms underlying PI4P and PI(4,5)P2 roles in signal transduction inresponse to extracellular stimuli, emphasizing common featuresand divergences between eukaryotic kingdoms. Functions of theother phosphoinositide species will not be addressed, and we referthe reader to recent reviews on this topic [5–7].

1.1. Spatio-temporal regulation of PI4P and PI(4,5)P2

Given the large range of functions regulated by PI4P andPI(4,5)P2, even in a same membrane compartment, an intriguingquestion is how one phosphoinositide species can be targeted todistinct physiological processes. This question has led to thehypothesis of phosphoinositide compartmentalization into inde-

pendent pools, dedicated to specific cellular activities [8]. Conse-quently, mapping phosphoinositide distribution within the celland understanding the spatio-temporal regulation of phosphoino-sitide subcellular pools have been a major issue of lipid signallingresearch in the last decades.

PI4P and PI(4,5)P2 are synthesized from PI, which typically rep-resents less than 15% of the total phospholipids found in eukaryoticcells [9]. Unlike structural lipids, PI4P and PI(4,5)P2 are only pres-ent in very small amounts (<1% of total cellular phospholipids ineach case) and have high turnover rates, making them ideal candi-dates for signalling functions [10,11]. If such a role is well estab-lished in all eukaryotic kingdoms, the relative abundance of PI4Pand PI(4,5)P2 differs markedly depending on the model. Indeed,the PI(4,5)P2:PI4P ratio is about 1:1 in yeast and mammalian cellswhereas it ranges from 1:10 to 1:100 in higher plants [12]. Such alow level suggests that PI(4,5)P2 might have different functions inplants compared to other eukaryotes. Regarding PI4P, it constitutesthe bulk of monophosphorylated PI derivatives in all eukaryoticcells [9,13,14]. If the overall contents of PI4P and PI(4,5)P2 do notvary widely upon stimulation in mammalian cells [13], they under-go rapid accumulation in plants exposed to heat or salt stress[12,15] and in nitrogen-starved yeasts in response to a nitrogensource [16].

1.2. Distribution over the cell

It is noteworthy that these phosphoinositides are not evenlydistributed in the cell. Their precursor, PI, is mainly synthesizedin the endoplasmic reticulum (ER) [17,18] or in ER-derived struc-tures [19]. It can be subsequently delivered to other cellular mem-branes through trafficking events or by action of phosphoinositide-specific lipid transfer proteins (PITPs), which play a central role incell signalling by coupling PI delivery to phosphoinositide synthe-sis [20–22]. It was initially assumed that most of the PI4P is pres-ent in the Golgi and in internal membranes, where it is thepredominant phosphoinositide [7,9,23,24]. However, recent stud-ies employing immunocytochemical techniques or specific phos-phoinositide-binding domains fused to fluorescent proteinshighlighted a major PI4P pool at the plasma membrane in mamma-lian, yeast and plant cells [14,25,26]. Polyphosphoinositides,including PI(4,5)P2, are mostly located in the inner leaflet of plasmamembrane [10,12,27]. In mammals small PI(4,5)P2 pools were alsodetected in intracellular membranes, including ER and mitochon-dria, suggesting a more complex pattern [27]. PI4P and PI(4,5)P2

presence in the nucleus will be discussed in Section 4.

E. Delage et al. / Progress in Lipid Research 52 (2013) 1–14 3

1.3. Lateral distribution in the plasma membrane

Although the overall PI(4,5)P2 level is relatively constant inmammalian cells, local increases are likely to occur, which mayserve to selectively regulate spatially restricted processes. Theexistence of PI(4,5)P2-rich microdomains within the plasma mem-brane has been evidenced. However, the exact nature of these do-mains remains controversial: if several studies point out PI(4,5)P2

association with the detergent-resistant membrane (DRM) ‘‘raft’’fraction, others highlight the biological importance of PI(4,5)P2

sequestration by proteins acting as PI(4,5)P2 reservoirs and thusdesignated as ‘‘pipmodulins’’ [28,29]. Rafts are lipid-ordered do-mains which can serve as ‘‘signalling platforms’’ in the plasmamembrane by concentrating substrates and enzymes together inthe same membrane microenvironment [30]. The observation thatup to half of the PI(4,5)P2 associates with the DRM fraction has ledto the hypothesis that rafts may enhance PI(4,5)P2-dependentpathways and production of second messengers [31,32]. By anelectron microscopic method that uses freeze-fracture replicas asa substrate for labelling, PI(4,5)P2 was recently shown to be highlyconcentrated at the rim of caveolae, small invaginated membranemicrodomains (50–100 nm) related to rafts [33]. Upon agoniststimulation, caveolae-associated PI(4,5)P2 displays distinct behav-iors from that in the undifferentiated membrane area, confirmingthe spatio-temporal heterogeneity of PI(4,5)P2 in the cell mem-brane. A pivotal role of PI(4,5)P2 in raft formation and maintenancehas been suggested [30]. Indeed, PI(4,5)P2 has a major role in theactin cytoskeleton organization and dynamic, and actin cytoskele-ton can promote membrane lipid ordering and raft protein cluster-ing. Interestingly, an enrichement of PI4P and PI(4,5)P2 in rafts hasalso been recently reported in Nicotiana tabacum by Furt et al. [34].Such a lateral segregation of these lipids may thus be a conservedfeature between mammalian and plant kingdom. In mammals localincreases in PI(4,5)P2 content could also involve several pipmodu-lins including the Myristoylated Alanine-Rich C Kinase Substrate(MARCKS) [28]. These proteins are able to electrostatically seques-ter (and thus concentrate) several PI(4,5)P2 molecules within theplane of the membranes [29,35,36]. MARCKS and related proteinsare highly abundant and could theorically buffer all PI(4,5)P2 pres-ent at the plasma membrane [8]. Furthermore, Golebiewska et al.(2008) evidenced a significant reduction of PI(4,5)P2 diffusion coef-ficient in the inner leaflet of the plasma membrane (compared toPI(4,5)P2 microinjected in the outer leaflet, giant unilamellar vesi-cles or blebs), and suggested that approximately two third of thePI(4,5)P2 in the inner leaflet may be reversibly bound to proteins[37]. Binding to calmodulin or phosphorylation by protein kinaseC (PKC) translocates MARCKS from membrane to cytosol in manycell types, thus releasing PI(4,5)P2 [29,38]. This suggests a Ca2+-dependent regulation by MARCKS of PI(4,5)P2 availability formetabolizing enzymes, such as phospholipase C (PLC; see Sec-

Fig. 1. Phosphoinositides synthesis and degrad

tion 2), or further interaction with effectors. Compartmentalizationinto rafts and sequestration by pipmodulins are not mutuallyexclusive. Indeed, Laux et al. (2000) reported localization of severalpipmodulins (including MARCKS) at rafts, where they appear topromote PI(4,5)P2 retention and clustering [27]. Proteins with anequivalent function have not been identified in yeast or plantshitherto.

