Structural and biochemical evaluation of the interaction of the phosphatidylinositol 3-kinase p85alpha Src homology 2 domains with phosphoinositides and inositol polyphosphates.

Src homology 2 (SH2) domains exist in many intracellular proteins and have well characterized roles in signal transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation is essential for protein-SH2 domain interactions, the binding specificity also derives from sequences C-terminal to the Tyr(P) residue. The high affinity and specificity of this interaction is critical for precluding aberrant cross-talk between signaling pathways. The p85alpha subunit of phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and it has been proposed that in competition with Tyr(P) binding they may also mediate membrane attachment via interactions with phosphoinositide products of PI 3-kinase. We used nuclear magnetic resonance spectroscopy and biosensor experiments to investigate interactions between the p85alpha SH2 domains and phosphoinositides or inositol polyphosphates. We reported previously a similar approach when demonstrating that some pleckstrin homology domains show binding specificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250). However, neither SH2 domain exhibited binding specificity for phosphoinositides in phospholipid bilayers. We show that the p85alpha SH2 domain Tyr(P) binding pockets indiscriminately accommodate phosphoinositides and inositol polyphosphates. Binding of the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these ligands do not bind p85alpha SH2 domains with high affinity or specificity. Moreover, we observed that although wortmannin blocks PI 3-kinase activity in vivo, it does not affect the ability of tyrosine-phosphorylated proteins to bind to p85alpha. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85alpha SH2 domains.     

High-affinity binding of a FYVE domain to phosphatidylinositol 3-phosphate requires intact phospholipid but not FYVE domain oligomerization.

FYVE domains are small zinc-finger-like domains found in many proteins that are involved in regulating membrane traffic and have been shown to bind specifically to phosphatidylinositol 3-phosphate (PtdIns-3-P). FYVE domains are thought to recruit PtdIns-3-P effectors to endosomal locations in vivo, where these effectors participate in controlling endosomal maturation and vacuolar protein sorting. We have compared the characteristics of PtdIns-3-P binding by the FYVE domain from Hrs-1 (the hepatocyte growth factor-regulated tyrosine kinase substrate) with those of specific phosphoinositide binding by Pleckstrin homology (PH) domains. Like certain PH domains (such as that from phospholipase C-delta(1)), the Hrs-1 FYVE domain specifically recognizes a single phosphoinositide. However, while phosphoinositide binding by highly specific PH domains is driven almost exclusively by interactions with the lipid headgroup, this is not true for the Hrs-1 FYVE domain. The phospholipase C-delta(1) PH domain shows a 10-fold preference for binding isolated headgroup over its preferred lipid (phosphatidylinositol 4,5-bisphosphate) in a membrane, while the Hrs-1 FYVE domain greatly prefers (more than 50-fold) intact lipid in a bilayer over the isolated headgroup (inositol 1,3-bisphosphate). By contrast with reports for certain PH domains, we find that this preference for membrane binding over interaction with soluble lipid headgroups does not require FYVE domain oligomerization.     

Green light for polyphosphoinositide signals in plants

Plant genomes lack homologues of the inositol 1,4,5-trisphosphate receptor and protein kinase C, which are important components of the canonical phospholipase C signalling system in animals. Instead, plants seem to utilize alternative downstream signalling molecules, that is, InsP(6) and phosphatidic acid. Inositol lipids may also function as second messengers themselves. By reversible phosphorylation of the inositol headgroup, five biologically active plant polyphosphoinositides can be detected. Protein targets interact with specific polyphosphoinositide isomers via selective lipid-binding domains, thereby altering their intracellular localization and/or enzymatic activity. Such lipid-binding domains have also been used to create GFP based-lipid biosensors to visualize PPIs dynamics in vivo. Here, we highlight some recent advances and ideas on PPIs' role in plant signalling.     

Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components

BACKGROUND: Phosphoinositides are required for the recruitment of many proteins to both the plasma membrane and the endosome; however, their role in protein targeting to other organelles is less clear. The pleckstrin homology (PH) domains of oxysterol binding protein (OSBP) and its relatives have been shown to bind to the Golgi apparatus in yeast and mammalian cells. Previous in vitro binding studies identified phosphatidylinositol (PtdIns) (4)P and PtdIns(4,5)P(2) as candidate ligands, but it is not known which is recognized in vivo and whether phosphoinositide specificity can account for Golgi-specific targeting. RESULTS: We have examined the distribution of GFP fusions to the PH domain of OSBP and to related PH domains in yeast strains carrying mutations in individual phosphoinositide kinases. We find that Golgi targeting requires the activity of the PtdIns 4-kinase Pik1p but not phosphorylation of PtdIns at the 3 or 5 positions and that a PH domain specific for PtdIns(4,5)P(2) is targeted exclusively to the plasma membrane. However, a mutant version of the OSBP PH domain that does not bind phosphoinositides in vitro still shows some targeting in vivo. This targeting is independent of Pik1p but dependent on the Golgi GTPase Arf1p. CONCLUSIONS: Phosphorylation of PtdIns at the 4 position but not conversion to PtdIns(4,5)P(2) contributes to recruitment of PH domains to the Golgi apparatus. However, potential phosphoinositide ligands for these PH domains are not restricted to the Golgi, and the OSBP PH domain also recognizes a second determinant that is ARF dependent, indicating that organelle specificity reflects a combinatorial interaction.     

Cell permeant polyphosphoinositide-binding peptides that block cell motility and actin assembly

Polyphosphoinositides (PPIs) affect the localization and activities of many cellular constituents, including actin-modulating proteins. Several classes of polypeptide sequences, including pleckstrin homology domains, FYVE domains, and short linear sequences containing predominantly hydrophobic and cationic residues account for phosphoinositide binding by most such proteins. We report that a ten-residue peptide derived from the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding region in segment 2 of gelsolin, when coupled to rhodamine B has potent PIP(2) binding activity in vitro; crosses the cell membrane of fibroblasts, platelets, melanoma cells, and neutrophils by a process not involving endocytosis; and blocks cell motility. This peptide derivative transiently disassembles actin filament structures in GFP-actin-expressing NIH3T3 fibroblasts and prevents thrombin- or chemotactic peptide-stimulated actin assembly in platelets and neutrophils, respectively, but does not block the initial [Ca(2+)] increase caused by these agonists. The blockage of actin assembly and motility is transient, and cells recover motility within an hour after their immobilization by 5-20 microm peptide. This class of reagents confirms the critical relation between inositol lipids and cytoskeletal structure and may be useful to probe the location and function of polyphosphoinositides in vivo.     

Activation of phosphatidylinositol 3-kinase in cells expressing abl oncogene variants.

A phosphoinositide kinase specific for the D-3 position of the inositol ring, phosphatidylinositol (PI) 3-kinase, associates with activated receptors for platelet-derived growth factor, insulin, and colony-stimulating factor 1, with products of the oncogenes src, fms, yes, crk, and with polyomavirus middle T antigen. Efficient fibroblast transformation by proteins of the abl and src oncogene families requires activation of their protein-tyrosine kinase activity and membrane association via an amino-terminal myristoylation. We have demonstrated that the PI 3-kinase directly associates with autophosphorylated, activated protein-tyrosine kinase variants of the abl protein. In vivo, this association leads to accumulation of the highly phosphorylated products of PI 3-kinase, PI-3,4-bisphosphate and PI-3,4,5-trisphosphate, only in myristoylated, transforming abl protein variants. Myristoylation thus appears to be required to recruit PI 3-kinase activity to the plasma membrane for in vivo activation and correlates with the mitogenicity of the abl protein variants.     

Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositide-protein co-operative mechanism

Pleckstrin homology (PH) domains are protein modules found in proteins involved in many cellular processes. The majority of PH domain-containing proteins require membrane association for their function. It has been shown that most PH domains interact directly with the cell membrane by binding to phosphoinositides with a broad range of specificity and affinity. While a highly specific binding of the PH domain to a phosphoinositide can be necessary and sufficient for the correct recruitment of the host protein to the membrane, a weaker and less specific interaction may be necessary but not sufficient, thus probably requiring alternative, co-operative mechanisms.     

Modular phosphoinositide-binding domains--their role in signalling and membrane trafficking.

