handbook of cell signaling || phosphoinositides and actin cytoskeletal rearrangement

Phosphoinositides and Actin Cytoskeletal Rearrangement

Paul A. Janmey1, Robert Bucki1 and Helen L. Yin2

1Institute for Medicine and Engineering, Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania2Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas

Chapter 141

Handbook of Cell Signaling, Three-Volume Set 2 ed.Copyright © 2010 Elsevier Inc. All rights reserved.

Historical perspective

Cytoskeletal proteins were the first proteins shown to be regulated by polyphosphoinositides (PPIs). This effort began with reports that profilin [1], alpha-actinin [2], vinculin [3, 4], and proteins comprising the erythrocyte cytoskel-eton [5] bound acidic phospholipids, and that phosphati-dylinositol 4,5-bisphosphate (abbreviated here as PIP2) dissociated complexes of profilin and actin [6]. There is now strong in vitro biochemical evidence that scores of cytoskeletal proteins bind to and are regulated by PPIs. These and subsequent findings suggested that, in most cases, increases in cellular PIP2 would drive the polymeri-zation of cytoskeletal actin and stabilize its interaction with the plasma membrane [7]. At that time, products of the PI3-kinase pathway had not yet been implicated in cell sig-naling, and it was thought that PIP2 was the primary lipid responsible for direct effects on profilin. This assumption is largely supported by many subsequent studies of other actin binding proteins, which are either activated or inhib-ited by PIP2, at least in vitro. Studies in the last few years confirm that at least some of these reactions occur in a simi-lar way within the cell (in vivo). The fundamental predic-tions that increased PPI synthesis leads to actin assembly and that depletion of PPIs triggers actin depolymerization have been borne out in studies where PPI levels in cells are altered by manipulation of expression of PPI kinases [8–11] or phosphatases [9, 12–18]. In addition, introduction of constructs like the PIP2-binding PH domains [14, 19] or PIP2-binding peptides [20–23] mimics the effects of endog-enous proteins whose cellular role appears to involve seques-tration of membrane PPIs [24–26].

In the past several years, the number of PPI-binding pro-teins such as those involved in membrane trafficking, ion transport, or spatial localization of signaling has increased

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enormously, and actin binding proteins are now a minority of the total ligands proposed for these lipids. Many of the newly reported proteins were identified by their possession of well-defined modular pleckstrin (PH), FYVE, PX or other PPI binding motifs, and their lipid binding potential was confirmed thereafter in vitro. Only a few of these mod-ules, such as the PLC1-PH domain and the Tubby domain bind preferentially to PI(4,5)P2, and they often bind exclu-sively to the lipid headgroup. In contrast, most PPI-bind-ing cytoskeletal proteins were first identified biochemically to interact with PIP2, and their specific binding sites were identified thereafter. It is particularly noteworthy that the PPI-binding domains common to proteins involved in vesi-cle traffic or spatial localization of signaling are conspicu-ously absent from the majority of actin binding proteins. Recently, a structure of cofilin complexed with a short acyl chain variant of PIP2 was solved by NMR, and it revealed a complex interface linking the protein with both the lipid headgroup and hydrophobic chains [27]. This mode of interaction with PPI is significantly different from that reported, for example, for the endocytic clathrin adaptor protein complex 2 (AP-2) [28].

This chapter will focus on recent advances showing how PPIs are involved in the regulation of actin polymeri-zation and the formation of cytoskeleton/membrane links, and how binding of cytoskeletal proteins to membrane PPIs may relate to the lateral or transverse movement of lipids to affect raft formation or lipid asymmetry.

