Spatial Segregation of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Signaling in Immune Cell Functions

doi:10.2174/187152208787169233

Immunol Endocr Metab Agents Med Chem. Author manuscript; available in PMC 2009 December 1.

Published in final edited form as:

Immunol Endocr Metab Agents Med Chem. 2008 December 1; 8(4): 349–357.

doi: 10.2174/187152208787169233

PMCID: PMC2771939

NIHMSID: NIHMS114350

Spatial Segregation of Phosphatidylinositol 4,5-Bisphosphate (PIP₂) Signaling in Immune Cell Functions

Corey M. Johnson¹ and William Rodgers^1,²

¹ Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation

² Departments of Microbiology and Immunology, & Pathology, University of Oklahoma Health Sciences Center

Correspondence should be addressed to: Dr. William Rodgers, Oklahoma Medical Research Foundation, Cardiovascular Biology Research Program, 825 NE 13^th St., MS 45, Oklahoma City, Oklahoma 73104, Email: William-Rodgers/at/omrf.ouhsc.edu, Phone: (405) 271-3550, Fax: (405) 271-8237

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP₂) is a prevalent phosphoinositide in the inner leaflet of the plasma membrane. PIP₂ associates with an ever-growing list of proteins, and participates in a variety of cellular processes. PIP₂ signaling to the actin cytoskeleton transduces specific signals necessary for changes in morphology, motility, endocytosis, exocytosis, phagocytosis, and cell activation. The mechanism(s) by which PIP₂ signaling pathways are specific is a topic of intense investigation. One working model is the compartmentalization of PIP₂-mediated signaling by concentrating PIP₂ in cholesterol-dependent membrane rafts, therefore providing spatial and temporal regulation. Here we discuss properties of PIP₂ signaling to the actin cytoskeleton in immune cell functioning, the association of PIP₂ cellular pools with membrane rafts, and recent work investigating models for compartmentalization of PIP₂-mediated signaling in membrane rafts to the actin cytoskeleton.

Keywords: membrane rafts, phosphatidylinositol 4,5-bisphoshpate (PIP₂), actin, immunological synapse

Background of PIP₂

Phosphatidylinositol 4,5-bisphosphate (PIP₂) is the most prevalent phosphoinositide in mammalian cell membranes, comprising approximately 1% of plasma membrane phospholipids [1, 2] and >99% of the doubly phosphorylated phosphoinositides. Structurally, PIP₂ consists of a negatively charged lipid headgroup, with phosphates at the 4 and 5 position of the inositol ring. Biologically, PIP₂ serves as both a precursor of important second messengers, as well as a co-factor for numerous membrane-associated enzymes. PIP₂ second messenger functions are often initiated by hydrolysis of PIP₂ by phospholipase C (PLC) to produce inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG) [3]. Once generated, IP₃ binds to an IP₃-specific receptor, which in turn leads to a release of intracellular Ca²⁺ stores and, in some cases, activation of store-operated Ca²⁺ channels in the plasma membrane [4]. DAG in turn regulates important membrane-associated enzymes, notably PKC. DAG functions are blocked by its phosphorylation by DAG kinase. In T cells, conversion of DAG to phosphatidic acid represents an important arm in regulating T cell receptor signaling and production of anergic, or nonresponsive, T cells [5].

Another critical function of PIP₂ is that of binding actin filaments to the plasma membrane [6], either by activating proteins that provide signals for actin polymerization, or by serving as a lipid co-factor for actin-binding membrane proteins. Consequently, PIP₂, and its PI3K product, phosphatidylinositol 3,4,5-trisphosphate (PIP₃), are essential for many of the actin-dependent properties of cells, including cell morphology, cell motility [7, 8], endocytosis and exocytosis [9–11], and phagocytosis [12, 13].