1.4. Distribution in biochemically different pools

Several recent studies in plants evidenced that fatty-acid mo-ities may also be involved in sorting phosphoinositides into func-tionally distinct pools. König et al. (2007) highlighted theformation of two different PI(4,5)P2 pools upon hyperosmoticstress. The major stress-induced pool is constituted of PI(4,5)P2

with polyunsaturated fatty acid and serves as substrate for PLCand the generation of second messengers [39]. The second pool,which contains mainly saturated and monounsaturated fatty acids,associates with clathrin-coated vesicles and may be involved in theformation and internalization of endocytic vesicles [40,41]. It hasbeen proposed that fatty acid composition influences phosphoino-sitide interactions with alternative protein partners by affecting li-pid lateral mobility [22]. Otherwise, the tertiary structure of thephosphoinositide molecule could affect enzymatic activities [42].Evidences that different molecular species of a same phosphoinosi-tide isomer can perform distinct physiological functions have alsobeen reported in invertebrate cells [43]. However, phosphoinosi-tide functions are generally studied according to their specific ino-sitol headgroup and the importance of their fatty-acid backbonestill has to be investigated in other eukaryotic models.

1.5. Synthesis and degradation of PI4P and PI(4,5)P2

A tight control of the balance between phosphoinositide syn-thesis and degradation is crucial to the formation and maintenanceof the different subcellular phosphoinositide pools and thus todetermine which function will be achieved [8]. Therefore the spa-tio-temporal regulation of phosphoinositide kinases and phospha-tases has been extensively studied. Phosphatidylinositol 4-kinases(PI4Ks) catalyse the phosphorylation of PI in D4-position of theinositol ring, generating PI4P (Fig. 1) [44]. This molecule can besubsequently phosphorylated in D5-position by PI4P 5-kinases(PI4P5Ks) to produce PI(4,5)P2 [3,45]. In metazoa exclusively,PI(4,5)P2 can also be produced from phosphatidylinositol 5-phos-phate (PI5P) phosphorylation in D4-position by PI5P 4-kinases,but the main function of this pathway may be PI5P elimination(Fig. 1) [46]. Since PI5P level is much smaller (at least ten-timesless) than PI4P level, it is commonly assumed that most of thePI(4,5)P2 is generated from PI4P [45,47,48]. PI4P and PI(4,5)P2

can be degradated by phospholipases, like PLC, or lipid phospha-

ation. Dashed arrows: in mammals only.

Table 1Isorforms of phosphoinositide kinases and phospholipases in eukaryotic models.

Enzyme S.cerevisiae

H. sapiens A. thaliana

Type IIPI4K

Lsb6 PI4KIIa (8 putative: PI4KIIc1–8)

PI4KIIb

Type IIIPI4K

Stt4 PI4KIIIa PI4KIIIa1

(+1 putative: PI4KIIIa2)Pik1 PI4KIIIb PI4KIIIb1

(+1 putative: PI4KIIIb2)

PI4P5K Mss4 PI4P5Ka (PIPKIa) Group A: PI4P5K10PI4P5Kb (PIPKIb)PI4P5Kc (PIPKIc)

(+1 putative: PI4P5K11)Group B: PI4P5K1–6(+3 putative: PI4P5K7–9)

PI5P4K ? PI5P4Ka (PIPKIIa)PI5P4Kb

?

(PIPKIIb) PI5P4Kc(PIPKIIc)

PLC Plc1 PLCb1–4 PLC1–5PLCc1–2PLCd1, PLCd3–4 (+4 putative: PLC6–9)PLCePLCfPLCg1–2

?: none identified hitherto.

Table 2Characterized phosphatases acting on PI4P or PI(4,5)P2.

Catalysed reaction S. cerevisiae H. sapiens A. thaliana

PI4P ? PI Sac1p SAC1 SAC7/RHD4Synaptojanin 1,2 SAC6 SAC8

PI(4,5)P2 ? PI4P Inp51/Sjl1 OCRL1 SAC9Inp52/Sjl2 INPP5B 5PTase7Inp53/Sjl3 INPP5J 5PTas11/FRA3Inp54 Synaptojanin1, 2 5PTase14

PI(4,5)P2 ? PI5P ? TMEM55A TMEM55B ?

?: none identified hitherto.


tases (Fig. 1). Phosphoinositide phosphatases are considered tohave broader substrate specificities than the corresponding ki-nases, since they can generally act upon more than one substrate,including soluble inositides [11,49,50]. PI(4,5)P2 signalling can beterminated via its conversion into PI4P catalysed by 5-phospha-tases [3,11,46]. In mammals PI(4,5)P2 can also be dephosphoryl-ated at D4-position by phosphoinositide 4-phosphatases, thusgenerating PI5P [46]. As to PI4P, it can be dephosphorylated bySAC1-domain phosphoinositide phosphatases, which can be foundin all eukaryotic models [3,11]. We will not attempt to cover ingreat details the current knowledge on the different phosphoinosi-tide kinases and phosphatases (for review: [3,11,44,46,47,51,52])and will rather focus on how these enzymes contribute to regulatethe different functions of PI4P and PI(4,5)P2.

1.6. Non-redundant functions of phosphoinositide-modifying enzymeisoforms

An intriguing common feature of phosphoinositide-modifyingenzymes in all eukaryotic kingdoms is their existence as multi-genic families. In mammals, yeast and plants, PI4Ks are sub-di-vided as type-II and type-III (according to their size, Km valuesand sensitivity to inhibitors), with a variable number of isoformsassigned to each type (Table 1) [51]. The three mammalian PI4P5Ksare also known as type-I PIPK (type-II PIPKs are PI5P 4-kinases) andare homologous of the unique yeast PI4P5K, Mss4 (Table 1) [46].Eleven PI4P5Ks have been identified in genome of the plant modelArabidopsis and classified as type-I/II PIPKs, since sequence com-parison failed to assigned them as either type-I or type-II [51].Comparison with mammalian PI4P5Ks revealed a much lowerVmax/Km ratio for two Arabidopsis PI4P5Ks isoforms, which may ex-plain the low PI(4,5)P2:PI4P ratio in terrestrial plants [53]. Numer-ous phosphoinositide phosphatases are also conserved in thevarious eukaryotic models (Table 2). Over the last decades evi-dences accumulated that the different isoforms of PI-modifying en-zymes synthesize discrete cellular pools, thus mediating non-redundant biological functions [51,54–56]. Type-III PI4Ks offer aparticularly interesting example of such non overlapping roles be-cause the spatial and functional specialization of the b sub-type