The membrane phospholipid phosphatidylinositol is the precursor of a family of lipid second-messengers, known as phosphoinositides, which differ in the phosphorylation status of their inositol group. A major advance in understanding phosphoinositide signalling has been the identification of a number of highly conserved modular protein domains whose function appears to be to bind various phosphoinositides. Such 'cut and paste' modules are found in a diverse array of multidomain proteins and recruit their host protein to specific regions in cells via interactions with phosphoinositides. Here, with particular reference to proteins involved in membrane traffic pathways, we discuss recent advances in our understanding of phosphoinositide-binding domains.     

Membrane and Actin Tethering Transitions Help IQGAP1 Coordinate GTPase and Lipid Messenger Signaling

The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific regulatory processes. This coupling often depends on the function of multidomain scaffolds that orchestrate transient interactions among multiple signaling intermediates and regulatory proteins on organelles. The number of possible scaffold interaction partners and the ability for these interactions to occur at different timescales makes investigations of scaffold functions challenging. This work employs live cell imaging to probe how the multidomain scaffold IQ motif containing GTPase activating protein 1 (IQGAP1) coordinates the activities of proteins affecting local actin polymerization, membrane processing, and phosphoinositide signaling. Using endosomes that are confined by a local actin network as a model system, we demonstrate that IQGAP1 can transition between different actin and endosomal membrane tethered states. Fast scaffold binding/disassociation transitions are shown to be driven by interactions between C-terminal scaffold domains and Rho GTPases at the membrane. Fluctuations in these binding modes are linked to negative regulation of actin polymerization. Although this control governs core elements of IQGAP1 dynamics, actin binding by the N-terminal calponin homology domain of the scaffold is shown to help the scaffold track the temporal development of endosome membrane markers, implying actin associations bolster membrane and actin coordination. Importantly, these effects are not easily distilled purely through standard (static) co-localization analyses or traditional pathway perturbations methods and were resolved by performing dynamic correlation and multiple regression analyses of IQGAP1 scaffold mutants. Using these capabilities with pharmacological inhibition, we provide evidence that membrane tethering is dependent on the activities of the lipid kinase phosphoinositide 3-kinase in addition to the Rho GTPases Rac1 and Cdc42. Overall, these methods and results point to a scaffold tethering mechanism that allows IQGAP1 to help control the amplitude of phosphoinositide lipid messenger signaling by coordinating signaling intermediate activities with the development and disassembly of local actin cytoskeletal networks.     

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.     

Signaling by distinct classes of phosphoinositide 3-kinases

Many signaling pathways converge on and regulate phosphoinositide 3-kinase (PI3K) enzymes whose inositol lipid products are key mediators of intracellular signaling. Different PI3K isoforms generate specific lipids that bind to FYVE and pleckstrin homology (PH) domains in a variety of proteins, affecting their localization, conformation, and activities. Here we review the activation mechanisms of the different types of PI3Ks and their downstream actions, with focus on the PI3Ks that are acutely triggered by extracellular stimulation.     

Pleckstrin homology domains and the cytoskeleton

Pleckstrin homology (PH) domains are 100-120 amino acid protein modules best known for their ability to bind phosphoinositides. All possess an identical core beta-sandwich fold and display marked electrostatic sidedness. The binding site for phosphoinositides lies in the center of the positively charged face. In some cases this binding site is well defined, allowing highly specific and strong ligand binding. In several of these cases the PH domains specifically recognize 3-phosphorylated phosphoinositides, allowing them to drive membrane recruitment in response to phosphatidylinositol 3-kinase activation. Examples of these PH domain-containing proteins include certain Dbl family guanine nucleotide exchange factors, protein kinase B, PhdA, and pleckstrin-2. PH domain-mediated membrane recruitment of these proteins contributes to regulated actin assembly and cell polarization. Many other PH domain-containing cytoskeletal proteins, such as spectrin, have PH domains that bind weakly, and to all phosphoinositides. In these cases, the individual phosphoinositide interactions may not be sufficient for membrane association, but appear to require self-assembly of their host protein and/or cooperation with other anchoring motifs within the same molecule to drive membrane attachment.     

Novel functional PI 3-kinase antagonists inhibit cell growth and tumorigenicity in human cancer cell lines.