stimulating site-specific actin polymerization in cells

There is increasing evidence for a localized increase in PIP2 at sites of actin polymerization and remodeling from

part | ii Transmission: Effectors and Cytosolic Events1142

studies using the fluorescent chimera GFP–PH–PLC1 [29] or GFP–Tubby [30] as a PIP2 reporter. Localized PIP2 increase may depend on small GTPases in the Rho fam-ily (Rac and Rho) and the ADP ribosylation factor fam-ily (Arf6, Arf1). These GTPases have profound effects on the actin cytoskeleton, and can either alter the activity of type I phosphatidylinositol 4 phosphate 5 kinases (PIP5Ks) that generate PIP2 from PI4P, or recruit them to sites of actin polymerization [11]. For example, Rac and Rho bind PIP5Ks and recruit them to the plasma membrane [31], while Arf6 acts downstream of Rac to activate PIP5Ks [32, 33]. Recently, it has been shown that PIP5K, one of the three major PIP5Ks, is localized to N-cadherin-medi-ated intercellular adhesions [34], and it regulates intercel-lular adhesion strength in part through PIP2-mediated regulation of gelsolin and the Arp2/3 actin regulatory pro-tein complex [35, 36]. Quantitative data characterizing PPI turnover and small GTPase activation have been combined in a recent theoretical model of how these signals can con-trol actin-dependent cell polarity and motility [37].

tHe mecHanisms of actin polymerization

Site-specific actin polymerization in vivo occurs prima-rily at the rapidly growing end () of actin nuclei [38]. The Wiskott-Aldrich syndrome family (such as WASP and N-WASP) and formin family proteins have been implicated in the coordination of cell protrusion and/or filopodia for-mation. Three mechanisms by which PPIs might control the location and rate at which () ends are generated have been proposed (reviewed in [16, 39, 40]): first, de novo actin nucleation by the Arp2/3 complex as a result of activation by WASP or other proteins; second, severing of pre-existing filaments by cofilin/actin depolymerizing protein (cofilin/ADF) or gelsolin family proteins; an third, uncapping of the () end by capping proteins such as the heterodimeric Capping Protein (CP)/CapZ or gCap39. Once the () ends are liberated, actin monomer delivery is accelerated by pro-filin and the funneling of actin monomers to the favored sites by selective () end capping at other, non-favored, sites. The supply of actin monomer is sustained by severing and facili-tated depolymerization from the () end by cofilin/ADF.

PIP2 alters in vitro the activity of critical proteins in each of these steps. It activates WASP family proteins, either synergistically with Cdc42 [41], or with SH3 adaptors such as Nck independently of Cdc42 [42]. The manner in which PIP2 and small GTPases act in concert or possibly antagonistically on WASP-related proteins is an actively studied and unresolved issue [43]. The prevail-ing hypothesis is that PIP2 activates WASP proteins by an unmasking mechanism as depicted in Figure 141.1c. This promotes interaction with the Arp2/3 complex to induce de novo nucleation.

In contrast to its activation of WASP family proteins, PIP2 inactivates cofilin/ADF, CP, profiling, and gelso-lin-related severing and capping proteins (reviewed in [7, 44, 45]) (Figure 141.2). Recently, it has been shown by total internal reflection fluorescence microscopy (TIRFM) that PIP2 induces CP uncapping from actin filament ends [46]. Another study has directly visualized the spatial and temporal regulation of cofilin by PIP2 in living cells, and concluded that EGF stimulated PIP2 hydrolysis by phos-pholipase C activates cofilin locally in carcinoma cells during chemotaxis [47]. The mechanism for PIP2 inhibi-tion of these proteins is not completely understood but may involve some of the conformation changes depicted in Figures 141.1a and b. For example, molecular modeling of CP shows that its PIP2 binding site overlaps the actin bind-ing site [46], and the NMR structure of cofilin complexed with PIP2 reveals secondary and tertiary changes in cofi-lin’s actin-binding sites [27].

There is increasing evidence that PIP2 is a major regula-tor of the actin cytoskeleton in vivo [7]. Genetic disruption of Mss4m (the only PIP5K gene in yeast), or skittles (one of two PIP5K genes in Drosophila) produces cytoskeletal defects. The role of mammalian PIP5Ks was initially exam-ined using non-genetic approaches. PIP5Ks [15, 35] and the PPI phosphatases that dephosphorylate PIP2 [16] were overexpressed. Other approaches include microinjection of an anti-PIP2 antibody [48], introduction of a cell-permeant gelsolin PIP2 binding peptide [20, 21, 49, 50], addition of PIP2 or a PIP2 binding peptide to semi-intact cells [11, 51],