seems to be conserved among eukaryotic kingdoms. Indeed, inyeast, plants and mammals, type-III PI4Kbs (i.e. Pik1 in yeast) havebeen mainly localized at the Golgi and are essential for Golgi struc-ture and functions [44,57–59]. On the other hand, cellular distribu-tion of a isoforms differ between organisms, therefore they arelikely to exert different functions. The yeast type-III PI4Ka Stt4 ismainly located at the plasma membrane, where it is necessaryfor cytoskeletal organization and various signalling pathways. Onthe contrary, in mammals and plants a isoforms of type-III PI4Ksappear to locate mostly at the ER/Golgi, where their functions arestill unknown [44,60]. Interestingly, even in a same membranecompartment distinct isoforms of the same phosphoinositide-modifying enzyme can be dedicated to different functions[58,61]. For instance, in the plasma membrane of stimulated mastcells PIP5KIb and PIP5KIc were recently shown to synthesize func-tionally distinguishable pools of PtdIns(4,5)P2 involved in store-operated Ca2+ entry and inositol 3,4,5-trisphosphate (IP3) genera-tion, respectively [61,62]. It implies the existence of finely tunedmechanisms governing the spatio-temporal regulation of theseenzymes.

1.7. Spatio-temporal regulation of phosphoinositide-modifyingenzymes

Most of the phosphoinositide-modifying enzymes are solubleand their recruitment to their site of action is a critical step inthe regulation of their subsequent biological functions. Their sub-cellular localization or catalytic activity can be altered by bindingto substrate and non-substrate lipids (for example phosphatidicacid (PA) is an important cofactor for the stimulation of PI4P5Ks[63]) or membrane anchored proteins [45,64,50]. In mammals,small G-proteins and their effectors are major recruiters and timersof PI4K and PI4P5K activities in response to extracellular signals[8,44,65]. An analogous interplay between phosphoinositide andsmall G-protein signalling pathways is also being unraveled inyeast and plants [65–68]. Existence of signalling complexes con-taining PI4Ks and/or PI4P5Ks along with their regulators anddownstream effectors is a tempting hypothesis for a coordinationof PI sequential phosphorylation and the channelling of phospho-inositide products toward specific signalling pathways. Evidencesfor such multimolecular complexes have been reported in the dif-ferent models [10,50,70]. Post-translational modifications alsocontribute to phosphoinositide kinases and phosphatases regula-tion. Their phosphorylation–dephosphorylation cycles by sharedprotein kinases and phosphatases provide a supplemental way tocoordinate their activities. It is illustrated by the control ofPI(4,5)P2 synthesis and degradation at the synapse by the cyclin-dependent kinase Cdk5 and the serine/threonine phosphatase cal-cineurin which regulates both PI4P5Kc and the phosphoinositidephosphatase synaptojanin 1 [2,70]. Examples of PI4P5K regulationby protein kinases have been reported in mammals, yeast andplant and phosphorylation generally has an inhibitory effect onPIPK activity [69,71].


The spatio-temporal regulation of PI4P and PI(4,5)P2 pools is inclose relation with their signalling functions and evident simili-tudes between eukaryotic kingdoms can be noticed. In yeast, plantand mammalian cells PI4P and PI(4,5)P2 are minority lipids with ahigh turnover and their distribution among membrane compart-ments is overall similar. Furthermore, their metabolizing enzymesare closely related in their primary sequence and, at least for someof them, in their regulation and cellular functions. However nota-ble differences also emerge, the most striking being the low abun-dance of PI(4,5)P2 in plants contrasting with the higher number ofPI4P5Ks in this model. On the other hand, mammals may possessunique means to locally regulate PI(4,5)P2 level and availability de-spite its relatively high and constant abundancy at the cellular le-vel, i.e. pipmodulins. It suggests that organisms may have evolveddifferent PI4P and PI(4,5)P2 functions over the course of eukaryoticdiversification.

2. PI4P and PI(4,5)P2 as precursors of second messengers

The best known function of PI(4,5)P2 in mammalian cells is assubstrate of two enzymes which produce major second messen-gers: PLC and Class I Phosphatidylinositol 3-kinases (PI3K). Inter-estingly, the PLC pathway is ubiquitous among eukaryotic cellswhereas signalling through Class I PI3K is a specific feature of met-azoa [6,72].

2.1. Biological functions of the PLC pathway

Historically, the discovery of the PLC pathway consitutes thefirst major insight into the importance of phosphoinositides in cellsignalling [73]. In mammals PLCs contribute to the regulation ofphysiological processes as diverse as cell motility, fertilizationand sensory transduction [74]. They are involved in cardiac andneuronal functions, as well as skin homeostasis, via regulation ofcalcium signalling, and also contribute to control the switch be-tween proliferation and differentiation [75]. In plants, PLCs areactivated in response to multiple abiotic stresses, such as heat,cold, osmotic and salt stresses but also in response to pathogens[76,77]. Furthermore, they are involved in light and gravity sens-ing, which both result in directional growth [78]. PLCs are alsoimportant for yeast response to stress, in addition to their rolesin nutrient sensing, growth control and differentiation [79].

2.2. Second messengers produced by the PLC pathway

In the ‘‘canonical’’ mammalian pathway, PLC activated by cellsurface receptors hydrolyses PI(4,5)P2, thus producing two secondmessengers: diacylglycerol (DAG) and IP3 [73,80]. DAG remains inthe membrane and activates several members of the PKC familyand other targets, including protein kinase D and ion channels[81,82]. On the other hand, IP3 binds to specific receptors (mainlylocated in the ER) which release Ca2+ from internal stores, thuspropagating the signal [83]. Although PLC activity has been ob-served in all eukaryotic models [72,79], clear differences with thecanonical mammalian pathway have emerged over the years. In-deed, neither yeast nor plant genomes contain any homologue ofthe mammalian IP3 receptor [5,84]. Furthermore no PKC has beenfound in plants and there is no direct evidence of a functional linkbetween yeast PKC and PLC homologues [5,85]. Compelling evi-dences are accumulating that plants rather use phosphorylationproducts of DAG, i.e. PA and diacylglycerol pyrophosphate (DGPP),and of IP3, i.e. IP4, IP5 and IP6, as second messengers [86–88]. Inyeast, cytoplasmic Ca2+ increase attributed to IP3 production byPLC has been reported in response to glucose but it seems to bea specificity of this particular signal transduction pathway [89].

Like in plants, IP3 phosphorylated derivatives rather appear to bethe major second messengers produced via the PLC pathway [90](see Section 4). As compelling evidence for signalling through IP3

receptor/Ca2+ channels is only available for metazoa, it has beensuggested that this pathway emerged during the later stages ofeukaryote diversification [6].