In the immune system, PIP₂-dependent properties in actin association with the plasma membrane are necessary for events that underlie both adaptive and innate immune responses. In the case of adaptive immune responses, this property is exemplified by the immunological synapse, which is a highly structured membrane complex of signaling and adhesion molecules that forms where activated lymphocytes bind to cells bearing antigen (Ag). Engagement of multichain immune recognition receptors (MIRR), such as T cell receptor (TCR) and B cell receptor (BCR), initiates signaling cascades that lead to increased PIP₂ hydrolysis and activation of PIP₂-dependent effectors that function in actin binding to the plasma membrane. These events result in a remodeling of the cell cytoskeleton and restructuring of the lymphocyte, and culminate in formation of the immunological synapse. Once established, the signals that maintain the immunological synapse are maintained for hours, a property that is necessary for effective cell activation; abbreviating signals necessary to maintain the immunological synapse results in disengagement of the lymphocyte and incomplete activation.

PIP₂-dependent functions in interactions between the outer membrane and actin filaments for innate immune responses includes phagocytosis by activated macrophages and neutrophils [14]. Specifically, PIP₂ acts as a second messenger in phagocytosis by regulating actin capping, nucleation of de novo F-actin, and targeting of actin-anchoring, crosslinking, and severing proteins [15]. It is suggested that local PIP₂ accumulation is involved in the initial recruitment of actin to the phagocytic cup, while disappearance of PIP₂ parallels the course of actin disassembly [16]. Furthermore, evidence suggests that localized synthesis of PIP₂ may play a role in its localized concentration for phagocytosis. As we discuss below, enrichment of PIP₂ in discrete membrane pools or domains is an attractive hypothesis in that it provides a mechanism for regulating the spatial and temporal functions of PIP₂. Importantly, recent experimental findings provide strong evidence for this model.

Mechanisms of PIP₂-regulated actin-membrane interactions

As the examples above illustrate, PIP₂ is central to the actin and membrane interactions that are necessary for efficient immune cell function. Examples of proteins ubiquitous to immune cells and that function in PIP₂-regulated interactions between cell membranes and the actin cytoskeleton are listed in Table 1. These factors have an array of functions, from providing signals for actin polymerization, to peptides that serve as structural links between actin filaments and cell membranes. Discussed below are the mechanisms by which PIP₂ regulates actin-binding proteins important for immune cell functioning.

Table 1

PIP₂-regulated actin proteins in immune cells.

Vav

Vav is a 95-kDa multidomain protein that serves as a guanine-nucleotide exchange factor (GEF) that activates the Rho family of small guanosine triphosphatases (GTPases), which include the Rho GTPases Cdc42 and Rac (Figure 1). Signaling from Vav is initiated by its activation by tyrosine phosphorylation, which occurs temporally proximal to engagement of the MIRR proteins. Similarly, inhibition of Vav or its effectors blocks receptor-dependent changes in the actin cytoskeleton and cell activation. Furthermore, alterations in lymphocyte activation due to disrupted Vav also affect lymphocyte development [17–19].

Figure 1

PIP₂-dependent T cell signaling pathways for regulation of the actin cytoskeleton

Vav is cytoplasmic, but localizes to the plasma membrane upon activation by binding to PIP₂ and PIP₃ via a pleckstrin homology (PH) domain. PH domains are small protein modules comprised of approximately 100–120 amino acids [20, 21] that bind specifically to phosphinositides, thereby anchoring the protein to the plasma membrane. Crystal structures of PH domains bound to IP₃ reveal their mechanism of binding to phosphoinositides [22, 23]. Typically, a set of lysine residues in the PH domain interacts with the phosphates at the 4 and 5 position of the inositol ring. These interactions lock the phosphatidylinositol phosphate into the binding pocket of the enzyme.

