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Mark A. Lemmon - One of the best experts on this subject based on the ideXlab platform.
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a possible effector role for the pleckstrin homology ph domain of dynamin
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Kelley A Bethoney, Michael E Ostap, Megan C King, Jenny E Hinshaw, Mark A. LemmonAbstract:The large GTPase dynamin plays a key role in clathrin-mediated endocytosis in animal cells, although its mechanism of action remains unclear. Dynamins 1, 2, and 3 contain a pleckstrin homology (PH) domain that binds Phosphoinositides with a very low affinity (KD > 1 mM), and this interaction appears to be crucial for function. These observations prompted the suggestion that an array of PH domains drives multivalent binding of dynamin oligomers to Phosphoinositide-containing membranes. Although in vitro experiments reported here are consistent with this hypothesis, we find that PH domain mutations that abolish dynamin function do not alter localization of the protein in transfected cells, indicating that the PH domain does not play a simple targeting role. An alternative possibility is suggested by the geometry of dynamin helices resolved by electron microscopy. Even with one phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] molecule bound per PH domain, these dynamin assemblies will elevate the concentration of PtdIns(4,5)P2 at coated pit necks, and effectively cluster (or sequester) this Phosphoinositide. In vitro fluorescence quenching studies using labeled Phosphoinositides are consistent with dynamin-induced PtdIns(4,5)P2 clustering. We therefore propose that the ability of dynamin to alter the local distribution of PtdIns(4,5)P2 could be crucial for the role of this GTPase in promoting membrane scission during clathrin-mediated endocytosis. PtdIns(4,5)P2 clustering could promote vesicle scission through direct effects on membrane properties, or might play a role in dynamin's ability to regulate actin polymerization.
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Pleckstrin homology (PH) domains and Phosphoinositides.
Biochemical Society symposium, 2007Co-Authors: Mark A. LemmonAbstract: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.
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determining selectivity of Phosphoinositide binding domains
Methods, 2006Co-Authors: Kartik Narayan, Mark A. LemmonAbstract:Abstract The burgeoning of Phosphoinositide-binding domains and proteins in cellular signaling and trafficking has drawn laboratories from a wide variety of fields into the study of lipid interactions with peripheral membrane proteins. Many different approaches have been developed to assess Phosphoinositide binding, some of which are more problematic than others, and some of which can be quantitated more readily than others. With a focus on the methods used in our laboratory, we describe here the considerations that need to be taken into account when establishing—and quantitating—the specific binding of a protein or domain to Phosphoinositides in membranes. We also discuss briefly a few examples in which no clear consensus has yet been reached as to the specificity of a given domain or protein because of discrepancies between different commonly used approaches.
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Pleckstrin homology domains: not just for Phosphoinositides
Biochemical Society Transactions, 2004Co-Authors: Mark A. LemmonAbstract:PH domains (pleckstrin homology domains) are the 11th most common domain in the human genome and are best known for their ability to target cellular membranes by binding specifically to Phosphoinositides. Recent studies in yeast have shown that, in fact, this is a property of only a small fraction of the known PH domains. Most PH domains are not capable of independent membrane targeting, and those capable of doing so (approx. 33%) appear, most often, to require both Phosphoinositide and non-Phosphoinositide determinants for their subcellular localization. Several recent studies have suggested that small GTPases such as ARF family proteins play a role in defining PH domain localization. Some others have described a signalling role for PH domains in regulating small GTPases, although Phosphoinositides may also play a role. These findings herald a change in our perspective of PH domain function, which will be significantly more diverse than previously supposed.