figure 141.1 Three models for regulation of protein function by membrane-bound phosphoinositides. Actin binding sites are shown as oval patches. Solid patches denote active sites and dotted patches inhibited sites. PIP2 is shown as large-headed domains within one leaflet of a bilayer composed of neutral lipid. (a) The actin site is occluded by the lipid without other structural change; (b) PIP2 binding reorients two protein domains such that structures required for actin binding can no longer function cooperatively; (c) protein binds and inserts in membrane to simultaneous stabilize membrane association and expose binding sites for actin and membrane proteins.

chapter | 141 Phosphoinositides and Actin Cytoskeletal Rearrangement 1143

Monomerbindersprofilin

MembranelinkersvinculintalinERM proteinsspectrin

Actin nucelationactivatorsWASP familyproteins

Filament cuttersFilament cappersCrosslinkers/bundlersfilaminalpha actinincortexillin

CappingProtein/CapZCapGgelsolin

Localization to membraneMyosin 1cMARCKSAnnexin 2

gelsolincofilin/ADF

PIP2

figure 141.2 Schematic summary of actin binding proteins that are regulated or localized by PIP2. Arrows denote activation, bars denote inhibition, and lines denote localization without necessarily change in actin binding function.

and overexpression of actin regulatory proteins with defec-tive PIP2 binding [52].

Mammals have three PIP5K genes encoding for the , , and isoforms. In addition, PIP5K has two major splice variants: a short 87-kDa protein (PIP5K87) and a slightly longer 90-kDa protein that has 28 additional amino acids at its COOH-terminal tail. The three isoforms have a highly conserved central lipid kinase core, but different amino- and carboxyl-termini extensions. With a few excep-tions, they are functionally similar in vitro and frequently generate similar changes in cells when overexpressed. Since high levels of overexpression are likely to overwhelm the normal mechanisms for specifying the unique functions of PIP2 pools generated by these PIP5K isoforms (see below), their isoform-specific roles cannot be identified system-atically in initial overexpression studies. This problem was confounded by the fact that some of the putative kinase dead PIP5K mutants are not actually kinase-dead, and most are not consistently dominant negative [7]. These complications are now overcome by using mammalian RNA interference (RNAi) and gene knockout by homologous recombination.

pip5K overexpression

Transient PIP5K overexpression has many effects. These include many actin-dependent processes, such as N-WASP-dependent actin comet tail formation [15], Rho and Rho-kinase dependent actin stress fiber formation [53], N-cadherin-mediated cell adhesion [35], Arf6-regu-lated plasma membrane-endosome recycling [54], neurite remodeling [55], and phagocytosis [33, 56]. Its impact on the actin cytoskeleton establishes a causal relation between PIP2 and actin cytoskeletal dynamics, and the identifica-tion of some of the actin modulating proteins involved

provides mechanistic insight into how PIP2 regulates the actin cytoskeleton in vivo. The diverse phenotypes and cell- specific responses are not surprising, given that PIP5Ks may be regulated by multiple small GTPases that induce distinct actin structures in a sometimes sequential and at other times mutually exclusive manner. Furthermore, the site of PIP2 generation and the particular subset of actin regulatory pro-teins at those sites will dictate the dominant response.

actin stress fiber formation

PIP5K overexpression in CV1 cells induces robust actin stress fiber formation and inhibition of membrane ruffling in response to growth factors due to an inability to generate () end actin nuclei [53]. These two effects are consistent with activation of Rho- and inhibition of Rac-dependent cytoskel-etal pathways, respectively, and are remarkably similar to that observed in fibroblasts isolated from gelsolin knockout animals [57]. Gelsolin binding to actin is inhibited in PIP5K overexpressing cells, suggesting that inhibition of severing by gelsolin and perhaps by cofilin/ADF may account for the formation of long actin filaments and the inability to generate () end nuclei to mount an actin polymerization response. Furthermore, profilin and CapZ are also inhibited, while the ezrin/radixin/moesin family of membrane-linker proteins (see below) are activated. Together, these changes can amplify the consequences of severing inhibition. In con-clusion, these studies show that several PIP2-sensitive actin-modulating proteins behave in vivo as predicted from their well characterized behavior in vitro.

