Human cofilin forms oligomers exhibiting actin bundling activity.

Human cofilin possesses the tendency for self-association, as indicated by the rapid formation of dimers and oligomers when reacted with water-soluble carbodiimide, Ellman's reagent, or glutathione disulfide. Intermolecular disulfide bonds involve Cys(39) and probably Cys(147) of two adjacent cofilin units. The disulfide-linked dimers and oligomers exhibit a biological activity distinct from the monomer. While monomeric cofilin decreased viscosity and light-scattering of F-actin solutions, dimers and oligomers caused an increase in viscosity and light scattering. Electron microscopy revealed that cofilin oligomers induce the formation of highly ordered actin bundles with occasionally blunt ends similar to actin-cofilin rods observed in cells under oxidative stress. Bundling activity of the disulfide-linked oligomers could be completely reversed into severing activity by dithiothreitol. Formation of cofilin oligomers occurred also in the presence of actin at pH 8, but not at pH 6.6, and was significantly enhanced in the presence of phosphatidylinositol 4,5-bisphosphate. Our data are consistent with the idea that cofilin exists in two forms in vivo also: as monomers exhibiting the known severing activity and as oligomers exhibiting actin bundling activity. However, stabilization of cofilin oligomers in cytoplasm is probably achieved not by disulfide bonds but by a local increase in cofilin concentration and/or binding of regulatory proteins.     

Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain

Radixin is a member of the ezrin/radixin/moesin (ERM) family of proteins, which play a role in the formation of the membrane-associated cytoskeleton by linking actin filaments and adhesion proteins. This cross-linking activity is regulated by phosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP2) in the downstream of the small G protein Rho. The X-ray crystal structures of the radixin FERM domain, which is responsible for membrane binding, and its complex with inositol-(1,4, 5)-trisphosphate (IP3) have been determined. The domain consists of three subdomains featuring a ubiquitin-like fold, a four-helix bundle and a phosphotyrosine-binding-like domain, respectively. These subdomains are organized by intimate interdomain interactions to form characteristic grooves and clefts. One such groove is negatively charged and so is thought to interact with basic juxta-membrane regions of adhesion proteins. IP3 binds a basic cleft that is distinct from those of pleckstrin homology domains and is located on a positively charged flat molecular surface, suggesting an electrostatic mechanism of plasma membrane targeting. Based on the structural changes associated with IP3 binding, a possible unmasking mechanism of ERM proteins by PIP2 is proposed.     

Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.     

Involvement of Elk-1 in L6E9 skeletal muscle differentiation

The binding of phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P(2)) to profilin at a region distinct from the actin interaction surface is demonstrated by experiments with covalently cross-linked profilin:beta-actin. The result is in agreement with observations made with several mutant profilins and provides strong evidence for two regions on mammalian profilin mediating electrostatic interaction with phosphatidylinositol lipids; one close to the binding site for poly(L-proline), and one partially overlapping with the actin-binding surface. Congruent with this, two plant profilins, which have a reduced number of positive amino acids in one of these regions, displayed a dramatically lower binding to PI(4,5)P(2) compared to human profilin I.     

The crystal structure of the actin binding domain from alpha-actinin in its closed conformation: structural insight into phospholipid regulation of alpha-actinin

Alpha-actinin is the major F-actin crosslinking protein in both muscle and non-muscle cells. We report the crystal structure of the actin binding domain of human muscle alpha-actinin-3, which is formed by two consecutive calponin homology domains arranged in a "closed" conformation. Structural studies and available biochemical data on actin binding domains suggest that two calponin homology domains come in a closed conformation in the native apo-form, and that conformational changes involving the relative orientation of the two calponin homology domains are required for efficient binding to actin filaments. The actin binding activity of muscle isoforms is supposed to be regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which binds to the second calponin homology domain. On the basis of structural analysis we propose a distinct binding site for PtdIns(4,5)P2, where the fatty acid moiety would be oriented in a direction that allows it to interact with the linker sequence between the actin binding domain and the first spectrin-like repeat, regulating thereby the binding of the C-terminal calmodulin-like domain to this linker.     

Polyphosphoinositides inhibit the interaction of vinculin with actin filaments.

