Allosteric activation of PTEN phosphatase by phosphatidylinositol 4,5-bisphosphate

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor that is lost in many human tumors and encodes a phosphatidylinositol phosphate phosphatase specific for the 3-position of the inositol ring. Here we report a novel mechanism of PTEN regulation. Binding of di-C8-phosphatidylinositol 4,5-P2 (PI(4,5)P2) to PTEN enhances phosphatase activity for monodispersed substrates, PI(3,4,5)P3 and PI(3,4)P2. PI(5)P also is an activator, but PI(4)P, PI(3,4)P2, and PI(3,5)P2 do not activate PTEN. Activation by exogenous PI(4,5)P2 is more apparent with PI(3,4)P2 as a substrate than with PI(3,4,5)P3, probably because hydrolysis of PI(3,4)P2 yields PI(4)P, which is not an activator. In contrast, hydrolysis of PI(3,4,5)P3 yields a potent activator, PI(4,5)P2, creating a positive feedback loop. In addition, neither di-C4-PI(4,5)P2 nor inositol trisphosphate-activated PTEN. Hence, the interaction between PI(4,5)P2 and PTEN requires specific, ionic interactions with the phosphate groups on the inositol ring as well as hydrophobic interactions with the fatty acid chains, likely mimicking the physiological interactions that PTEN has with the polar surface head groups and the hydrophobic core of phospholipid membranes. Mutations of the apparent PI(4,5)P2-binding motif in the PTEN N terminus severely reduced PTEN activity. In contrast, mutation of the C2 phospholipid-binding domain had little effect on PTEN activation. These results suggest a model in which a PI(4,5)P2 monomer binds to PTEN, initiates an allosteric conformational change and, thereby, activates PTEN independent of membrane binding.     

A mutant form of PTEN linked to autism

The tumor suppressor, phosphatase, and tensin homologue deleted on chromosome 10 (PTEN), is a phosphoinositide (PI) phosphatase specific for the 3‐position of the inositol ring. PTEN has been implicated in autism for a subset of patients with macrocephaly. Various studies identified patients in this subclass with one normal and one mutated PTEN gene. We characterize the binding, structural properties, activity, and subcellular localization of one of these autism‐related mutants, H93R PTEN. Even though this mutation is located at the phosphatase active site, we find that it affects the functions of neighboring domains. H93R PTEN binding to phosphatidylserine‐bearing model membranes is 5.6‐fold enhanced in comparison to wild‐type PTEN. In contrast, we find that binding to phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P₂) model membranes is 2.5‐fold decreased for the mutant PTEN in comparison to wild‐type PTEN. The structural change previously found for wild‐type PTEN upon interaction with PI(4,5)P₂, is absent for H93R PTEN. Consistent with the increased binding to phosphatidylserine, we find enhanced plasma membrane association of PTEN‐GFP in U87MG cells. However, this enhanced plasma membrane association does not translate into increased PI(3,4,5)P₃ turnover, since in vivo studies show a reduced activity of the H93R PTEN‐GFP mutant. Because the interaction of PI(4,5)P₂ with PTEN's N‐terminal domain is diminished by this mutation, we hypothesize that the interaction of PTEN's N‐terminal domain with the phosphatase domain is impacted by the H93R mutation, preventing PI(4,5)P₂ from inducing the conformational change that activates phosphatase activity.     

Activation of plant phospholipase Dbeta by phosphatidylinositol 4,5-bisphosphate: characterization of binding site and mode of action