Rac and Cdc42

The Rho GTPases Rac and Cdc42 serve are molecular switches that cycle between two conformational states: one bound to GTP (active state), and the other bound to GDP (inactive state). Following activation, Rac and Cdc42 provide signals for actin polymerization (Figure 1). Rac activation produces lamellipodia and membrane ruffles [24], while Cdc42 activation produces long, finger-like protrusions called filopodia [25, 26]. A principal effector of Cdc42 is the Wiskott-Aldrich syndrome protein (WASP). WASP is expressed exclusively in haematopoietic cells [27] and N-WASP is expressed in neurons [28]. WASP is activated in part by binding PIP₂ via a PH domain. WASP also contains a Cdc42/Rac-binding (CRIB) domain and a VCA domain (Verprolin homology, cofilin homology, and highly acidic), which activates the Arp 2/3 complex [29]. When WASP is not bound to PIP₂ or Cdc42, it assumes an auto-inhibited conformation [30]. However, binding of PIP₂ and Cdc42 induces conformational changes that activate WASP, and subsequently the Arp 2/3 complex, which nucleates actin [31–33]. Finally, similar to the Cdc42-WASP pathway, Rac activates actin polymerization through a related protein WASP family verprolin-homologous protein (WAVE). WAVE, like all WASP family proteins, activates the Arp 2/3 complex through its VCA domain [34].

ERM proteins

The ezrin-radixin-moesin (ERM) proteins are a separate group of PIP₂-regulated group of proteins that function in tethering actin filaments to the plasma membrane. By doing so, ERM proteins control cell shape, cell adhesion, cytokinesis and cytoskeleton anchoring in various cell types [35]. The ERM family proteins bind PIP₂ through an N-terminal FERM domain (band 4.1, ezrin, radixin, moesin) [36], which forms a PIP₂ binding pocket from a basic cleft between two subdomains [36]. The N-terminal and C-terminal domain of ERM proteins interact and stabilize a dormant folded state. PIP₂ binding produces the “active” conformation of ERM proteins, allowing the C-terminal domain to interact with the actin cytoskeleton [36, 37]. In addition to regulation by PIP₂ binding, the ERM proteins are also regulated through phosphorylation of a conserved threonine residue [38]. Once activated, the ERM proteins bind simultaneously to membrane-associated proteins and F-actin via their N-terminal FERM domain and their C-terminus, respectively.

One effect of the regulated tethering of actin filaments to the plasma membrane by ERM proteins is modifying membrane fluidity during cell activation [39, 40]. In resting cells, the ERM proteins are in the active conformation, bound to PIP₂ and phosphorylated. The constitutive binding of the plasma membrane to the rigid actin cytoskeleton reduces membrane flexibility. However, upon cell activation, ERM proteins are rapidly dephosphorylated and assume the “inactive” conformation. Inactivation of ERM proteins reduces tethering of the bilayer to the cytoskeleton, which increases membrane flexibility in order to assist the cell adopt changes in shape and structure necessary for adhesion [41].

ERM-regulated interactions between actin filaments and the plasma membrane are also important for cell movement/migration, which is necessary for immune response, wound repair, metastasis, cell survival and development [42, 43]. For example, adherence and migration of T cells into inflamed tissue require an ERM dependent polarization of the T cell [44]. The formation of a leading edge protrusion, or lamellipodium, begins the migration process, followed by a formation of new adhesion sites at the front, cell body contraction, and detachment of adhesion sites at the rear. The entire process requires tight, spatial and temporal regulation of actin cytoskeleton dynamics, including that of binding of ERM proteins to the plasma membrane and actin filaments [42, 45].

Talin and vinculin

Another membrane-tethering protein for actin filaments is talin, which binds to integrins, including the T cell β2 integrin LFA-1. Integrins represent important adhesion receptors that undergo a change from an inactive to active conformation as a result of “inside-out” signaling. Talin binding to cytoplasmic tail of the β subunit through an N-terminal FERM domain represents a final step in the integrin activation [46–48]. Talin binding to actin filaments occurs mostly through a C-terminal rod domain. T cells require talin for regulation of chemokine induced adhesion and migration [49, 50] and TCR-induced up-regulation of LFA-1 at the Immunological synapse [51].