2.3. Substrate supply to PLC

PI(4,5)P2 is only present in small amounts at the plasma mem-brane and it has long been recognized that sustained PLC activationrequires constant replenishment (i.e. de novo synthesis) of thePI(4,5)P2 pool [10,79]. In both mammals and plants, IP3 productionattributed to PLC is increased by the overexpression of PI4P5K[91,92]. Therefore substrate supply, via sequential phosphorylationof PI by PI4Ks and PI4P5Ks, could be a key regulatory step in PLCpathway. Hence, identification of PI4K and PI4P5K isoforms in-volved in PLC supply is critical to further understand PLC signal-ling. Several studies indicated that PI(4,5)P2 generated byPI4P5Kc is hydrolysed by PLC in mammals [61,93]. However, theidentity of PI4P5K isoforms that provide substrate to PLC may varyaccording to cell types or physiological stimuli. For instance bothPI4P5Ka and PI4P5Kb, but not PI4P5Kc contribute to IP3 produc-tion in thrombin-stimulated platelets [53]. Metabolic and pharma-cological studies have shown that PI4Ks are also stimulated duringagonist activation of PLC [53]. In this context, Balla et al. (2008)highlighted the predominant role of type-III PI4Ka in the mainte-nance of plasma membrane PI4P and PI(4,5)P2 pools [94]. Thesedata were quite surprising since mammalian type-III PI4Ka ismostly found in the ER/Golgi and not at the plasma membrane.The authors hypothesize the presence of a small amount of activetype-III PI4Ka at the plasma membrane, below the detection limitof immunocytochemical methods [94]. Alternatively, PI4P synthe-sis might occur at ER-plasma membrane contact zones [10]. Inter-estingly, using a recruitable SAC1 phosphatase to rapidly depletePI4P from Golgi membranes, the same group also reported a size-able contribution of the Golgi PI4P to the maintenance of plasmamembrane PI(4,5)P2 [95]. These data suggest probable communi-cations between distinct cellular pools of phosphoinositides. Theimportance of substrate availability in the regulation of the PLCpathway was further evidenced recently in mammals by An et al.(2011), who showed that the protein kinase WNK1 stimulatesPLC signalling by promoting the synthesis of PI(4,5)P2 via stimula-tion of type-III PI4Ka [96]. Interestingly, in addition to its role asPLC substrate PI(4,5)P2 was shown to be an important recruiterand regulator of several mammalian PLC isoforms (see Section 3).

The question of substrate supply to PLC is even more relevant inhigher plants, given the much lower amount of PI(4,5)P2 in thismodel. As mentioned above, heterologous expression of the mam-malian PIP5Ka in plant cells triggers elevation of plasma mem-brane PI(4,5)P2 and increases IP3 production by PLC in steadystate [92]. The authors consequently suggest that PI4P phosphory-lation by PI4P5K may be the limiting step of plant PLC pathway.This is consistent with the observations showing that plantPI4P5Ks are considerably less active than their mammalian homo-logues [53]. Since PI4P is the most abundant phosphoinositide inplant models, and because PLCs can hydrolyse it in vitro, PI4P hasbeen suggested to be also an in vivo substrate of plant PLCs[12,72,97]. We recently highlighted the activation of a PI4K in par-allel to the PLC activation triggered by cold exposure, and therequirement of type-III PI4Ks to supply the PLC pathway in thiscontext [98]. Since in yeast PI(4,5)P2 synthesis by the uniquePI4P5K Mss4 requires Stt4 [5], the involvement of type-III PI4Ksin the generation of PLC substrate appears to be a conserved fea-ture between eukaryotic kingdoms.


2.4. PI3K signalling pathway

In addition to be the PLC substrate, in metazoan cells PI(4,5)P2

can be metabolized into phosphatidylinositol 3,4,5-trisphophate(PI(3,4,5)P3) by Class I PI3Ks [99]. This major signalling pathwayis involved in the regulation of various biological processes suchas immune response, wound healing, cell directional movements,neuronal patterning and embryogenesis [100]. After stimulationPI(3,4,5)P3 accumulates at the plasma membrane where it bindsand recruits signalling proteins [101]. Its best characterized effec-tors are the phosphoinositide-dependent kinase 1 (PDK1) and theserine/threonine protein kinase Akt (also known as protein kinaseB, PKB), which subsequently regulate numerous downstream tar-gets [102]. PI(3,4,5)P3 can be dephosphorylated at the D3-positionof the inositol ring by PTEN (phosphatase and tensin homolog de-leted on chromosome 10) to produce PI(4,5)P2 or at the D5-posi-tion by SHIP (SH2-containing inositol phosphatase) familyphosphatases to produce PI(3,4)P2, which also acts as signallingmolecule [103]. No Class I PI3K has been found in yeast nor plantgenomes (the only PI3K identified so far in these organisms is aClass III PI3K which only phosphorylates PI) [51]. SurprisinglyPI(3,4,5)P3 has been detected in fission yeast (Schizosaccharomycespombe) but it is not produced by phosphorylation of PI(4,5)P2

[104]. It would rather originate from PI3P synthesis. If PI(3,4,5)P3

functions remain to be determined in yeast, these results suggestthat a PI(3,4,5)P3 synthesis pathway evolved before the appearanceof Class I PI3K. So far PI(3,4,5)P3 has never been isolated in plants.Strikingly although PDK1 has been reported in Arabidopsis it isregulated by PA, which may be the major signalling molecule pro-duced by PI(4,5)P2 metabolism in the plant kingdom [105]. TheClass I PI3K pathway thus consitutes a marked divergence betweenmetazoa phosphoinositide system and other eukaryotes.

By generating several second messengers having various signal-ling functions from a single molecule, PLC and Class I PI3K path-ways represent important hubs in mammalian cell signalling.Interestingly, although PLCs seem conserved in all eukaryoticorganisms both substrate and end products of this pathway differin their nature and amount depending on the model. It has beensuggested that PLC pathway may have more prominent roles inmammals than in yeast and plants [6,106]. Signalling viaPI(4,5)P2-derived second messengers may thus have gained impor-tance with the increase of organismic complexity.

3. Downstream effectors of PI4P and PI(4,5)P2 signalling

Over the last decades, it became increasingly clear that PI4P andPI(4,5)P2 are not only precursors of second messengers but can alsoserve as membrane-bound signalling molecules per se, by interact-ing with effector proteins (Fig. 2) [107,108]. In mammals theseinteractions are crucial for a wide variety of functions, includingthe organization and regulation of signal transduction pathways.In plants and yeast similar signalling mechanisms begin to beunraveled [109–111].