Hydrolysis of phospholipids by plant phospholipase Dbeta (PLDbeta) requires phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Here we show that PLDbeta is stimulated by different polyphosphoinositides, among which PI(4,5)P2 is most effective. On the basis of amino acid sequence analysis, PI(4,5)P2 binding assay, and protein engineering studies, we have identified in the catalytic region of PLDbeta a new PI(4,5)P2 binding region (PBR1), which is conserved in eukaryotic PLDs. PBR1 is a second domain besides the previously characterized N-terminal C2 domain of PLDbeta which also binds PI(4,5)P2. Submillimolar levels of calcium ions, while inhibiting PI(4,5)P2 binding by the C2 domain, enhanced the affinity of PBR1 for that phosphoinositide. Substrate binding by PLDbeta was promoted by PI(4,5)P2-bound PBR1. Isolated, recombinant PBR1 bound PI(4,5)P2 specifically and in a saturable manner. Deletion of PBR1 from PLDbeta or mutation of the conserved basic amino acid residues in PBR1 (K437G/K440G) abolished the enzymatic activity. Circular dichroism spectroscopy revealed a conformational change caused by PI(4,5)P2 binding to the catalytic region of PLD. The conformational change apparently helps in the recruitment of the substrate to the active site of the enzyme. The results taken together allow us to describe an anchorage-scooting model for the synergistic activation of PLDbeta by PI(4,5)P2 and Ca2+.     

Phospholipid-binding Sites of Phosphatase and Tensin Homolog (PTEN): EXPLORING THE MECHANISM OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE ACTIVATION*

The lipid phosphatase activity of the tumor suppressor phosphatase and tensin homolog (PTEN) is enhanced by the presence of its biological product, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). This enhancement is suggested to occur via the product binding to the N-terminal region of the protein. PTEN effects on short-chain phosphoinositide ³¹P linewidths and on the full field dependence of the spin-lattice relaxation rate (measured by high resolution field cycling ³¹P NMR using spin-labeled protein) are combined with enzyme kinetics with the same short-chain phospholipids to characterize where PI(4,5)P₂ binds on the protein. The results are used to model a discrete site for a PI(4,5)P₂ molecule close to, but distinct from, the active site of PTEN. This PI(4,5)P₂ site uses Arg-47 and Lys-13 as phosphate ligands, explaining why PTEN R47G and K13E can no longer be activated by that phosphoinositide. Placing a PI(4,5)P₂ near the substrate site allows for proper orientation of the enzyme on interfaces and should facilitate processive catalysis.     

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.     

Phospholipase C regulation of phosphatidylinositol 3,4,5-trisphosphate-mediated chemotaxis

Generation of a phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] gradient within the plasma membrane is important for cell polarization and chemotaxis in many eukaryotic cells. The gradient is produced by the combined activity of phosphatidylinositol 3-kinase (PI3K) to increase PI(3,4,5)P(3) on the membrane nearest the polarizing signal and PI(3,4,5)P(3) dephosphorylation by phosphatase and tensin homolog deleted on chromosome ten (PTEN) elsewhere. Common to both of these enzymes is the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is not only the substrate of PI3K and product of PTEN but also important for membrane binding of PTEN. Consequently, regulation of phospholipase C (PLC) activity, which hydrolyzes PI(4,5)P(2), could have important consequences for PI(3,4,5)P(3) localization. We investigate the role of PLC in PI(3,4,5)P(3)-mediated chemotaxis in Dictyostelium. plc-null cells are resistant to the PI3K inhibitor LY294002 and produce little PI(3,4,5)P(3) after cAMP stimulation, as monitored by the PI(3,4,5)P(3)-specific pleckstrin homology (PH)-domain of CRAC (PH(CRAC)GFP). In contrast, PLC overexpression elevates PI(3,4,5)P(3) and impairs chemotaxis in a similar way to loss of pten. PI3K localization at the leading edge of plc-null cells is unaltered, but dissociation of PTEN from the membrane is strongly reduced in both gradient and uniform stimulation with cAMP. These results indicate that local activation of PLC can control PTEN localization and suggest a novel mechanism to regulate the internal PI(3,4,5)P(3) gradient.     

Phosphoinositides specify polarity during epithelial organ development

The molecular mechanisms that integrate cellular polarity with tissue architecture during epithelial morphogenesis are poorly understood. Using a three-dimensional model of epithelial morphogenesis, report that the phosphatase PTEN and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] regulate the GTPase Cdc42 and the kinase aPKC to generate the apical plasma membrane domain and maintain apical-basolateral polarity.     