Talin binds to acidic phospholipids and it is activated by binding PIP₂ within its FERM domain [33]. However, PIP₂-dependent regulation of integrin activation also includes vinculin binding to talin. Vinculin is a component of focal adhesions that localizes to cell-cell junctions, where it is thought to regulate the assembly and stabilization of the junction [52]. Focal adhesions are modulated by PIP₂ [53, 54] and consist of structural and signaling molecules that participate in both inside-out and outside-in signal transduction [55]. Vinculin, similar to WASP, exists in an inactive conformation previous to PIP₂ binding where its head and tail domains interact with each other [56]. However, binding of PIP₂ to the vinculin D5 domain opens a talin-binding site in its D1 domain [57, 58]. Once activated, binds to actin-associated proteins that include α-actinin, α-catenin, and Arp2/3. Complexes containing vinculin and associated proteins therefore modulate focal adhesion assembly [33].

Gelosin

PIP₂ can also control filament structure by regulating proteins that function in actin filament disassembly, such as gelsolin, villin and severin [33, 59–61]. In the case of gelsolin, which is a potent actin filament-severing protein, PIP₂ binding dissociates gelsolin-actin complexes and inhibits gelosin-dependent actin severing [59, 62]. PIP₂ binding occurs through separate clusters of basic residues located in a P1 and P2 regions of the N-terminus [63]. A third PIP₂-binding site has also been identified at the gelosin C-terminal region. Interestingly, rather than directly competing for the actin binding site, PIP₂ induces conformational changes in gelsolin which disrupt its association with actin [64].

Spatial regulation of PIP₂ functions

The central role that PIP₂ plays in cytoskeleton structure and cell physiology underscores the necessity of its proper regulation. Further complicating this paradigm is that separate PIP₂ functions are often simultaneous and sometimes competing. One mechanism for providing necessary regulation of PIP₂ functions is by establishing and maintaining separate and discrete pools of PIP₂ in the plasma membrane. Spatially segregating separate PIP₂ pools, such by concentrating the PIP₂ in discrete domains, is hypothesized to provide a spatio-temporal control of PIP₂-dependent functions. For example, a localized accumulation of PIP₂ is predicted to augment PIP₂-dependent by increasing the effective concentration of PIP₂ in these regions. Association of specific PIP₂ effectors with these regions would therefore spatially concentrate effector functions.

Multiple mechanisms exist to form localized pools or concentrations of PIP₂ in the plasma membrane. One relates to sequestering of PIP₂ through electrostatic interactions with poly-basic proteins such as myristoylated alanine rich C-kinase substrate (MARCKS) [65, 66], GAP43 (growth-associated protein 43), and CAP23 (cytoskeleton-associated protein 23). MARCKS is ubiquitously expressed [67], while GAP43 and CAP23 are expressed primarily in neurons. The poly-basic proteins are anchored to the plasma membrane by an N-terminal acylation and binding to PIP₂ via a cluster of basic residues known as a basic effector domain [1, 66]. MARCKS, which is present at similar concentrations as PIP₂ (10μM), can laterally sequester three tetravalent acidic PIP₂. Accordingly, a cluster of MARCKS proteins can sequester a large number of PIP₂ molecules into spatially segregated membrane rafts.

To make the localized concentration of PIP₂ bioavailable, the electrostatic sequestration of PIP₂ by MARCKS must be interrupted. Calcium/calmodulin (Ca/CaM) interacts directly with the basic residues of the effector domain and allows MARCKS to be released from the membrane [68, 69]. This action makes the electrostatically sequestered raft PIP₂ pools available for PIP₂ signaling to the actin cytoskeleton. Another mechanism for reversing PIP₂ sequestration is by phosphorylation of MARCKS by PKC. For example, phosphorylation of serine residues in the basic effector domain cause a reduced electrostatic attraction for the membrane and allow MARCKS to translocate to the cytoplasm [1, 70].