Binding of vinculin to adhesion plaque proteins is restricted by an intramolecular association of vinculin's head and tail regions. Results of previous work suggest that polyphosphoinositides disrupt this interaction and thereby promote binding of vinculin to both talin and actin. However, data presented here show that phosphatidylinositol 4,5-bisphosphate (PI4,5P2) inhibits the interaction of purified tail domain with F-actin. Upon re-examining the effect of PI4,5P2 on the actin and talin-binding activities of intact vinculin, we find that when the experimental design controls for the effect of magnesium on aggregation of PI4,5P2 micelles, polyphosphoinositides promote interactions with the talin-binding domain, but block interactions of the actin-binding domain. In contrast, if vinculin is trapped in an open confirmation by a peptide specific for the talin-binding domain of vinculin, actin binding is allowed. These results demonstrate that activation of the actin-binding activity of vinculin requires steps other than or in addition to the binding of PI4,5P2.     

Identification and characterization of a phosphoinositide phosphate kinase homolog

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) plays a central role in regulating the actin cytoskeleton as a substrate for phosphoinositide 3-kinase and phospholipase C as well as by binding directly to proteins that control the processes of actin monomer sequestration, filament severing, capping, nucleation, cross-linking, and bundling (Ma, L., Cantley, L. C., Janmey, P. A., and Kirschner, M. W. (1998) J. Cell Biol. 140, 1125-1136; Hinchliffe, K. (2000) Curr. Biol. 10, R104-R1051). Three related phosphatidylinositol 4-phosphate 5-kinases (PI(4)P 5-kinases) have been identified in mammalian cells (types Ialpha, Ibeta, and Igamma) and appear to play distinct roles in actin remodeling. Here we have identified a fourth member of this family by searching the human genome and EST data bases. This new protein, which we have designated phosphatidylinositol phosphate kinase homolog (PIPKH), is expressed at relatively high levels in brain and testis. Immunoprecipitates of PIPKH expressed in mammalian cells contain PI(4)P 5-kinase activity, but this activity is not affected by mutations in residues that inactivate other type I PI(4)P 5-kinases. We show that the PI(4)P 5-kinase activity in PIPKH immunoprecipitates can be explained by the ability of PIPKH to heterodimerize with other type I PI(4)P 5-kinases. Transfection of 293t cells with PIPKH resulted in >8-fold increase in total phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) without a significant net increase in total PI(4,5)P(2). When coexpressed with PIPKH, green fluorescent protein (GFP) fusion construct of the pleckstrin homology domain from Bruton's tyrosine kinase (GFP-BTK-PH) localized in intracellular vesicular structures, suggesting an unusual intracellular site of PI(3,4,5)P(3) production. Finally, expression of PIPKH induced the reorganization of actin from predominantly stress fibers to predominantly foci and comets similar to those observed previously in cells infected with the intracellular pathogen Listeria or transfected with recombinant PIPKIalpha. These results suggest that PIPKH acts as a scaffold to localize and regulate type I PI(4)P 5-kinases and the synthesis of PI(3,4,5)P(3).     

Novel mechanism of PTEN regulation by its phosphatidylinositol 4,5-bisphosphate binding motif is critical for chemotaxis

In chemotaxing cells, localization of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) to the leading edge of the cell sets the direction and regulates the formation of pseudopods at the anterior. We show that the lipid phosphatase activity of PTEN mediates chemotaxis and that the sharp localization of PI(3,4,5)P3 requires localization of PTEN to the rear of the cell. Our data suggest that a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) binding motif at the N terminus of PTEN serves the dual role of localizing the enzyme to the membrane and regulating its activity. Mutations in this motif enhance catalytic activity but render the enzyme inactive in vivo by preventing membrane association. The key role of this motif may explain the heretofore puzzling tumor-suppressing mutations occurring within the PI(4,5)P2 binding motif. On the other hand, the localization of PTEN does not depend on its phosphatase activity, the actin cytoskeleton, or the intracellular level of PI(3,4,5)P3, suggesting that events controlling localization are upstream of phosphoinositide signaling.     