3.1. Regulation of transmembrane proteins

Numerous transmembrane proteins are regulated by PI(4,5)P2

in mammals, including a striking number of ion channels andtransporters which are essential for many signal transductionpathways (Fig. 2A) [112,113]. Although much less is known inother models, recent examples of ion channel regulation byPI(4,5)P2 have been recently reported in plants [114–116]. SincePI(4,5)P2 is mostly found in the plasma membrane, PI(4,5)P2

dependence is thought to restrict channel/transporter activity intothis compartment [112]. Channel binding to PI(4,5)P2 can result,

for instance, in a shift of their voltage dependence (e.g. CardiacNa+–Ca2+ exchange (NCX1)) or a stabilization of their open state(e.g. Kir channels) ([117]; voltage-gated Ca2+ channels,[112,118]). Besides channels, Michailidis et al. (2011) recently re-ported a novel requirement for PI(4,5)P2 in the activation of theepidermal growth factor receptor (EGFR) and its downstream sig-nalling to ion channels. The precise mechanism of action ofPI(4,5)P2 on the EGFR is unknown, but it may help maintainingthe receptor in a proper active conformation. Since EGFR activationleads to PI(4,5)P2 hydrolysis via PLC activation, its functionaldependence on PI(4,5)P2 has been suggested to serve as a mecha-nism of self-desensitization [119].

3.2. Membrane anchorage of signalling proteins

Another possible consequence of protein interaction with phos-phoinositides is their recruitment to specific locations in mem-branes, where they exert their functions or become colocalizedwith signalling partners (Fig. 2B) [120]. A recent example has beenhighlighted by Garrenton et al. (2010) in yeast response to peptidemating pheromone [121]. In this context, PI(4,5)P2 was shown toconcentrate at the shmoo tip (i.e. the cellular projection resultingfrom mating pheromone) and the disruption of the resultingPI(4,5)P2 gradient markedly reduces the mating response. This accu-mulation of PI(4,5)P2 turned out to be required for the restrictedlocalization at the plasma membrane of Ste5, a mitogen activatedprotein Kinase (MAPK) scaffold protein [122]. Ste5 interaction withPI(4,5)P2 is essential for downstream signal propagation, since it al-lows Ste11 (a MAPKKK bound to Ste5) to be activated by the mem-brane-bound Ste20 (a MAPKKKK) [121,122]. Similar examples havebeen reported in mammalian cells. For instance PI(4,5)P2 directlyregulates Ca2+-induced PKCa translocation to the plasma mem-brane which is crucial in the signalling network controlling neuraldifferentiation [123,124]. Besides it is noteworthy that domainsmediating phosphoinositide binding have been found in a substan-tial number of phosphoinositide-modifying enzymes in all eukary-otic models and may target their catalytic activity to substrate-rich membranes [60,82,125]. For instance PI(4,5)P2 anchors themammalian PLCd1 to the plasma membrane via a non-catalytic do-main, thus allowing the enzyme to hydrolyse its substrate in a pro-cessive manner [74,126]. PLCd1 has a greater affinity for IP3 and ithas been shown that accumulation of IP3 can displace PLCd1 fromPI(4,5)P2-containing membranes in vitro. A similar function for IP3

as a non-competitive inhibitor of PLCd1 in vivo has been proposedand may be important for the regulation of this PLC isoform [127].Recently, interaction of the mammalian PLCf with PI(4,5)P2 via a re-gion situated between the X and Y catalytic domains was also shownto be crucial for PLCf physiological function [128]. Although yeastand plant PLC are related to mammalian PLCd and f, the PI(4,5)P2-interacting domains are not conserved [79,129]. Mechanisms allow-ing their recruitment to membrane thus remain to be determined.

3.3. Regulation of enzymatic activities

In addition to translocate cytosolic enzymes to membranes, inclose proximity with their substrate or protein partners, phosphoin-ositide binding can also directly modify enzymatic activities(Fig. 2C). For instance PI(4,5)P2 regulates in several ways anotherfamily of phospholipases: the phospholipases D (PLDs). PLDs hydro-lyze phospholipids to PA and free alcohols and are involved in multi-ple signalling networks [63,130]. Mammalian, yeast, and most of theplant PLDs are PI(4,5)P2-dependent [131,132]. In mammals andyeast PI(4,5)P2 is required for PLD recruitment to their site of actionbut also induces a conformational change which stimulates theircatalytic activity [131,133]. Similar mechanisms have been reportedfor plant PLDb [134,135]. Furthermore, all the activators of the hu-

Fig. 2. Protein regulation by PI(4,5)P2. (A) Ion channel regulation, (B) recruitment and colocalization with signalling partner, (C) recruitment to substrate-rich membrane andenzymatic regulation by conformational change and (D) sequestration under steady state and release upon stimulation.


man PLD1 (such as PKCa and ADP-ribosylation factor 1) are them-selves activated by PI(4,5)P2 and their synergistic interactions isdependent on PI(4,5)P2 concentration [136]. Since stimulation ofPI4P5K activity is a key downstream effect of PLD activation inmammals, it may form a positive feed-back loop that leads to a rapidgeneration of PA and PI(4,5)P2 [137]. Unlike mammalian PLDs, sev-

eral plant PLDs are also activated by PI4P, albeit to a lesser extentthan by PI(4,5)P2 [138–140]. Given the relative abundance of PI4Pcompared to PI(4,5)P2 in plant cells, this particular feature couldbe physiologically relevant and it has been suggested that the sumof these two lipids is in fact responsible for PLD activation [141].In mammals, PI(4,5)P2 can also interact with a large number of small


GTPases and their regulators (guanine nucleotide exchange factors(GEFs) and GTPase activating proteins (GAPs)) [142], thus regulatingtheir activity through both translocation and conformationalchanges [9,143]. Although much less evidences for such a tightinterplay between phosphoinositides and small G-protein signal-ling are available in other models, examples of PI(4,5)P2 regulationof small GTPases have been reported in yeast and plants[144,145]. Since small GTPases regulate both signalling, cytoskeletaland transport processes, it suggests that PI(4,5)P2 can act as a signal-ling hub in cellular control systems. Furthermore, in mammals,yeast, and plants, PI4P- and PI(4,5)P2-interacting partners also com-prise regulators of actin cytoskeleton and membrane trafficking. Itincludes many different actin binding proteins (profilin, gelsolin,ADP/cofilin...) [4,146,147] and components of the molecularmachineries involved in membrane bending and fission, and in ves-icle movement, tethering and fusion [3,24,148]. Thus PI4P andPI(4,5)P2 are likely major coordinators of membrane traffickingand cytoskeleton dynamic with signal transduction pathways.

3.4. Protein sequestration

Finally, apart from attracting effectors to their site of action inresponse to upstream stimuli, PI(4,5)P2 can also sequester proteinsfrom their downstream targets under steady state and releasethem upon stimulation (Fig. 2D). An elegant example is the regula-tion of the transcription factor Tubby highlighted by Santagataet al. (2001) in mammals [149]. Upon receptor stimulationPI(4,5)P2 is hydrolysed by a PLC and consequently releases Tubby,which subsequently translocates from the plasma membrane tothe nucleus where it regulates gene expression. Several tubby-likeproteins have been identified in mammals and plants and mightprovide a direct link between plasma membrane phosphoinositidesignalling and gene regulation [150,151]. Existence of such a link iscorroborated by the identification of several other transcriptionfactors as potential PI(4,5)P2 targets by Catimel et al. (2008) intheir recent analysis of PI(4,5)P2 interactome [142].