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the p21 activated protein kinase related kinase cla4 is a coincidence detector of signaling by cdc42 and phosphatidylinositol 4 phosphate
Journal of Biological Chemistry, 2004Co-Authors: Angela C Wild, Mark A. Lemmon, Kendall J BlumerAbstract:Abstract Signal transduction pathways that co-regulate a given biological process often are organized into networks by molecules that act as coincidence detectors. Phosphoinositides and the Rho-type GTPase Cdc42 regulate overlapping processes in all eukaryotic cells. However, the coincidence detectors that link these pathways into networks remain unknown. Here we show that the p21-activated protein kinase-related kinase Cla4 of yeast integrates signaling by Cdc42 and phosphatidylinositol 4-phosphate (PI4P). We found that the Cla4 pleckstrin homology (PH) domain binds in vitro to several Phosphoinositide species. To determine which Phosphoinositides regulate Cla4 in vivo, we analyzed phosphatidylinositol kinase mutants (stt4, mss4, and pik1). This indicated that the plasma membrane pool of PI4P, but not phosphatidylinositol 4,5-bisphosphate or the Golgi pool of PI4P, is required for localization of Cla4 to sites of polarized growth. A combination of the Cdc42-binding and PH domains of Cla4 was necessary and sufficient for localization to sites of polarized growth. Point mutations affecting either domain impaired the ability of Cla4 to regulate cell morphogenesis and the mitotic exit network (localization of Lte1). Therefore, Cla4 must retain the ability to bind both Cdc42 and Phosphoinositides, the hallmark of a coincidence detector. PI4P may recruit Cla4 to the plasma membrane where Cdc42 activates its kinase activity and refines its localization to cortical sites of polarized growth. In mammalian cells, the myotonic dystrophy-related Cdc42-binding kinase possesses p21-binding and PH domains, suggesting that this kinase may be a coincidence detector of signaling by Cdc42 and Phosphoinositides.
Gary J Fisher - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of thromboxane a2 receptor stimulated Phosphoinositide hydrolysis in isolated platelet membranes involvement of Phosphoinositide specific phospholipase c β and gtp binding protein gq
Biochemical Journal, 1993Co-Authors: Joseph J Baldassare, Alan Tarver, Patricia A Henderson, W M Mackin, B Sahagan, Gary J FisherAbstract:Activation of human platelets by the arachidonic acid metabolite thromboxane A2 and the thromboxane A2 mimic U46619 is mediated through Phosphoinositide-specific phospholipase C-catalysed hydrolysis of Phosphoinositides. We have established conditions to reconstitute U46619-stimulated Phosphoinositide breakdown by addition of guanine nucleotides and soluble platelet phospholipase C activities to isolated 32P-labelled membranes. Receptor-activated Phosphoinositide hydrolysis was observed in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 microM and 500 nM respectively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet cytosol and their ability to reconstitute GTP[S]-dependent and GTP/U46619-dependent Phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not phospholipase C-gamma 1 or -gamma 2, catalysed Phosphoinositide breakdown in the presence of GTP[S]. In contrast, only phospholipase C-beta was able to reconstitute GTP-dependent U46619-induced hydrolysis. The participation of GTP-regulatory proteins in the reconstitution of GTP[S]- and GTP/U46619-induced Phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeric GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reconstitution of Phosphoinositide hydrolysis with phospholipase C-beta. In contrast GTP[S]-stimulated hydrolysis by phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the functional specificity of GTP-binding proteins and phospholipase C isoenzymes in mediating agonist-induced Phosphoinositide hydrolysis in human platelets.
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reconstitution of thromboxane a2 receptor stimulated Phosphoinositide hydrolysis in isolated platelet membranes involvement of Phosphoinositide specific phospholipase c beta and gtp binding protein gq
Biochemical Journal, 1993Co-Authors: Joseph J Baldassare, Alan Tarver, Patricia A Henderson, W M Mackin, B Sahagan, Gary J FisherAbstract:Activation of human platelets by the arachidonic acid metabolite thromboxane A2 and the thromboxane A2 mimic U46619 is mediated through Phosphoinositide-specific phospholipase C-catalysed hydrolysis of Phosphoinositides. We have established conditions to reconstitute U46619-stimulated Phosphoinositide breakdown by addition of guanine nucleotides and soluble platelet phospholipase C activities to isolated 32P-labelled membranes. Receptor-activated Phosphoinositide hydrolysis was observed in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 microM and 500 nM respectively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet cytosol and their ability to reconstitute GTP[S]-dependent and GTP/U46619-dependent Phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not phospholipase C-gamma 1 or -gamma 2, catalysed Phosphoinositide breakdown in the presence of GTP[S]. In contrast, only phospholipase C-beta was able to reconstitute GTP-dependent U46619-induced hydrolysis. The participation of GTP-regulatory proteins in the reconstitution of GTP[S]- and GTP/U46619-induced Phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeric GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reconstitution of Phosphoinositide hydrolysis with phospholipase C-beta. In contrast GTP[S]-stimulated hydrolysis by phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the functional specificity of GTP-binding proteins and phospholipase C isoenzymes in mediating agonist-induced Phosphoinositide hydrolysis in human platelets.