Differential association of phosphatidylinositol 3-kinase, SHIP-1, and PTEN with forming phagosomes

In macrophages, enzymes that synthesize or hydrolyze phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P(3)] regulate Fcgamma receptor-mediated phagocytosis. Inhibition of phosphatidylinositol 3-kinase (PI3K) or overexpression of the lipid phosphatases phosphatase and tensin homologue (PTEN) and Src homology 2 domain-containing inositol phosphatase (SHIP-1), which hydrolyze PI(3,4,5)P(3) to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)], respectively, inhibit phagocytosis in macrophages. To examine how these enzymes regulate phagosome formation, the distributions of yellow fluorescent protein (YFP) chimeras of enzymes and pleckstrin homology (PH) domains specific for their substrates and products were analyzed quantitatively. PTEN-YFP did not localize to phagosomes, suggesting that PTEN regulates phagocytosis globally within the macrophage. SHIP1-YFP and p85-YFP were recruited to forming phagosomes. SHIP1-YFP sequestered to the leading edge and dissociated from phagocytic cups earlier than did p85-cyan fluorescent protein, indicating that SHIP-1 inhibitory activities are restricted to the early stages of phagocytosis. PH domain chimeras indicated that early during phagocytosis, PI(3,4,5)P(3) was slightly more abundant than PI(3,4)P(2) at the leading edge of the forming cup. These results support a model in which phagosomal PI3K generates PI(3,4,5)P(3) necessary for later stages of phagocytosis, PTEN determines whether those late stages can occur, and SHIP-1 regulates when and where they occur by transiently suppressing PI(3,4,5)P(3)-dependent activities necessary for completion of phagocytosis.     

Profilin binding to sub-micellar concentrations of phosphatidylinositol (4,5) bisphosphate and phosphatidylinositol (3,4,5) trisphosphate

Profilin is a small (12-15 kDa) actin binding protein which promotes filament turnover. Profilin is also involved in the signaling pathway linking receptors in the cell membrane to the microfilament system within the cell. Profilin is thought to play critical roles in this signaling pathway through its interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] (P.J. Lu, W.R. Shieh, S.G. Rhee, H.L. Yin, C.S. Chen, Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity, Biochemistry 35 (1996) 14027-14034). To date, profilin's interaction with polyphosphoinositides (PPI) has only been studied in micelles or small vesicles. Profilin binds with high affinity to small clusters of PI(4,5)P(2) molecules. In this work, we investigated the interactions of profilin with sub-micellar concentrations of PI(4,5)P(2) and PI(3,4,5)P(3). Fluorescence anisotropy was used to determine the relevant dissociation constants for binding of sub-micellar concentrations of fluorescently labeled PPI lipids to profilin and we show that these are significantly different from those determined for profilin interaction with micelles or small vesicles. We also show that profilin binds more tightly to sub-micellar concentrations of PI(3,4,5)P(3) (K(D)=720 microM) than to sub-micellar concentrations of PI(4,5)P(2) (K(D)=985 microM). Despite the low affinity for sub-micellar concentration of PI(4,5)P(2), profilin was shown to bind to giant unilamellar vesicles in presence of 0.5% mole fraction of PI(4,5)P(2) The implications of these findings are discussed.     

PDZ domains of Par-3 as potential phosphoinositide signaling integrators

Multiple PDZ domain scaffold protein Par-3 and phosphoinositides (PIPs) are required for polarity in diverse cell types. We show that the second PDZ domain of Par-3 binds to phosphatidylinositol (PI) lipid membranes with high affinity. We further demonstrate that a large subset of PDZ domains in mammalian genomes are capable of binding to PI lipid membranes, indicating that lipid binding is the second most prevalent interaction mode of PDZ domains known to date. The biochemical and structural basis of Par-3 PDZ2-mediated membrane interaction is characterized in detail. The membrane binding capacity of Par-3 PDZ2 is critical for epithelial cell polarization. Interestingly, the lipid phosphatase PTEN directly binds to the third PDZ domain of Par-3. The concatenation of the PIP-binding PDZ2 and the lipid phosphatase PTEN-binding PDZ3 endows Par-3 as an ideal scaffold protein for integrating PIP signaling events during cellular polarization.     

Membrane association of the PTEN tumor suppressor: molecular details of the protein-membrane complex from SPR binding studies and neutron reflection

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P₂) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P₂ were determined to be K_d∼12 µM and 0.4 µM, respectively, and K_d∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P₂. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P₂ and an increased apparent affinity to PI(3,4,5)P₃, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P₂ and no synergy in its binding with PS and PI(4,5)P₂. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.     