Actin and phosphoinositide binding by the ActA protein of the bacterial pathogen Listeria monocytogenes

The surface protein ActA of the pathogenic bacterium Listeria monocytogenes induces actin-driven movement of bacteria in the cytoplasm of infected host cells and serves as a model for actin-based motility in general. We generated and purified soluble recombinant fragments of ActA and assessed their ability to interact with the acidic phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, both implicated in the regulation of actin polymerization. Purified ActA consisted of biologically active, elongated molecules with an alpha-helix and beta-sheet content of 11 and 32%, respectively. In the presence of either phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate, but not phosphatidylcholine, ActA molecules underwent a structural change that raised the alpha-helix content to 19% and lowered the beta-sheet content to 27%. Co-sedimentation experiments with phosphatidylcholine vesicles containing different acidic phospholipids demonstrated that ActA binds preferentially to D-3 phosphoinositides. The D-3 phosphoinositide binding activity was mapped to a small subregion in the N-terminal domain of ActA. This subregion comprised 19 amino acids and showed homology to cecropins. In addition, we found that amino acids 33 to 74 of ActA mediated actin binding by the whole, folded ActA molecule. These findings shed new light on ActA function.     

The interaction of skeletal myosin subfragment 1 with the polyanion, heparin

The association between chymotryptic skeletal muscle myosin subfragment 1 (S1) and the polyanion, heparin, was investigated as an experimental approach in probing the functional importance of the cationic sites on S1 and their involvement in ionic interactions within the myosin head during energy transduction. The direct binding of heparin, used at micromolar concentrations, and its influence on the structural and functional properties of S1 were followed by gel chromatography, electron microscopy, chemical cross-linking techniques and limited digestion studies. 1. The limited tryptic digestion of S1 showed that the presence of heparin, as well as of the homopolymer, poly-(L-glutamic acid) causes a specific structural change in the 50-kDa heavy chain region of S1 and accelerates the breakdown of this segment into a 45-kDa species by a proteolytic cleavage restricted to its COOH-terminal portion. Under similar experimental conditions, the binding of MgATP and MgADP to S1 led also to the 50-kDa----45-kDa conversion, suggesting that the S1-nucleotide interactions exhibit some resemblances to the polyanion-S1 binding of polyanionic ligands to S1. This particular area is adjacent to the actin site containing the 45-kDa and 20-kDa segments of the S1 heavy chain. On the other hand, the polyanions as well as nucleotides induced changes in the interface between the heavy chain and the alkali light chains. 2. Moreover, the binding of heparin to S1 resulted in the self-association of the enzyme and the production of stable small S1 oligomers, most likely dimers, which were demonstrated by the alteration of the size of the S1 particles examined by electron microscopy and their freezing by chemical cross-linking agents. These findings are relevant to the recently reported property of skeletal chymotryptic S1 to form dimers under convenient ionic conditions, in particular in the presence of Mg-nucleotides. The interaction of cationic sites on S1 and possibly on the 50-kDa region of the heavy chain with polyanions promotes the dimerization of the S1 molecules. The binding of S1 to F-actin abolished S1 aggregation.     

The vinculin C-terminal hairpin mediates F-actin bundle formation, focal adhesion, and cell mechanical properties

Vinculin is an essential and highly conserved cell adhesion protein, found at both focal adhesions and adherens junctions, where it couples integrins or cadherins to the actin cytoskeleton. Vinculin is involved in controlling cell shape, motility, and cell survival, and has more recently been shown to play a role in force transduction. The tail domain of vinculin (Vt) contains determinants necessary for binding and bundling of actin filaments. Actin binding to Vt has been proposed to induce formation of a Vt dimer that is necessary for cross-linking actin filaments. Results from this study provide additional support for actin-induced Vt self-association. Moreover, the actin-induced Vt dimer appears distinct from the dimer formed in the absence of actin. To better characterize the role of the Vt strap and carboxyl terminus (CT) in actin binding, Vt self-association, and actin bundling, we employed smaller amino-terminal (NT) and CT deletions that do not perturb the structural integrity of Vt. Although both NT and CT deletions retain actin binding, removal of the CT hairpin (1061-1066) selectively impairs actin bundling in vitro. Moreover, expression of vinculin lacking the CT hairpin in vinculin knock-out murine embryonic fibroblasts affects the number of focal adhesions formed, cell spreading as well as cellular stiffening in response to mechanical force.     