actin comet formation

Actin comets formed around pathogens, such as Listeria, Shigella, and vaccinia, have contributed significantly to our


understanding of the mechanisms of actin polymerization in cells. These pathogens introduce their own membrane pro-teins to either mimic or hijack the host’s WASP machinery to initiate actin assembly. Since WASP also stimulates actin comet formation around intracellular vesicles and lipid vesi-cles in cell extracts [58] and in Xenopus eggs [59], the pos-sibility that PIP2 promotes vesicle trafficking by generating actin comets is particularly attractive. Indeed, tiny comets that form spontaneously have been sighted, and much more robust comets are found in cells that overexpress PIP5K [15]. Overexpressed PIP5K is enriched at the head of the comets, establishing that the in situ generation of PIP2 at the vesicle may recruit and activate N-WASP to promote de novo actin nucleation by Arp2/3. These actin comets are formed from endocytic and Golgi-derived exocytic vesicles, particularly those with cholesterol:sphinogolipid-enriched membrane microdomains (rafts) [15]. The relation between PIP2 and membrane rafts will be discussed in the final section.

Interestingly, actin comets are also induced by over-expression of Arf6 [60], which activates PIP5K in vitro and in vivo [32, 54]. In addition, the PH domain of an Arf6-spe-cific exchange factor binds PIP2 and actin, suggesting that there is a complex feedback circuit that regulates PIP2 pro-duction and actin polymerization in a site-specific manner [61]. Significantly, inappropriately high level overexpres-sion of either constitutively active Arf6 or PIP5K gener-ate a similar phenotype in which recycling endosomes are trapped by polymerized actin into vesicular aggregates that cannot recycle back to the plasma membrane [54]. The requirement for dynamic cycles of PIP2 synthesis and dissipation has now been established in many impor-tant physiological systems, including endocytosis [62, 63], phagocytosis [56], and neuronal remodeling [64] It high-lights the importance of regulating PIP2 homeostasis in a spatially and temporally defined manner.

ppi pHospHatase manipulations

PIP2 levels can be decreased in many ways. These include inactivation of PIP5Ks, hydrolysis by PLC, and dephospho-rylation by phosphoinositide 5 phosphatases [16], such as synaptojanin 1 (Synj1) [65] and Ocrl [66]. Overexpression of Synj1 or similar phosphatases decreases actin stress fib-ers [12, 67] or induces actin arborization [68].

Disruption of the Synj1 gene results in an accumula-tion of clathrin-coated vesicles and polymerized actin in the endocytic zones of nerve terminals, which is due to defective post-endocytic uncoating [62]. In addition, there are problems with endocytosis [69]. These changes are cor-related with an increase in PIP2 concentration. Synj1 and PIP5K are both concentrated at synapses, and they antag-onize each other in the recruitment of clathrin coats to lipid membranes in vitro [9]. These results strongly suggest that the PIP2 level at the synapse is critically dependent on the balance PIP2 synthesis and dissipation, and that PIP2 has a

pivotal role in the regulation of actin and endocytic vesicle formation at the synapse [70]. Multicolor TIRFM showed that the neuron-specific and ubiquitous forms of Synj1 are recruited to clathrin-coated pits sequentially, raising the possibility that dynamic PPI metabolism occurs throughout the lifecycle of a coated vesicle [65].