Association of the polymorphisms in the 5'-untranslated region of PTEN gene with type 2 diabetes in a Japanese population

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known to act as a lipid phosphatase hydrolyzing phosphatidylinositol (PI)(3,4,5)P(3) to PI(4,5)P(2). Since the PI3-kinase product, PI(3,4,5)P(3), is an important second messenger leading to the metabolic action of insulin, PTEN functions as a potent negative regulator of insulin signaling and its gene is one of the possible candidates involved in susceptibility to the development of type 2 (non-insulin-dependent) diabetes. In the present study, we investigated the polymorphisms of the PTEN gene in Japanese patients with type 2 diabetes and non-diabetic control subjects. We identified three mutations of the gene in the type 2 diabetes patients. Among these mutations, the frequency of the substitution of C with G at position -9 (-9C-->G) (SNP1), located in the untranslated region of exon 1, was significantly higher in type 2 diabetic patients than in control subjects. In addition, transfection of the PTEN gene with SNP1 resulted in a significantly higher expression level of PTEN protein compared with that of the wild-type PTEN gene in Cos1 and Rat1 cells. Furthermore, insulin-induced phosphorylation of Akt in HIRc cells was decreased more greatly by transfection of SNP1 PTEN gene than that of wild-type PTEN gene. These findings suggest that the change of C to G at position -9 of the PTEN gene is associated with the insulin resistance of type 2 diabetes due possibly to a potentiated hydrolysis of the PI3-kinase product.     

Overexpression of Arabidopsis thaliana PTEN caused accumulation of autophagic bodies in pollen tubes by disrupting phosphatidylinositol 3-phosphate dynamics

Autophagy is a pathway in eukaryotes by which nutrient remobilization occurs through bulk protein and organelle turnover. Autophagy not only aides cells in coping with harsh environments but also plays a key role in many physiological processes that include pollen germination and tube growth. Most autophagic components are conserved among eukaryotes, but phylum-specific molecular components also exist. We show here that Arabidopsis thaliana PTEN, a protein and lipid dual phosphatase homologous to animal PTENs (phosphatase and tensin homologs deleted on chromosome 10), regulates autophagy in pollen tubes by disrupting the dynamics of phosphatidylinositol 3-phosphate (PI3P). The pollen-specific PTEN bound PI3P in vitro and was localized at PI3P-positive vesicles. Overexpression of PTEN caused accumulation of autophagic bodies and resulted in gametophytic male sterility. Such an overexpression effect was dependent upon its lipid phosphatase activity and was inhibited by exogenous PI3P or by expression of a class III phosphatidylinositol 3-kinase (PI3K) that produced PI3P. Overexpression of PTEN disrupted the dynamics of autophagosomes and a subpopulation of endosomes, as shown by altered localization patterns of respective fluorescent markers. Treatment with wortmannin, an inhibitor of class III PI3K, mimicked the effects by PTEN overexpression, which implied a critical role for PI3P dynamics in these processes. Despite sharing evolutionarily conserved catalytic domains, plant PTENs contain regulatory sequences that are distinct from those of animal PTENs, which might underlie their differing membrane association and thereby function. Our results show that PTEN regulates autophagy through phylum-specific molecular mechanisms.     

Molecular mechanism of an oncogenic mutation that alters membrane targeting: Glu17Lys modifies the PIP lipid specificity of the AKT1 PH domain