3.5. Phosphoinositide binding domains

Our understanding of the molecular mechanisms and structuralbasis underlying protein targeting to membrane and phosphoino-sitide recognition have greatly expanded over the last decades.All phosphoinositide-interacting proteins possess specific domainswhich bind the inositol headgroup of phosphoinositides with var-ious degrees of affinity and selectivity. These domains consist ofstretches of clustered basic and hydrophobic residues or foldedmodules. They vary widely in their binding mechanisms and thesedifferences can be exploited for distinct modes of control (e.g. reg-ulation by soluble second messengers, membrane curvature orcomposition, oligomerization, etc.) [13,152]. Folded modules iden-tified within mammalian PI(4,5)P2 effectors include members ofPleckstrin Homology (PH), Phox homology (PX), PKC homology-2(C2), FERM (acronym of band Four.1, Ezrin, Radixin, Moesin), EpsinN-Terminal Homology (ENTH), AP180 N-terminal Homology(ANTH), and Tubby domain families [127,153,154]. Most of thesedomains are also present in yeast and plant [13,110].

PH domains constitute the best characterized family of phos-phoinositide-interacting modules and are the most frequentlyfound in PI(4,5)P2 effectors with signalling function. These domainscontain about 120 residues, harboring low degree of primary se-quence homology, but a highly conserved three-dimensional struc-ture [154]. Many of the PH domains that have been characterizedweakly bind to phosphoinositides, with little or no specificity[155]. However, a subset (10–20% of the PH domains) binds indi-vidual phosphoinositides with high affinity and specificity, due tostereospecific interaction with the inositol headgroup [13]. The

best example is the PH domain of the mammalian PLCd1, whichbinds strongly to PI(4,5)P2 and its isolated headgroup, IP3 [156](see Section 2). Other PH domains displaying high affinity for bothPI(4,5)P2 and PI(3,4,5)P3 are found (for instance, in b-adrenergicreceptor kinase, insulin receptor substrate-1, RasGAP, b-spectrinand diacylglycerol kinase (DAGK) d [157]. PH domains that prefer-entially bind to PI4P have also been characterized in mammals,yeast and plants [158–161].

High affinity and specific binding is far from being a commonfeature of phosphoinositide-binding domains [162,163]. For mostof them, phosphoinositide-interaction is non-selective and in-volves attraction to physical properties of the membrane (charge,amphiphilicity, curvature) [164]. Indeed, PI4P and PI(4,5)P2 havehigh net negative charges of -3 and -5, respectively, and their inter-action with proteins is often mediated by clusters of positivelycharged amino acids. The partitioning of exposed hydrophobic sur-face within the interfacial region of the bilayer can also provide thedriving force to the plasma membrane [153].

A consequence of this weak affinity and selectivity is that manydomains cannot act alone as localization signals. This raises thequestion of how phosphoinositide interaction with cytosolic pro-teins can mediate specific signal transduction. As a general rule,both stereospecific recognition (3D structure specificity) andnon-specific interactions (such as electrostatic interactions) arecoupled to allow the selective binding of phosphoinositide targets[153]. Indeed, the specific phosphoinositide-binding sites areflanked by basic or hydrophobic residues, whose electrostaticinteraction with the membrane can add to the stereospecific inter-action and greatly increase the net binding energy [157]. Besides,several PI(4,5)P2-binding domains in the same protein or oligomercan cooperate to drive multivalent membrane binding. It is thecase for mammalian dynamin-1 and -2, whose affinity forPI(4,5)P2 is much higher when they are oligomerized [165]. Alter-natively, one protein can possess several distinct domains havingdifferent lipid or protein targets, which cooperate to allow highaffinity binding to membranes containing both targets [13]. For in-stance the human p120-RasGAP possesses a PH domain and a C2domain, along with two Src Homology 2 (SH2) domains whichinteract with phosphorylated tyrosines [166].

Since the phosphomonoester groups of PI(4,5)P2 have a veryhigh turnover, PI(4,5)P2-binding proteins may continuously en-gage-disengage and, hence, could rapidly react to local changesin inositide levels [167]. Therefore it has been suggested that an-other key to specificity is the localized production and eliminationof the phosphoinositide in close vicinity to its effector molecules.As mentionned above, this could involve the recruitment of kinasesand phosphatases along with the effector protein. Finally, it shouldbe noted that the requirements for PI(4,5)P2 targeting are lessstringent than for D3-phosphoinositides in mammalian cells, sinceit constitutes the most abundant species [127].

The ability of PI(4,5)P2 (and to a lesser extent PI4P) to regulatesignalling proteins via their lipid-binding domains is clearly a con-served feature in eukaryotic cells. However most of our actualknowledge is derived from mammals and yeast, and only a fewphosphoinositide targets have been identified in plants hitherto.Nevertheless, existence of similar effectors and mechanisms of reg-ulation has been evidenced. The numerous proteins with charac-teristic lipid-binding domains that have been identified inArabidopsis genome thus offer great future prospects in lipid sig-nalling research [110].

4. PI4P and PI(4,5)P2 in nuclear signalling

Recent studies in both mammals and plants have provided evi-dences of nucleus autonomy in terms of calcium signalling [167–


170]. Interestingly, a similar paradigm is emerging for nuclearphosphoinositide signalling in mammals. Indeed, in many casescytosolic and nuclear phosphoinositide systems are not inducedby the same agonists, or, if they are, it is in a temporally distinctmanner [171,172]. Furthermore, astonishing peculiarities of thenuclear phosphoinositide system have been revealed.

4.1. Nuclear pools of PI(4,5)P2 in mammalian cells

Most of the enzymes involved in phosphoinositide signallingare also found in the nucleus, eventually shuttling upon stimula-tion of cells [172]. Nuclear phosphoinositides and phosphoinosi-tide-modifying enzymes have a dual localization: they arepresent in the inner nuclear enveloppe and, much more surpris-ingly, in the nucleoplasm and nucleolus, separated from any mem-brane system [172,173]. For instance, PI(4,5)P2 and PI4P5Ks arepresent in nuclear speckles (or interchromatin granule clusters),along with enzymes of the phosphoinositide cycle, such as PLCs,DAGKs, PI3Ks and phosphatases [171,174–176]. Nuclear specklesare non-membranous nuclear structures involved in mRNA pro-cessing [175]. PI(4,5)P2 presence without membrane anchorageraises the question of what mechanism shield the hydrophobicacyl chains from solvent. It has been proposed that phosphoinosi-tides and other nuclear lipids might form micellar structures [172].Otherwise, they might associate with carrier proteins containingphosphoinositide acyl chain-binding pockets, similar to the hydro-phobic pocket of the yeast PITP Sec14 [172,176]. Thus their hydro-phobic tails would be hidden while their inositol head group wouldremain accessible for metabolizing enzymes.