Recently, the rapamycin-inducible FKBP and FRB dimerization system has been used to target a PPI 5-phos-phatase to the plasma membrane [18]. This novel approach clearly establishes that a drop in PM PIP2 level rapidly inhibits receptor-mediated endocytosis [36, 71].

pip5K rnai and gene KnocKout

The three PIP5K isoforms have divergent amino- and car-boxyl-terminal extensions that are likely to be important in specifying isoform-specific functions and regulation. RNAi and gene knockout show that these PIP5Ks indeed have dif-ferent roles in the regulation of the actin cytoskeleton. In this review, we will use the human PIP5K isoform designa-tion, even when referring to a gene that is knocked out in mice [7]. The dominant theme that emerges is that PIP5K isoforms have non-overlapping roles in the regulation of the actin cytoskeleton and actin-dependent processes. This led to the inevitable conclusion that the PIP2 pools generated by these PIP5Ks are somehow segregated into distinct microdo-mains that are not necessarily interchangeable. The question of how this is achieved is a major challenge for the future, and the answer holds the key to understanding how PIP2 regulates the actin cytoskeleton spatially and temporally.

pip5K rnai

Depletion of PIP5K in HeLa cells inhibits receptor-medi-ated endocytosis of transferrin [72], while depletion of PIP5K decreases histamine-induced IP3 generation [73]. Recently, in a human kinase RNAi library screen, PIP5K has been identified as an apical player in the activation of the canonical Wnt3a signaling pathway at the plasma mem-brane [74]. PIP5K binds to a component of the Wnt3a LRP6 transmembrane receptor complex, and is activated to induce LRP6 clustering to trigger the downstream cascade. Since the cortical actin cytoskeleton is known to partition transmembrane receptors by restricting their diffusion, as well as dynamically influencing their long-range mobility, sequestration, and response to ligand binding [75], these results suggest that PIP2 activation of LRP6 may also be mediated in part through actin rearrangements.

pip5K gene Knockout

Recently, knockout animals for each of the three PIP5K isoforms have been generated and they are beginning to be characterized. PIP5K/ mice are viable and are almost completely normal. However, they exhibit enhanced passive


cutaneous and systemic anaphylaxis in response to hista-mine stimulation. The underlying problem is that their mast cells have less cortical actin and degranulate exuberantly. PIP5K gene knockout is either perinatally lethal [76] or embryonic lethal [77], depending on how the lines were generated. In both cases, there are significant actin defects. Mice from the perinatally lethal PIP5K/ line have no gross developmental defects, but the synaptosomes prepared from their brains do not increase PIP2 synthesis in response to K depolarization, and their neurons exhibit severe defects in synaptic transmission that correlate with abnor-mal exocytosis and clathrin-mediated endocytosis [76]. Mice from the embryonically lethal PIP5K/ line have severe defects in cardiovascular and neuronal development that are consistent with problems with defective cell migra-tion and adherens junction formation [78]. Furthermore, their megakaryocytes have abnormal membrane blebbing that is correlated with a decrease in the association of the membrane cytoskeleton with the plasma membrane [77]. Blebbing can be rescued by the introduction of PIP5K90 but not PIP5K87. Since these two splice variants are functionally identical except that PIP5K90 is localized in focal adhesions by binding the focal adhesion protein talin [79, 80], the blebbing defect may be due to loss of a talin-dependent function. PIP5K gene knockout decreases the fertility and intrauterine survival [81]. However, PIP5K/ mice that survived to birth have no apparent abnormality, and live to adulthood. Their platelets are, however, unable to generate IP3 in response to thrombin stimulation, and they fail to properly form experimentally induced arterial thrombi in vivo. These results suggest that PIP5K gener-ates the bulk of the PIP2 pool that is required for G-protein-coupled receptor-mediated second messenger generation to activate stable platelet adhesion.

actin-membrane linKers localized or activated by pip2

In contrast to actin monomer-binding or severing proteins that are generally inactivated by PPIs, proteins that crosslink actin filaments to each other or link them to the cell mem-brane are usually activated by PPIs to bind actin or to link actin to transmembrane receptors [82] (Figure 141.2). Pharmacologic disruption of cellular PIP2 destabilizes the link between the cytoskeleton and the plasma membrane [14]. Significantly, a recent study shows that PIP5K gene knockout disrupts the integrity of the membrane cytoskel-eton in megakaryocytes by perturbing a talin-dependent pathway [77].