A separate mechanism for establishing spatially discrete pools of PIP₂ is through its association with specific lipid-enriched domains, such as the cholesterol-dependent membrane rafts. In general, membrane rafts serve as a platform for signaling events in the plasma membrane, including many that do involve PIP₂. In principal, rafts are modeled as membrane “reaction chambers” that increase the kinetics of signaling reactions by concentrating substrates and enzymes together in the same membrane microenvironment [71]. In immune cells, such effectors will include PLC and Vav, which are concentrated in rafts through their association with raft-associated linker molecules, such as linker for activation of T cells (LAT) (Figure 1).

Structurally, membrane rafts are heterogeneous in nature, ranging from nanoclusters that are 10 nm in size and contain only a few protein molecules, to nanodomain complexes that are tens of nanometers in diameter, and even larger micron-size raft macrodomains [72]. Examples of raft macrodomains include the Immunological synapse in stimulated lymphocytes, and raft-enriched adhesion complexes. Furthermore, separate raft nanodomains likely have distinct compositions, thereby adding another level of complexity. Indeed, one important outcome from production of raft macrodomains may be to bring together proteins and lipids into the same membrane environment from one that is sequestered between separate raft nanodomains.

Mechanisms that distinguish protein clustering into macrodomains versus the nanodomains and smaller nanoclusters represent an ongoing topic of study. One important factor is the cell cytoskeleton and its association with the membrane bilayer. For example, the raft macrodomains often co-enrich with actin filaments, and they form in an actin-dependent manner. Interestingly, one recent study, using FRET, demonstrated the actin cytoskeleton was at least as important as cholesterol in a specific co-clustering of raft-associated membrane fluorescent proteins [73]. The mechanism by which the actin cytoskeleton promotes clustering of raft proteins is unclear, but it may include establishing an actin-dependent ordering of membrane lipids such as that recently demonstrated in liposomes [74]. Altogether, the role of actin cytoskeleton in forming membrane rafts together with the properties of PIP₂ in establishing linkages between actin filaments and the plasma membrane suggest a pivotal role for PIP₂ in forming and maintaining plasma membrane rafts.

Localization of PIP₂ functions to membrane rafts evidenced using targeted phosphatases

Earlier biochemical experiments have provided evidence of a compartmentalization of PIP₂ in membrane rafts [75–80]. For example, measurement of Triton X-100 (TX 100)-resistant membranes, which historically have been a working definition of rafts, showed up to half of the total cellular PIP₂ was present in the detergent-resistant membranes [76]. Furthermore, disrupting membrane rafts by depleting membrane cholesterol using methyl-β-cyclodextrin (MβCD) inhibited epidermal growth factor- and bradykinin-stimulated phosphatidylinositol turnover [78], thus suggesting raft integrity is necessary for efficient PIP₂-dependent signaling. Replacing the cholesterol rescued hormone-stimulated PIP₂ hydrolysis [78]. Similarly, heterotrimeric G-proteins, seven transmembrane domain receptors, and inositol triphosphate receptors are enriched in membrane caveolae [77], which share many of the properties of membrane rafts.

As these examples illustrate, data demonstrating raft-specific properties of PIP₂ often utilized methods that are disruptive in nature in characterizing cell membranes. Accordingly, some investigators contend that deduced roles of rafts in PIP₂ functions are the outcome of experimental artifacts arising from these invasive approaches [81, 82]. Importantly, however, one recent study from our group demonstrated compartmentalization of PIP₂ signaling in intact, viable T cells [83]. Specifically, the yeast PIP₂-specific phosphatase Inp54p was targeted to either membrane fraction using separate membrane anchoring signals (Figure 2A, B, and C). Both phosphatases altered the phenotype of transfected T cells (Figure 2D). Interestingly, an increase in the raft pool of PIP₂ resulted in an activation phenotype, represented by an increase in membrane ruffling (Figure 2D) and cell spreading on poly-L-lysine. In contrast, decreasing raft PIP₂ pools resulted in cells that were smooth in appearance and lacking the background ruffling evidenced in control cells, as well as an inhibited capping when stimulated by crosslinking the TCR. Importantly, neither phosphatase altered the total PIP₂ levels in the cells, thus showing that the changes in phenotype can be attributed to the alterations in the relative amount of PIP₂ in each membrane pool.