Phosphoinositide specificity of and mechanism of lipid domain formation by annexin A2-p11 heterotetramer

Annexin A2 is a phospholipid-binding protein that forms a heterotetramer (annexin II-p11 heterotetramer; A2t) with p11 (S100A10). It has been reported that annexin A2 is involved in binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and in inducing membrane microdomain formation. To understand the mechanisms underlying these findings, we determined the membrane binding properties of annexin A2 wild type and mutants both as monomer and as A2t. Our results from surface plasmon resonance analysis showed that A2t and annexin A2 has modest selectivity for PtdIns(4,5)P2 over other phosphoinositides, which is conferred by conserved basic residues, including Lys279 and Lys281, on the convex surface of annexin A2. Fluorescence microscopy measurements using giant unilamellar vesicles showed that A2t of wild type, but not (K279A)2-(p11)2 or (K281A)2-(p11)2, specifically induced the formation of 1-microm-sized PtdIns(4,5)P2 clusters, which were stabilized by cholesterol. Collectively, these studies elucidate the structural determinant of the PtdIns(4,5)P2 selectivity of A2t and suggest that A2t may be involved in the regulation of PtdIns(4,5)P2 clustering in the cell.     

Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.     

Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation

Profilin is a conserved, widely distributed actin monomer binding protein found in eukaryotic cells. Mammalian profilin reversibly sequesters actin monomers in a high affinity profilactin complex. In vitro, the complex is dissociated in response to treatment with the polyphosphoinositides, phosphatidylinositol monophosphate, and phosphatidylinositol 4,5-bisphosphate. Here, we demonstrate the ultrastructural immunolocalization of profilin in human leukocytes and platelets. In both cell types, a significant fraction of profilin is found associated with regions of cell membrane devoid of actin filaments and other discernible structures. After platelet activation, the membrane association of profilin reversibly increases. This study represents the first direct evidence for an interaction between profilin and phospholipids in vivo.     

Inhibition of actin regulatory activity of the 74-kDa protein from bovine adrenal medulla (adseverin) by some phospholipids

A 74-kDa protein (adseverin) derived from adrenal medulla severs actin filaments and nucleates actin polymerization in a Ca2(+)-dependent manner but does not form an EGTA-resistant complex with actin monomers, which is different from the gelsolin-actin interaction. The dissociation of gelsolin-actin complexes by phosphatidylinositol 4,5-bisphosphate (PIP2) and the inhibitory effect on actin filament severing by gelsolin was recently reported. This study shows that the activity of adseverin is inhibited not only by PIP2 but also by some common phospholipids including phosphatidylinositol (PI) and phosphatidylserine (PS). Other phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) showed no effect. The addition of PC or PE to PI diminished the inhibitory effect of PI. Triton X-100 and neomycin were also found effective in suppressing the effect of PI, suggesting that the arrangement of polar head groups is important in exerting the inhibitory effect. Ca2(+)-dependent binding of adseverin to PS liposomes but not to PC or PE liposomes was observed by a centrifugation assay.     

Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides

Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P(2). This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P(2)-induced talin activation. Finally, sequestration of PI4,5P(2) by a specific pleckstrin homology domain confirms that PI4,5P(2) is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P(2) exposes the integrin-binding site on talin. We propose that PI4,5P(2)-dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrin-binding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P(2) to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.     

Overexpression of murine phosphatidylinositol 4-phosphate 5-kinase type Ibeta disrupts a phosphatidylinositol 4,5 bisphosphate regulated endosomal pathway