ezrin/radixin/moesin

ERM proteins are among the best currently characterized PPI-activated proteins. Their actin and membrane protein binding sites are both inactive in the dormant state because

of self-association between the two domains responsible for these separate activities. The mechanism of their self-inactivation has now been determined from the 3D crystal structures of radixin [83] and moesin [84, 85]. Self-inac-tivation is a common feature of several other actin-mem-brane linkers, including talin and vinculin. In retrospect, this explains why their in vitro actin binding was so diffi-cult to characterize compared to proteins like cofilin or fil-amin, whose actin binding sites appear to be constitutively exposed. Biochemical and cell localization studies showed that ERM proteins co-localize with transmembrane pro-teins in activated cells and that in vitro this association was stimulated by PPIs. The PIP2-dependent linkage of ezrin to ICAMs [86, 87] involves reordering of the FERM domain, which contains an acidic loop distinct from the IP3-binding site that may also participate in binding to the basic jux-tamembrane regions present in adhesion receptors such as CD44 [83]. The importance of the PIP2-binding regions for cellular localization and function of ERM proteins has been increasingly well demonstrated in recent studies. Mutation of four basic residues found in the PIP2 binding site prevented localization of ezrin to actin-rich membrane structures [88]. The affinity was found to be approximately 5 M for large unilammelar vesicles (LUVs) containing PIP2, and 20- to 70-fold lower for phosphatidylserine-LUVs. The interaction between ezrin and PIP2-LUVs is not cooperative, but a single ezrin FERM domain with a cross-sectional area of approximately 30 nm2 binding to a single PIP2 with a cross-sectional area of 0.5 nm2 [89,90] can block access to neighboring PIP2 molecules, and thus contributes to lowering the accessible PIP2 concentration [91]. These results emphasize the importance of the lipid particle or membrane patch in which the PPIs are located for the strength of the association between the protein and its lipid ligand.

alpha-actinin

Recent studies of alpha-actinin provide a good example of how activation of actin or other ligand binding may occur, and the complexity of this protein’s interactions with differ-ent PPIs. In this case, an actin- and titin-binding motif of the anti-parallel alpha-actinin dimer is occluded in the inactive state because it binds a complementary domain within the same homodimer [92]. When PIP2 binds to alpha-actinin, self-association is disrupted, exposing the actin- and titin-binding motifs so that they can potentially bind their tar-gets (See Figure 141.1c). However, the situation may less straightforward because although initial studies concluded that PIP2 enhanced the actin bundling activity of skeletal muscle alpha-actinin [93], more recent studies show that PIP2 inhibits the bundling activity of smooth muscle alpha-actinin [94]. Alpha-actinin is also differentially affected by PIP2 and PIP3. Whereas PIP3 strongly promotes cal-pain-mediated degradation of alpha-actinin, PIP2 inhibits


this reaction [95]. Here, as in other contexts, sequential or competitive effects of different PPIs might orchestrate a cycle of cytoskeletal changes in vivo. Similar activation switches have been proposed for ERM family members, and for the focal adhesion proteins talin and vinculin.

talin/vinculin

Talin, which binds vinculin, actin, and integrin, has a key role in coupling the cytoskeleton to integrins. Like ezrin, talin is also activated by PIP2 to increase membrane asso-ciation. In this case, one consequence of PIP2 binding is an increased affinity of the intact protein for the cyto-plasmic domain of beta 1 integrins [96]. The relevance of this interaction in a cellular context is reinforced by evi-dence that PIP2 co-sediments after immunoprecipitation with anti-talin antibodies, and the amount of PIP2 shows a strong transient increase after suspended cells are plated on fibronectin, reaching a maximum 15 minutes after engagement of integrins that is 5 times higher than the ini-tial state or the levels 1 hour after plating. The finding that only PIP2, but not PIP or PI, shows this transient change rules out the possibility that the lipid in the immunopre-cipitates results from non-specific contaminating mem-branes, but also raises the question of the state of the lipid in these lipid–protein complexes. In addition, there is now strong evidence that talin has a direct role in increasing PIP2 at focal adhesions by recruiting PIP5K90 to focal adhesion [79, 80]