Kathryn M Ferguson - One of the best experts on this subject based on the ideXlab platform.
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Pleckstrin homology domains and the cytoskeleton
FEBS letters, 2001Co-Authors: Mark A. Lemmon, Kathryn M Ferguson, Charles S. AbramsAbstract:Pleckstrin homology (PH) domains are 100–120 amino acid protein modules best known for their ability to bind Phosphoinositides. All possess an identical core β-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.
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signal dependent membrane targeting by pleckstrin homology ph domains
Biochemical Journal, 2000Co-Authors: Mark A. Lemmon, Kathryn M FergusonAbstract:Pleckstrin homology (PH) domains are small protein modules of around 120 amino acids found in many proteins involved in cell signalling, cytoskeletal rearrangement and other processes. Although several different protein ligands have been proposed for PH domains, their only clearly demonstrated physiological function to date is to bind membrane Phosphoinositides. The PH domain from phospholipase C-δ 1 binds specifically to PtdIns(4,5) P 2 and its headgroup, and has become a valuable tool for studying cellular PtdIns(4,5) P 2 functions. More recent developments have demonstrated that a subset of PH domains recognizes the products of agonist-stimulated Phosphoinositide 3-kinases. Fusion of these PH domains to green fluorescent protein has allowed dramatic demonstrations of their independent ability to drive signal-dependent recruitment of their host proteins to the plasma membrane. We discuss the structural basis for this 3-phosphoinoistide recognition and the role that it plays in cellular signalling. PH domains that bind specifically to Phosphoinositides comprise only a minority (perhaps 15%) of those known, raising questions as to the physiological role of the remaining 85% of PH domains. Most (if not all) PH domains bind weakly and non-specifically to Phosphoinositides. Studies of dynamin-1 have indicated that oligomerization of its PH domain may be important in driving membrane association. We discuss the possibility that membrane targeting by PH domains with low affinity for Phosphoinositides could be driven by alteration of their oligomeric state and thus the avidity of their membrane binding.
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Signal-dependent membrane targeting by pleckstrin homology (PH) domains
Biochemical Journal, 2000Co-Authors: Mark A. Lemmon, Kathryn M FergusonAbstract:Pleckstrin homology (PH) domains are small protein modules of around 120 amino acids found in many proteins involved in cell signalling, cytoskeletal rearrangement and other processes. Although several different protein ligands have been proposed for PH domains, their only clearly demonstrated physiological function to date is to bind membrane Phosphoinositides. The PH domain from phospholipase C-delta(1) binds specifically to PtdIns(4,5)P(2) and its headgroup, and has become a valuable tool for studying cellular PtdIns(4,5)P(2) functions. More recent developments have demonstrated that a subset of PH domains recognizes the products of agonist-stimulated Phosphoinositide 3-kinases. Fusion of these PH domains to green fluorescent protein has allowed dramatic demonstrations of their independent ability to drive signal-dependent recruitment of their host proteins to the plasma membrane. We discuss the structural basis for this 3-phosphoinoistide recognition and the role that it plays in cellular signalling. PH domains that bind specifically to Phosphoinositides comprise only a minority (perhaps 15%) of those known, raising questions as to the physiological role of the remaining 85% of PH domains. Most (if not all) PH domains bind weakly and non-specifically to Phosphoinositides. Studies of dynamin-1 have indicated that oligomerization of its PH domain may be important in driving membrane association. We discuss the possibility that membrane targeting by PH domains with low affinity for Phosphoinositides could be driven by alteration of their oligomeric state and thus the avidity of their membrane binding.
Michael D. Waterfield - One of the best experts on this subject based on the ideXlab platform.
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synthesis and function of 3 phosphorylated inositol lipids
Annual Review of Biochemistry, 2001Co-Authors: Bart Vanhaesebroeck, Sally J. Leevers, Paul C Driscoll, Rudiger Woscholski, Peter J Parker, Khatereh Ahmadi, John F Timms, Roy Katso, Michael D. WaterfieldAbstract:The 3-phosphorylated inositol lipids fulfill roles as second messengers by interacting with the lipid binding domains of a variety of cellular proteins. Such interactions can affect the subcellular localization and aggregation of target proteins, and through allosteric effects, their activity. Generation of 3-Phosphoinositides has been documented to influence diverse cellular pathways and hence alter a spectrum of fundamental cellular activities. This review is focused on the 3-Phosphoinositide lipids, the synthesis of which is acutely triggered by extracellular stimuli, the enzymes responsible for their synthesis and metabolism, and their cell biological roles. Much knowledge has recently been gained through structural insights into the lipid kinases, their interaction with inhibitors, and the way their 3-Phosphoinositide products interact with protein targets. This field is now moving toward a genetic dissection of 3-Phosphoinositide action in a variety of model organisms. Such approaches will reveal the true role of the 3-Phosphoinositides at the organismal level in health and disease.