The type I phosphatidylinositol 4-phosphate 5-kinases (PI4P5K) phosphorylate phosphatidylinositol 4-phosphate [PI(4)P] to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 has been implicated in signal transduction, receptor mediated endocytosis, vesicle trafficking, cytoskeletal structure, and membrane ruffling. However, the specific type I enzymes associated with the production of PI(4,5)P2 for the specific cellular processes have not been rigorously defined. Murine PI4P5K type Ibeta (mPIP5K-Ibeta) was implicated in receptor mediated endocytosis through the isolation of a truncated and inactive form of the enzyme that blocked the ligand-dependent downregulation of the colony-stimulating factor-1 receptor. The present study shows that enforced expression of mPIP5K-Ibeta in 293T cells resulted in the accumulation of large vesicles that were linked to an endosomal pathway. Similar results were obtained after the expression of the PI(4,5)P2-binding pleckstrin homology (PH) domain of phospholipase-Cdelta (PLC-delta). Analysis of the conserved domains of mPIP5K-Ibeta led to the identification of dimerization domains in the N- and C-terminal regions. Enforced expression of the individual dimerization domains interfered with the proper subcellular localization of mPIP5K-Ibeta and the PLC-delta-PH domain and blocked the accumulation of the endocytic vesicles induced by these proteins. In addition to regulating early steps in endocytosis, these results suggest that mPIP5K-Ibeta acts through PI(4,5)P2 to regulate endosomal trafficking and/or fusion.     

Modification of gelsolin with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole.

Pig plasma gelsolin was modified with the fluorescent reagent 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F) for lysyl residues. The relationship between the gelsolin activity and the degree of NBD labeling suggested that a single lysyl residue, which reacted five times slower than the other reactive lysyl residues, was essential for the activity. Taking advantage of the slow reactivity of the essential residue, active NBD-gelsolin was prepared. Limited cleavage of NBD-gelsolin by chymotrypsin indicated that the fluorescent reagents were randomly incorporated into all fragments observed. When NBD-gelsolin formed a gelsolin/actin (1:2) complex in the presence of micromolar Ca2+, the fluorescence spectra of NBD-gelsolin were red-shifted by 5 nm and the intensity decreased by 30%. However, on binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), the fluorescence spectra were blue-shifted by 5 nm with a concomitant increase in intensity by 20%. The addition of PtdIns(4,5)P2 to the NBD-gelsolin actin (1:2) complex restored the fluorescence spectra to that obtained in the presence of PtdIns(4,5)P2 alone. These results indicated that NBD-gelsolin, selectively labeled on lysyl residues not essential for activity, can be a useful probe to monitor the binding of PtdIns(4,5)P2 and actin.     

Nitration of profilin effects its interaction with poly (L-proline) and actin

Profilin from bovine spleen was nitrated with peroxynitrite; immunoblotting and spectrophotometric quantitation of nitrotyrosine residues suggested nitration of a single tyrosine residue in profilin with a stoichiometry of 0.6 mol of nitrotyrosine/mole of profilin. A decrease in the nitrotyrosine immunoreactivity of nitroprofilin during digestion with carboxypeptidase Y indicated that nitrotyrosine is located at the C-terminus of profilin. Nitroprofilin interaction with ligands such as phosphatidylinositol 4,5-bisphosphate, actin and poly (l-proline) was analyzed by monitoring the tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no significant difference in affinity of nitroprofilin to phosphatidylinositol 4,5-bisphosphate (K(d) of 4.8 +/- 0.5 muM for profilin, and K(d) of 5.7 +/- 0.6 muM for nitroprofilin), while poly (l-proline) binding studies revealed a twenty-fold increase in the affinity of profilin to poly (l-proline) upon nitration (K(d) of 21.8 +/- 1.7 muM for profilin, and K(d) of 1.1 +/- 0.1 muM for nitroprofilin). Actin polymerization studies involving pyrene-labeled actin indicated that profilin nitration inhibits the actin sequestering property of profilin. The critical actin monomer concentration (C(c)) was 150 and 250 nM in the presence of nitroprofilin and profilin, respectively. Thus, nitric oxide and free radicals produced under different conditions could alter the functions of profilin through nitration, such as its interaction with actin and poly (l-proline).     

Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain

The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by protein-protein and/or protein-lipid interactions. Here we show that Dbl PH domain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the PH domain significantly inhibits the Cdc42 interactive activity of the DH domain suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed that whereas the PH domain localizes to actin stress fibers and plasma membrane, the PH mutants are no longer detectable on the plasma membrane. These results suggest that modulation of PIPs in both the GEF catalytic activity and the targeting to plasma membrane determines the outcome of the biologic activity of Dbl.