Vinculin also appears to be regulated by a PIP2-sensitive conformational switch, but the functional consequence is not necessarily activation of ligand binding. In vitro, PIP2 binds to two sites in the vinculin tail domain, leading to vin-culin oligomerization as well as enhanced binding to VASP protein [97] and talin [98]. However, actin binding to the vinculin tail domain is inhibited by PIP2 [99]. Therefore, while PPIs may initiate activation of vinculin and promote ligand binding, the final linkage to the actin cytoskeleton may require additional or alternative signals. Recent studies show that although a PIP2 binding vinculin is still recruited to the focal adhesion, these focal adhesions turn over slowly [100, 101]. Therefore, vinculin is likely to act as a sensor of PIP2 in the focal adhesion to regulate dynamic cycles of focal adhesion assembly and disassembly.

spectrin

Erythrocyte spectrin was among the first proteins found to bind PIP2. PIP2 helps localize it to the cytoplasmic face of the plasma membrane and other organelles. Formation or destruction of PIP2-enriched membrane domains alters the binding of actin to the membrane. In addition, recent work shows that PIP2 also enhances the binding of protein 4.1r to the N-terminal domain of spectrin [102].

different mechanisms of ppi-actin binding protein regulation and global effects on actin assembly

There are at least three distinct mechanisms and several variations by which membranes containing PPIs can alter actin binding protein function. The simplest mechanism, shown in Figure 141.1a, is that an actin binding site coin-cides with a PIP2 binding site, and therefore targeting of the protein to PPI-rich membranes dissociates actin com-petitively without necessarily changing the protein struc-ture. However, even for small monomer sequestering proteins like cofilin/ADF/actophorin, careful mapping of the actin and PIP2 binding sites shows that they are not pre-cisely coincident, and that selected residues can be altered to perturb one but not the other activity [52, 103]. Profilin, likewise, appears to have an extensive surface that inter-acts with PIP2, and binding to the lipid promotes increased -helix in the protein [104].

A different model for inhibition of actin binding func-tion, shown in Figure 141.1b, is that PIP2 binding induces a rearrangement of the actin binding domains or a local unfolding of polypeptide within these domains to derange the surface required to bind actin. This model appears to account for the effects on gelsolin and related proteins [105, 106]. This type of allosteric regulation may occur either with or without the protein inserting into the hydro-phobic domain of the membrane

The third mode of binding (Figure 141.1c) involves docking of the protein to the membrane in a manner that disrupts interactions between domains within mono-mers or homo-oligomers that mask binding sites for actin or membrane anchors. This model, which may apply to ERM proteins, talin, alpha-actinin N-WASP, and vincu-lin, would result in activation rather than inhibition of the protein functions. In the model drawn, both sites are acti-vated after PIP2 binding, but it is also plausible that one of the sites remains occupied by the lipid and is available only after PIP2 hydrolysis or reorganization of the mem-brane. Such a mechanism would explain how PIP2 binding can work to activate vinculin in vivo while purified PIP2 apparently inhibits vinculin-actin binding in vitro [99], and would allow for sequential activation of two sites as PIP2 is turned over at the cell membrane. This model has recently been validated for ezrin, in a study that shows that actin binding of ezrin is activated by specific recognition of PIP2-functionalized lipid bilayers [107].

Considering the number of proteins affected by PIP2 and the different ways in which protein function can be affected by PPIs, there is a striking pattern to the functions that are activated or inhibited by these lipids, as summarized in Figure 141.2. All PIP2-sensitive actin monomer binding fac-tors, and proteins that sever actin filaments, are inactivated by PIP2. In contrast, proteins that promote actin assembly or that link F-actin to the membrane are activated by PIP2.