The protein kinase AKT1 regulates multiple signaling pathways essential for cell function. Its N-terminal PH domain (AKT1 PH) binds the rare signaling phospholipid phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)], resulting in plasma membrane targeting and phosphoactivation of AKT1 by a membrane-bound kinase. Recently, it was discovered that the Glu17Lys mutation in the AKT1 PH domain is associated with multiple human cancers. This mutation constitutively targets the AKT1 PH domain to the plasma membrane by an unknown mechanism, thereby promoting constitutive AKT1 activation and oncogenesis. To elucidate the molecular mechanism underlying constitutive plasma membrane targeting, this work compares the membrane docking reactions of the isolated wild-type and E17K AKT1 PH domains. In vitro studies reveal that the E17K mutation dramatically increases the affinity for the constitutive plasma membrane lipid PI(4,5)P(2). The resulting PI(4,5)P(2) equilibrium affinity is indistinguishable from that of the standard PI(4,5)P(2) sensor, PLCdelta1 PH domain. Kinetic studies indicate that the effects of E17K on PIP lipid binding arise largely from electrostatic modulation of the dissociation rate. Membrane targeting analysis in live cells confirms that the constitutive targeting of E17K AKT1 PH to plasma membrane, like PLCdelta1 PH, stems from PI(4,5)P(2) binding. Overall, the evidence indicates that the molecular mechanism underlying E17K oncogenesis is a broadened target lipid selectivity that allows high-affinity binding to PI(4,5)P(2). Moreover, the findings strongly implicate the native Glu17 side chain as a key element of PIP lipid specificity in the wild-type AKT1 PH domain. Other PH domains may employ an analogous anionic residue to control PIP specificity.     

Point mutations in the split PLC-gamma1 PH domain modulate phosphoinositide binding

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal (PH(1)) and a split PH domain (nPH(2) and cPH(2)). We previously reported that the split PH domain of PLC-gamma1 binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P(2), we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-gamma1 nPH(2) domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-gamma1 nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-gamma1 molecules showed reduced PI(4,5)P(2) hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH(1) and nPH(2) domains are responsible for membrane-targeted translocation of PLC-gamma1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro(500) and His(503) are critical for binding of PLC-gamma1 to one of its substrates, PI(4,5)P(2) in the membrane.     

Structural and membrane binding analysis of the Phox homology domain of phosphoinositide 3-kinase-C2alpha

Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX domains, we determined the crystal structure of the PX domain from phosphoinositide 3-kinase C2alpha (PI3K-C2alpha), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). To delineate the mechanism by which this PX domain interacts with membranes, we measured the membrane binding of the wild type domain and mutants by surface plasmon resonance and monolayer techniques. This PX domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P(2) binding. The membrane binding of the PX domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P(2). Furthermore, the PX domain displayed significantly higher PtdIns(4,5)P(2) membrane affinity and specificity when compared with the PI3K-C2alpha C2 domain, demonstrating that high affinity PtdIns(4,5)P(2) binding was facilitated by the PX domain in full-length PI3K-C2alpha. Together, these studies provide new structural insight into the diverse PI specificities of PX domains and elucidate the mechanism by which the PI3K-C2alpha PX domain interacts with PtdIns(4,5)P(2)-containing membranes and thereby mediates the membrane recruitment of PI3K-C2alpha.     

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.     

Phosphorylation of the PTEN tail acts as an inhibitory switch by preventing its recruitment into a protein complex.

PTEN is a tumor suppressor protein that functions, in large part, by dephosphorylating the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate and by doing so antagonizing the action of phosphoinositide 3-kinase. PTEN structural domains include an N-terminal phosphatase domain, a lipid-binding C2 domain, and a 50-amino acid C-terminal tail that contains a PDZ binding sequence. We showed previously that phosphorylation of the PTEN tail negatively regulates PTEN activity. We now show that phosphorylated PTEN exists in a monomeric "closed" conformation and has low affinity for PDZ domain-containing proteins. Conversely, when unphosphorylated, PTEN is in an "open" conformation, is recruited into a high molecular weight complex (PTEN-associated complex), and strongly interacts with PDZ-containing proteins such as MAGI-2. As a consequence, when compared with wild-type PTEN, the phosphorylation-deficient mutant form of PTEN strongly cooperates with MAGI-2 to block Akt activation. These results indicate that phosphorylation of the PTEN tail causes a conformational change that results in the masking of the PDZ binding domain. Consequently, the ability of PTEN to bind to PDZ domain-containing proteins is reduced dramatically. These data suggest that phosphorylation of the PTEN tail suppresses the activity of PTEN by controlling the recruitment of PTEN into the PTEN-associated complex.     

Enzyme domain affects the movement of the voltage sensor in ascidian and zebrafish voltage-sensing phosphatases

The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN).The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.