New efforts in cancer therapy are being focused at various levels of signaling pathways. With phosphoinositide 3-kinase (PI3-K) potentially being necessary for a range of cancer-related functions, we have investigated the influence of selected inositol tris- to hexakisphosphates on cell growth and tumorigenicity. We show that micromolar concentrations of inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P(4)] inhibit IGF-1-induced [(3)H]-thymidine incorporation in human breast cancer (MCF-7) cells and the ability to grow in liquid medium and form colonies in agarose semisolid medium by small cell lung cancer (SCLC) cells, a human cancer cell line containing a constitutively active PI3-K. In an ovarian cancer cell line that also contains a constitutively active PI3-K (SKOV-3 cells), Ins(1,4,5,6)P(4) again inhibited liquid medium growth. Furthermore, when applied extracellularly, inositol 1,3,4,5-tetrakisphosphate was shown indeed to enter SCLC cells. These effects appeared specifically related to PH domains known to bind to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], indicating involvement of the PI3-K downstream target protein kinase B (PKB/Akt). This was further supported by inhibition of PKB/Akt PH domain membrane targeting in COS-7 cells by Ins(1,4,5,6)P(4). Thus, we propose that specific inositol polyphosphates inhibit PI3-K by competing with PtdIns(3,4, 5)P(3)-binding PH domains and that this occurs mainly at the level of the downstream PI3-K target, PKB/Akt.     

Phosphoinositide 3-kinase γ has Multiple Phospholipid Binding Sites

Phosphoinositide 3-kinase γ is a multifunctional enzyme with lipid and protein kinase activities that also acts as a scaffold protein in many diverse signalling processes. The enzyme contains five different domains, but their individual contributions to membrane binding are not fully understood. Here, using in vitro liposome binding assays of individual domains and deletion constructs of human phosphoinositide 3-kinase γ, we show that each domain is capable of binding anionic phospholipids to varying degrees, depending on the charge of the anionic substrate. Moreover, with the exception of the C2-domain, deletion of any single protein domain results in a complete loss of kinase activity toward both lipids and proteins.     

Pleckstrin homology (PH) domains and phosphoinositides

PH (pleckstrin homology) domains represent the 11th most common domain in the human proteome. They are best known for their ability to bind phosphoinositides with high affinity and specificity, although it is now clear that less than 10% of all PH domains share this property. Cases in which PH domains bind specific phosphoinositides with high affinity are restricted to those phosphoinositides that have a pair of adjacent phosphates in their inositol headgroup. Those that do not [PtdIns3P, PtdIns5P and PtdIns(3,5)P2] are instead recognized by distinct classes of domains including FYVE domains, PX (phox homology) domains, PHD (plant homeodomain) fingers and the recently identified PROPPINs (b-propellers that bind polyphosphoinositides). Of the 90% of PH domains that do not bind strongly and specifically to phosphoinositides, few are well understood. One group of PH domains appears to bind both phosphoinositides (with little specificity) and Arf (ADP-ribosylation factor) family small G-proteins, and are targeted to the Golgi apparatus where both phosphoinositides and the relevant Arfs are both present. Here, the PH domains may function as coincidence detectors. A central challenge in understanding the majority of PH domains is to establish whether the very low affinity phosphoinositide binding reported in many cases has any functional relevance. For PH domains from dynamin and from Dbl family proteins, this weak binding does appear to be functionally important, although its precise mechanistic role is unclear. In many other cases, it is quite likely that alternative binding partners are more relevant, and that the observed PH domain homology represents conservation of structural fold rather than function.     

Polyphosphoinositide hydrolysis is associated with exocytosis in adrenal medullary cells

[3H]inositol-labelled products are released from adrenal medullary cells during exocytotic secretion. In 'leaky' cells in which small molecules readily enter and leave the cytoplasm, addition of micromolar calcium ions in the presence of ATP stimulates exocytosis and causes the release of inositol polyphosphates. These data support the idea that hydrolysis of plasma membrane polyphosphoinositides may be an essential step in exocytotic secretion.     

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices.

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.     