Figure 2

Alteration of plasma membrane PIP₂ pools by targeted PIP₂-specific phosphatase Inp54p

Another interesting finding from the experiments with targeted phosphatases was that a specific reduction of nonraft-associated PIP₂ coincided with a large increase in raft-associated PIP₂ (Figure 2B and C). These data therefore suggest that consumption of nonraft PIP₂ is offset by an increase in the synthesis of PIP₂ in membrane rafts (Figure 3A). Alternatively, depleting nonraft PIP₂ may result in an increase in the expression of PIP₂-binding proteins such as MARCKS, which will increase the affinity of PIP₂ for ordered membrane environments such as the rafts (Figure 3B) [84].

Figure 3

Mechanisms for membrane compartmentalization of PIP₂

Segregation of PIP₂ by localized synthesis

Consistent with the model illustrated in Figure 3A regarding compartmentalized synthesis of PIP₂, earlier experimental data show PIP₂ synthesis occurs within specific regions of the plasma membrane. For example, PIP₂ is synthesized through phosphorylation of PI4P by PI4P5K [85], and both the PI4P5K and PI3K are localized to sites of actin polymerization occurs [86–90]. In the case of PI4P5K, three isoforms of this enzyme occur: α, β, and γ [91–94]. The structural variation of the PI4P5K isoforms, along with small G-proteins and their regulators, may determine the intracellular location of PIP₂ synthesis [95, 96].

Some evidence for localized synthesis as a means of affecting actin polymerization exists. Expressing PI4P5K in Cos-7 cells induced massive actin polymerization and produced a “pine needle” cell morphology [97], which is also similar to what was observed by artificially increasing the raft pool of PIP₂ using targeted PIP₂ phosphatase. Localized synthesis has also been shown to regulate actin-dependent membrane ruffle formation [98] and phagocytosis [90]. Upon agonist stimulation, the small GTPase ADP-ribosylation factor 6 (Arf 6) is activated. PI4P5Kα is a downstream effector of Arf 6 and colocalizes with the GTPase in membrane ruffles [98]. These studies also show that the activation of PI4P5Kα is dependent upon PA, the product of phospholipase D (PLD). PIPKIα also accumulates transiently on forming phagosomes [90]. These studies show the localization of PIP₂ synthesis at the phagasomal cup and that a mutant PIPKIα, lacking kinase activity, impairs phagocytosis.

Summary and Future Directions

In summary, the importance of PIP₂ and its regulation is reflected in its multiplicity of functions. PIP₂ signaling to the actin cytoskeleton transduces specific signals necessary for changes in morphology, motility, endocytosis, exocytosis, phagocytosis and T cell activation. We have discussed separate models of PIP₂ signaling from the plasma membrane to the actin cytoskeleton and how, specifically, PIP₂ may be spatially segregated in order to provide the localized concentration of PIP₂ necessary to regulate its various cellular functions. One working model is the compartmentalization of PIP₂-mediated signaling by concentrating PIP₂ into membrane rafts through binding of raft-associated proteins. There is also evidence of localized synthesis of PIP₂ by PI4P5K in the raft fraction of the membrane. Additionally, specific depletion of raft-associated PIP₂ by targeted phosphatases produces specific and distinct phenotypes in T cells. These phenotypes include changes in actin-dependent processes such as changes in morphology, cell spreading, and actin capping.

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