4.2. Nuclear PI(4,5)P2 as a precursor of second messengers inmammalian cells

Like its cytosolic counterpart nuclear PI(4,5)P2 is the substrateof PLCs and Class I PI3Ks, and the resultant products are importantsecond messengers. They serve major functions in the regulation ofcell growth, differentiation and proliferation in response to extra-cellular stimuli [175].

Several PLC isoforms have been identified in the nucleus [177].The produced DAG attracts to the nucleus and subsequently acti-vates PKCs, which in turn regulate numerous nuclear targets[178,179]. Although it has long been a controversial issue, evi-dences are accumulating that nuclear IP3 participates in calciumhomeostasis, by mobilizing Ca2+ from the nuclear enveloppe store[178,180–182]. Furthermore, nuclear IP3 serves as a precursor forhigher inositol polyphosphates, whose role in mRNA export inmammalian cell have been suggested [183]. Among the PLCs de-tected in nucleus, PLCb1 is the isoform whose nuclear functionshave been the most consistently documented. Upon short termstimulation by the insulin growth factor IGF-1, PLCb1 is phosphor-ylated by extracellular signal-regulated kinases/mitogen-activatedprotein kinases (ERKs/MAPKs) and evidences implicate this phos-phorylation as part of the growth response to IGF-1 [184]. Besides,recent studies reported the involvement of PLCb1 in the progres-sion of myelodysplastic syndromes (MDS) to acute myeloid leuke-mia, via both genetic and epigenetic processes in hematopoieticstem cells [185]. Indeed, PLCb1 promoter is hypermethylated inhigh-risk MDS patients, resulting in gene silencing, and is a specifictarget for demethylating therapy, alone or in combination with his-tone deacetylase inhibitors [186,187].

In addition to the components of the PLC pathway, Class I PI3Ks,their product PtdIns(3,4,5)P3, and the PtdIns(3,4,5)P3 effector Akt/PKB have also been localized within the nucleus. This nuclear path-way might be involved in cell survival, through a block of caspase-activated DNase and the inhibition of chromatin condensation[188]. Okada et al. (2008) recently identified the mRNA export fac-

tor Aly as a direct target of activated nuclear Akt [189]. Akt-phos-phorylation of Aly regulates its capacity to bind PI(4,5)P2 andPI(3,4,5)P3, which is essential for Aly nuclear speckles localization.The authors showed that cell cycle progression, cell proliferation,and mRNA export activities of Aly are regulated by nuclear PI3Ksignalling, via both Akt-phoshorylation and PI(3,4,5)P3 binding.

4.3. Nuclear PI(4,5)P2 downstream effectors in mammalian cells

Nuclear PI(4,5)P2 is not only the precursor of second messen-gers but also contributes, by itself, to the regulation of variousimportant functions including gene expression, via mRNA process-ing and chromatin remodeling [172,175]. Mechanisms underlyingthe role of PI(4,5)P2 in the nucleus are starting to emerge[172,175,190,191]. As mentionned above, PI4P5Ks and PI(4,5)P2

localize at nuclear speckles (or interchromatin granule clusters)in the nucleus of mammalian cells [173]. Nuclear speckles are nu-clear domains enriched in pre-mRNA processing factors, located inthe interchromatin regions of the nucleoplasm [192]. They arethought to act as storage/assembly/modification compartmentsthat can supply splicing factors to active transcription sites.PI4P5Ks and PI(4,5)P2 reorganize along with speckle morphologyupon inhibition of mRNA transcription [173]. Osborne et al.(2001) highlighted that structures resembling interchromatingranule clusters contain PI(4,5)P2, present in a complex togetherwith proteins (including RNA polymerase II and mRNA splicing fac-tors), lipids and nucleic acids, and are essential for pre-mRNA splic-ing in vitro [173]. Moreover, nuclear speckles contain PI(4,5)P2

effectors. Mortier et al. (2005) demonstrated that PDZ (Postsynap-tic density protein, Disc large, Zona occludens) domain of syntenin-2 binds PI(4,5)P2 with high affinity and targets the protein to nu-clear speckles and nucleoli [193]. Depletion of syntenin-2 disruptsthe nuclear speckle PI(4,5)P2 pattern and affects cell survival andcell division, suggesting that syntenin-2 functions as an organizerof nuclear PI(4,5)P2. The authors propose that it could stabilizespeckle structure through its propensity to self-associate and bindPI(4,5)P2.

Recently, nuclear PI(4,5)P2 was shown to bind to and regulatethe activity of a poly(A) polymerase named Star-PAP (nuclearspeckle targeted PI4PK5a regulated poly(A) polymerase; [194]).In addition, Star-PAP also binds directly to PI4P5Ka in vitroand in vivo. PI(4,5)P2 enhances the processivity of Star-PAP, thusregulating the 30-end formation of a subset of specific mRNAs.Star-PAP activity and the production of specific Star-PAP mes-sages also result from Star-PAP activation via phosphorylationby the casein kinase Ia (CKIa) [195,196]. Interestingly, CKIa it-self is a PI(4,5)P2-sensitive enzyme. Many of the genes whosemRNA processing is controlled by PI4P5Ka and Star-PAP encodeproteins involved in oxidative stress response [194]. These datapoint out a phosphoinositide pathway that controls the expres-sion of specific genes via mRNA 30-end processing of their in re-sponse to oxidative stress.

Additionally, PI(4,5)P2 may also impact gene expression bycontributing to chromatin unfolding and transcriptional regula-tion [197]. Indeed, histones H1 and H3 were also identified asphosphoinositide-binding proteins. In vitro, PI(4,5)P2 binding tohistone H1 C-terminal tail releases histone from DNA and there-fore counteracts the H1-mediated repression of basal transcrip-tion by RNA polymerase II [198]. Another mechanism forPI(4,5)P2 regulation of chromatin structure could involvePI(4,5)P2 interaction with Brahma-related gene associated factor(BAF) complex, the prototypical mammalian chromatin-remodel-ing complex [199]. In T-lymphocytes, antigen receptor signallinginduces the rapid translocation of BAF complex from a solublefraction to an insoluble fraction by association with chromatin[172,200]. PI(4,5)P2 was shown to enhance BAF complex


interaction with both chromatin and actin filaments and there-fore was suggested to constitute a matrix localization signal forBAF complex [200,201]. As a consequence of nuclear matrixinteraction, BAF could be stabilized or, alternatively, may stabi-lize actin branch points resulting in force generation whichmay participate in the resolution of higher order chromatinstructure [201]. Additionally, PI4P5K interacts with and may beregulated by the retinoblastoma protein Rb, which recruits theBAF complex to transcription sites [202]. This could constitutea mechanism for regulation of localized PI(4,5)P2 synthesis atsites where it can specifically affect BAF activity [195,202,203].