Effects of CapG overexpression on agonist-induced motility and second messenger generation

Actin modulating proteins that bind polyphosphoinositides, such as phosphatidylinositol 4, 5-bisphosphate (PIP2), can potentially participate in receptor signaling by restructuring the membrane cytoskeleton and modulating second messenger generation through the phosphoinositide cycle. We examined these possibilities by overexpressing CapG, an actin filament end capping, Ca(2+)- and polyphosphoinositide-binding protein of the gelsolin family. High level transient overexpression decreased actin filament staining in the center of the cells but not in the cell periphery. Moderate overexpression in clonally selected cell lines did not have a detectible effect on actin filament content or organization. Nevertheless, it promoted a dose-dependent increase in rates of wound healing and chemotaxis. The motile phenotype was similar to that observed with gelsolin overexpression, which in addition to capping, also severs and nucleates actin filaments. CapG overexpressing clones are more responsive to platelet-derived growth factor than control- transfected clones. They form more circular dorsal membrane ruffles, have higher phosphoinositide turnover, inositol 1,4,5-trisphosphate generation and Ca2+ signaling. These responses are consistent with enhanced PLC gamma activity. Direct measurements of PIP2 mass showed that the CapG effect on PLC gamma was not due primarily to an increase in the PIP2 substrate concentration. The observed changes in cell motility and membrane signaling are consistent with the hypothesis that PIP(2)-binding actin regulatory proteins modulate phosphoinositide turnover and second messenger generation in vivo. We infer that CapG and related proteins are poised to coordinate membrane signaling with actin filament dynamics following cell stimulation.     

Inhibition of plant plasma membrane phosphoinositide phospholipase C by the actin-binding protein, profilin

A key event in signal transduction in many eukaryotes is activation of polyphosphoinositide-specific phospholipase C (PIC). This enzyme hydrolyses the plasma membrane-associated lipid, phosphatidylinositol(4,5)bisphosphate (Ptdlns(4,5)P₂) which leads to the production of the two second messenger molecules: inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃) and 1,2-diacylglycerol (DG). In plants, an enzyme which functionally resembles mammalian PIC is known to exist in the plasma membrane, but little is understood about how its activity is regulated. The recent discovery of several plant proteins with 30–40% homology to the mammalian actin- and phosphoinositide-binding protein, profilin, has prompted an investigation as to whether these proteins (plant profilins) are able to interact with polyphosphoinositides and, if so, whether such interactions have physiological relevance for signal transduction via the plant phosphoinositide system.
In this study it is demonstrated that a direct and highly specific interaction does exist between plant profilin and polyphosphoinositides and that these interactions dramatically affect the ability of plant plasma membrane phosphoinositide phospholipase C to utilize phosphoinositides for second messenger production. These data are the first to demonstrate a functional role of plant profilin in controlling polyphosphoinositide turnover and also provide the first evidence for a direct effect of an actin-binding protein on a membrane-associated signalling enzyme. These findings indicate a novel mechanism for control of plant phosphoinositide turnover, and suggest a possible link between plant cell activation, second messenger production and modulation of cytoskeletal dynamics.     

Nuclear phosphoinositides and their roles in cell biology and disease

Since the late 1980s, a growing body of evidence has documented that phosphoinositides and their metabolizing enzymes, which regulate a large variety of cellular functions both in the cytoplasm and at the plasma membrane, are present also within the nucleus, where they are involved in processes such as cell proliferation, differentiation, and survival. Remarkably, nuclear phosphoinositide metabolism operates independently from that present elsewhere in the cell. Although nuclear phosphoinositides generate second messengers such as diacylglycerol and inositol 1,4,5 trisphosphate, it is becoming increasingly clear that they may act by themselves to influence chromatin structure, gene expression, DNA repair, and mRNA export. The understanding of the biological roles played by phosphoinositides is supported by the recent acquisitions demonstrating the presence in the nuclear compartment of several proteins harboring phosphoinositide-binding domains. Some of these proteins have functional roles in RNA splicing/processing and chromatin assembly. Moreover, recent evidence shows that nuclear phospholipase Cβ1 (a key phosphoinositide metabolizing enzyme) could somehow be involved in the myelodysplastic syndrome, i.e. a hematopoietic disorder that frequently evolves into an acute leukemia. This review aims to highlight the most significant and updated findings about phosphoinositide metabolism in the nucleus under both physiological and pathological conditions.