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Signalling through Phosphoinositide 3-kinases: the lipids take centre stage.
Current opinion in cell biology, 1999Co-Authors: Sally J. Leevers, Bart Vanhaesebroeck, Michael D. WaterfieldAbstract:Phosphoinositide 3-kinases (PI3Ks) phosphorylate inositol lipids at the 3' position of the inositol ring to generate the 3-Phosphoinositides PI(3)P, PI(3,4) P2 and PI(3,4,5) P3. Recent research has shown that one way in which these lipids function in signal transduction and membrane trafficking is by interacting with 3-Phosphoinositide-binding modules in a broad variety of proteins. Specifically, certain FYVE domains bind PI(3)P whereas certain pleckstrin homology domains bind PI(3,4) P2 and/or PI(3,4,5) P3. Also in 1998, PTEN - a major tumour suppressor in human cancer - was also shown to antagonise PI3K signalling by removing the 3-phosphate from 3-Phosphoinositides.
Philip N Tsichlis - One of the best experts on this subject based on the ideXlab platform.
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akt pkb and other d3 Phosphoinositide regulated kinases kinase activation by Phosphoinositide dependent phosphorylation
Annual Review of Biochemistry, 1999Co-Authors: Tung O Chan, Susan E Rittenhouse, Philip N TsichlisAbstract:The protein kinase Akt/PKB is activated via a multistep process by a variety of signals. In the early steps of this process, PI-3 kinase-generated D3-phosphorylated Phosphoinositides bind the Akt PH domain and induce the translocation of the kinase to the plasma membrane where it co-localizes with Phosphoinositide-dependent kinase-1. By binding to the PH domains of both Akt and Phosphoinositide-dependent kinase-1, D3-phosphorylated Phosphoinositides appear to also induce conformational changes that permit Phosphoinositide-dependent kinase-1 to phosphorylate the activation loop of Akt. The paradigm of Akt activation via Phosphoinositide-dependent phosphorylation provided a framework for research into the mechanism of activation of other members of the AGC kinase group (p70S6K, PKC, and PKA) and members of the Tec tyrosine kinase family (TecI, TecII, Btk/Atk, Itk/Tsk/Emt, Txk/Rlk, and Bm/Etk). The result was the discovery that these kinases and Akt are activated by overlapping pathways. In this review, we present our current understanding of the regulation and function of the Akt kinase and we discuss the common and unique features of the activation processes of Akt and the AGC and Tec kinase families. In addition, we present an overview of the biosynthesis of Phosphoinositides that contribute to the regulation of these kinases.
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akt pkb and other d3 Phosphoinositide regulated kinases kinase activation by Phosphoinositide dependent phosphorylation
Annual Review of Biochemistry, 1999Co-Authors: Tung O Chan, Susan E Rittenhouse, Philip N TsichlisAbstract:▪ Abstract The protein kinase Akt/PKB is activated via a multistep process by a variety of signals. In the early steps of this process, PI-3 kinase-generated D3-phosphorylated Phosphoinositides bind the Akt PH domain and induce the translocation of the kinase to the plasma membrane where it co-localizes with Phosphoinositide-dependent kinase-1. By binding to the PH domains of both Akt and Phosphoinositide-dependent kinase-1, D3-phosphorylated Phosphoinositides appear to also induce conformational changes that permit Phosphoinositide-dependent kinase-1 to phosphorylate the activation loop of Akt. The paradigm of Akt activation via Phosphoinositide-dependent phosphorylation provided a framework for research into the mechanism of activation of other members of the AGC kinase group (p70S6K, PKC, and PKA) and members of the Tec tyrosine kinase family (TecI, TecII, Btk/Atk, Itk/Tsk/Emt, Txk/Rlk, and Bm/Etk). The result was the discovery that these kinases and Akt are activated by overlapping pathways. In this r...