In addition, some motor proteins, such as myosin 1, are tar-geted to PIP2-enriched membranes, where they presumably retain their ability to move on membrane-proximal actin fil-aments. Filamin bundling proteins are generally inactivated by PIP2. The overall effect in the cell, assuming that all activities are equally affected, is increased actin polymeriza-tion and linkage to the membrane, with dissociation of actin bundles to promote more open network structures as actin assembles at the membrane interface. When PIP2 levels are depleted, actin stops polymerizing, and is actively depolym-erized while factors that stabilize bundling become active. This constellation of in vitro activities is consistent with the effects of increasing or decreasing PIP2 levels in the cell, although there are few if any studies to determine which of the many possible PIP2-mediated reactions is preferentially stimulated under different cellular conditions.

context-dependent interaction of ppis witH cytosKeletal proteins

Perhaps the main challenge in understanding how PPIs reg-ulate cytoskeletal or other proteins is the sheer number of PPI (usually PIP2) binding proteins that have been reported and generally well characterized in vitro as specific and high-affinity ligands for these lipids. When only a hand-ful of proteins, mostly actin binding proteins, were shown to be regulated by PIP2, it was a plausible hypothesis that such proteins with M concentrations in the cell could be all be regulated by PIP2, present at 10s to 100s of M con-centrations, when specific signals were initiated. Currently however, over 100 proteins are reported to bind PIP2 with similar affinity and there are very few studies of how differ-ent PIP2-binding proteins might compete with each other. There is also good evidence that proteins such as annexin 2, which bind only one PIP2 molecule, can prevent the bind-ing of multiple PIP2s in a lipid bilayer from their protein ligands, either by occluding their access to soluble proteins or by changing the structure of the membrane. The reports cited above, that the different PIP5Ks which all produce the same lipid have very different cellular effects, also sug-gest that access of PIP2 to its target to its multiple possible targets depends strongly on the location and environment in which the PIP2 is placed. As a result, perhaps the main unresolved issue is how PIP2 distributes laterally within the plasma membrane, and whether all PIP2 molecules within a membrane are equally effective at binding their targets. Two critical issues, for which there are conflicting reports and no consensus, are the relation of PIP2 to formation of cholesterol-dependent lipids rafts, and whether PIP2 can self-associate to form clusters independent of (or at least not requiring) cholesterol.

It has been reported that approximately half of the cell’s PIP2 is synthesized preferentially in cholesterol/sphingoli-pid-enriched caveolar light membrane fractions (“rafts”)

[108–110], and that these PIP2-enriched microdomains exhibit locally regulated PIP2 turnover and restricted dif-fusion-mediated exchanges with their environment [108]. There are also reports that PIP2 is enriched in non-caveo-lar microdomains that are the staging platforms for cho-reographing signaling and cytoskeletal dynamics. The existence of PIP2 microdomains is confirmed by immun-ofluorescence staining of PM sheets prepared from PC12 cells [111, 112]. The plasma membrane PIP2 microdo-mains are heterogeneous; some contains conventional raft markers, while others are enriched for syntaxin, which is involved in Ca2-mediated exocytosis and mostly excluded from the low-density raft fraction [111]. Other PIP2 clus-tering proteins have also been identified. These include MARCKS, which sequesters PIP2 under basal conditions, and is induced by agonist signaling to release PIP2 for interaction with other PIP2 targets [113]. An additional fac-tor that would modulate the formation of spatially distinct pools of PIP2 is suggested by reports that a combination of electrostatic repulsion and hydrogen bonding can influence PIP2 packing [89, 90, 114, 115] and work together with peripheral membrane binding proteins to organize local domains. On the other hand, some studies conclude that formation of domains enriched for PIP2 in both pure lipid systems [116] or the cell membrane [117] are induced by the manipulations used to visualize them, and imply that a system of dilute randomly distributed PIP2 in a fluid mem-brane is the most physiologically relevant system by which to evaluate the relative ability of different proteins to com-pete for scarce membrane PIP2 in vivo.

Specific functional regulation by PIP2 has now been confirmed for multiple proteins, and manipulation of PIP2 levels in cell consistently shows large effects on the actin-base cytoskeleton that are consistent with many observa-tions from in vitro biochemical studies. A challenge for future studies is to delineate more clearly the localization and diffusivity of PIP2 in the different membranes within the cell, and to evaluate how the large number of PIP2 lig-ands is selectively regulated when changes in PIP2 levels or localization are generated in vivo.

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handbook of cell signaling || phosphoinositides and actin cytoskeletal rearrangement

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