Till now, only a few nuclear phosphoinositide effectors havebeen identified. Employing a proteomic approach based on neomy-cin extraction of intact nuclei, Lewis et al. (2011) recently identi-fied 28 nuclear PI(4,5)P2-interacting proteins [191]. Functionalclassification of these proteins pointed to roles in mRNA transcrip-tion regulation, mRNA splicing and protein folding. They provide apromising resource for future investigations on PI(4,5)P2 functionsin the nucleus.

4.4. Existence of a nuclear phosphoinositide system in other eukaryoticmodels?

Although phosphoinositides have been detected in yeast [175]and plant nucleus [15], the existence of a similar nuclear phospho-inositide system in these organisms remains highly speculative.Very few data are available concerning nuclear PI4P and PI(4,5)P2

in plants [204]. In Arabidopsis, PI4KIIIb1 and PI4P5K9 are foundin the nucleus [205,206] and nuclear PI4K and PI4P5K activitieshave been reported [204,207]. Moreover, heat stress triggersPI(4,5)P2 accumulation in the nuclear envelope and nucleolus[15], suggesting that nuclear phosphoinositide signalling pathwaysmight also take place in plant nucleus. In yeast, both the PI4K Pik1and the PI4P5K Mss4 undergo nucleo-cytoplasmic shuttling. Theresulting nuclear PI(4,5)P2 pool is assumed to be necessary for cellviability, although its precise localization within the nucleus is stillcryptic [113]. No direct signalling function of this nuclear PI(4,5)P2

has been reported thus far and its main role may be to serve as pre-cursor of inositol polyphosphates, which are important nuclearsecond messengers [175]. Indeed, Plc1 is also found in the nucleusand, along with the inositol polyphosphate kinases Ipk1 and Ipk2,is involved in various aspects of DNA and RNA metabolism[208,209].

Existence of an independent nuclear phosphoinositide systemwith crucial roles in the regulation of nuclear functions has beena major breakthrough of the lipid research in the last decade. De-spite the accumulation of data obtained from the nucleus of mam-malian cells several aspects remain cryptic. In particular, the wayphosphoinositides reach nucleoplasmic structures and are main-tained there in the absence of a membrane environment is stillan enigma. Furthermore, a major issue will be to determine if sim-ilar nuclear phosphoinositide systems also exist in other eukary-otic organisms, and, if it be so, what are their physiologicalsignificances.

5. Concluding remarks

The picture emerging from our current knowledge reveals anevolutionary conserved signalling system centered aroundPI(4,5)P2. This system is based on very similar machineries in thedifferent eukaryotic kingdoms. Indeed, homologues of most ofthe enzymes which synthesize and hydrolyse PI(4,5)P2 and its pre-cursor, PI4P, in mammals have been found in yeast and plants.They are clearly related to their mammalian counterpart in boththeir primary structure and physiological functions, although the

number of isoforms for each enzyme varies between organisms.Furthermore, similar lipid-binding domains promote the interac-tion of protein effectors with PI(4,5)P2 and PI4P in all eukaryoticmodels. The signalling events involving these phosphoinositidesin response to extracellular stimuli have also much in common:production of second messengers, spatio-temporal organizationof signalling complexes, regulation of ion channels and signallingenzymes.

Nevertheless, each model has its peculiarities. Although PLCpathway is conserved among eukaryotes the nature of the resul-tant second messengers differs, and so differ their downstream tar-gets. In metazoa exclusively PI(4,5)P2 performs the additionalcrucial role of being substrate for the Class I PI3K pathway. Fur-thermore, it is involved in many different aspects of nuclear signal-ling and thus have functions which have not been reported in otherorganisms, such as regulation of mRNA processing and chromatinremodeling. Thus, one may assume that PI(4,5)P2 is more impor-tant in animal cells than in other eukaryotes. This hypothesis isapparently confirmed by the minor amount of this phosphoinosi-tide in higher plants. In this model it has been suggested thatPI4P may assume functions attributed to PI(4,5)P2 in other organ-isms. Interestingly, recent evidences indicate a role of PI4P as anextracellular signalling molecule involved in plant defence re-sponses [210,211]. Such a role has not been reported in otherorganisms and could constitute an additional unique feature ofthe plant system. However, our overview of convergences anddivergences between eukaryotic kingdoms is still limited by thelack of data in plants. The modest progress in deciphering phos-phoinositide functions in this model can be explained, notably,by the difficulties encountered in the past to identify phosphoino-sitides from plant tissues and to obtain protein sequences frompurified phosphoinositide-modifying enzymes [51]. Thus it is likelythat only the tip of the iceberg has been discovered and the roles ofplant PI4P and PI(4,5)P2 in cell signalling may prove to be as wide-spread as in mammals and yeast.

Our present knowledge of upstream regulators and down-stream effectors of PI4P and PI(4,5)P2 is still fragmentary andthese phosphoinositides likely regulate more functions than wecurrently know. Furthermore, future work should be directedto the elucidation of the complex interplay existing betweentheir multiple functions in the different eukaryotic kingdomsand especially between their constitutive and signalling func-tions. With this aim in view, it is crucial to improve our knowl-edge on the spatio-temporal regulation of phosphoinositides atthe subcellular scale. Today, a lot of questions remain to be an-swered. The most intriguing are probably those raised by thediscovery in the nucleus of PI(4,5)P2 pools which are not associ-ated with any known membrane. PI(4,5)P2 may exist there with-in composite complexes, whose components remain to bedetermined. It is tempting to speculate that such complexesmight also exist in other cellular compartments but are not de-tected in classical localization studies.

Better understanding of phosphoinositide functions and regula-tion is a major issue since they control organism development,metabolism and interactions with their environment. It could thusenable new possibilities to improve plant growth and pathogenresistance, including for staple food crops. Furthermore a growinglist of human diseases of major societal importance have beenlinked to phosphoinositide kinases and phosphatases. It includeshepatitis C, Alzheimer’s Disease, and cancer [11,56,212]. Conse-quently, these enzymes as drug targets represent an emerging to-pic for therapeutical research.

Given the universal importance and overall conservation of thephosphoinositide system in the different eukaryotic kingdoms, webet that interdisciplinary studies will be the key for a more com-prehensive view of this fascinating signalling system.


Acknowledgements

We thank Dr. Sébastien Mongrand (CNRS, UMR 5200, Bor-deaux), and Emmanuel Baudouin (CNRS, EAC 7180, Paris) for crit-ical reading of the manuscript. We apologize to all colleagueswhose work was not cited.

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