PIP2 Binding Site Research Articles

Structural Basis of TRPV4 N Terminus Interaction with Syndapin/PACSIN1-3 and PIP2

Transient receptor potential (TRP) channels are polymodally regulated ion channels. TRPV4 (vanilloid 4) is sensitized by PIP2 and desensitized by Syndapin3/PACSIN3, which bind to the structurally uncharacterized TRPV4N terminus. We determined the nuclear magnetic resonance structure of the Syndapin3/PACSIN3 SH3 domain in complex with the TRPV4 N-terminal proline-rich region (PRR), which binds as a class I polyproline II (PPII) helix. This PPII conformation is broken by a conserved proline in a cis conformation. Beyond the PPII, we find that the proximal TRPV4N terminus is unstructured, a feature conserved across species thus explaining the difficulties in resolving it in previous structural studies. Syndapin/PACSIN SH3 domain binding leads to rigidification of both the PRR and the adjacent PIP2 binding site. We determined the affinities of the TRPV4N terminus for PACSIN1, 2, and 3 SH3 domains and PIP2 and deduce a hierarchical interaction network where Syndapin/PACSIN binding influences the PIP2 binding site but not vice versa.

Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels.

TREK/TRAAK channels are polymodal K+ channels that convert very diverse stimuli, including bioactive lipids, mechanical stretch and temperature, into electrical signals. The nature of the structural changes that regulate their activity remains an open question. Here, we show that a cytoplasmic domain (the proximal C-ter domain, pCt) exerts antagonistic effects in TREK1 and TRAAK. In basal conditions, pCt favors activity in TREK1 whereas it impairs TRAAK activity. Using the conformation-dependent binding of fluoxetine, we show that TREK1 and TRAAK conformations at rest are different, and under the influence of pCt. Finally, we show that depleting PIP2 in live cells has a more pronounced inhibitory effect on TREK1 than on TRAAK. This differential regulation of TREK1 and TRAAK is related to a previously unrecognized PIP2-binding site (R329, R330, and R331) present within TREK1 pCt, but not in TRAAK pCt. Collectively, these new data point out pCt as a major regulatory domain of these channels and suggest that the binding of PIP2 to the pCt of TREK1 results in the stabilization of the conductive conformation in basal conditions.

Abstract 682: The mechanism of PI3Kα activation by SH2 release

Abstract Phosphoinositide 3-kinase α (PI3Kα) is a ubiquitous kinase mediating signaling cascades important for cell survival, proliferation and protein synthesis. It phosphorylates PIP2 to PIP3, to which downstream Akt kinase binds to execute singling. PI3Kα dysfunction has been extensively implicated in cancers. The kinase activity of PI3Kα is controlled by the interactions between its catalytic p110 and regulatory p85 subunits. The interactions of the SH2 domains in the p85 subunit, particularly nSH2, with p110 are key factors in PI3Kα inhibition. Release of the SH2 domains from the p110 subunit, is promoted by factors including phosphotyrosine peptides (RTK), calmodulin (CaM), and oncogenic mutations, thereby activating PI3Kα. Our recent data show that both phosphorylated and unphosphorylated CaMs are capable of binding to the SH2 domains, nSH2 and cSH2, thus promoting the PI3K activation. Despite numerous seminal publications over the past two decades, the mechanism of PI3Kα activation is still a mystery. The large distance between the PIP2 binding sites and the ATP pocket in the crystal structure suggests that the solved PI3Kα structure represents the inactive conformation. What is the activated conformation of PI3Kα, and how PI3Kα switches from the inactive to the active conformation in response to stimulus are the significant mechanistic questions. We employ the large-scale molecular dynamics (MD) simulations to visualize the PI3Kα activation stimulated by the release of SH2 domains in the p85 subunit at the atomic resolution. Our trajectories show that release of the nSH2 domain initiates a “switch-like” conformational change of the iSH2 domain, extracting the ATP from its pocket to the PIP2 binding sites, eventually leading to an activated conformation. This dynamic process is initiated by the break of salt-bridges between iSH2 and helical domains induced by the nSH2 release, and further facilitated by the various residues in activation loop of p110 subunit including His-917, Lys-802 and Lys-776 and in iSH2 of p85 subunit including Arg-461 and Arg-465. The release of the cSH2 domain in PI3Kα has a minor effect. This work, which for the first time uncovers the mechanism of PI3Kα activation at atomic resolution, is expected to promote cancer drug design from a mechanistic dynamic standpoint view. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Citation Format: Mingzhen Zhang, Hyunbum Jang, Ruth Nussinov. The mechanism of PI3Kα activation by SH2 release [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 682.

Phosphatidylinositol (4, 5)‐bisphosphate coordinates functional interactions in the dopamine transporter to promote amphetamine behaviors

The psychostimulant amphetamine (AMPH) mainly mediates its pharmacological and behavioral effects by increasing extracellular dopamine (DA) availability. DA homeostasis is maintained by the dopamine transporter (DAT), a presynaptic membrane protein that mediates the high‐affinity reuptake of released DA from the synaptic cleft. We have previously demonstrated that AMPH induces N‐term phosphorylation of the DAT, which leads to transport‐mediated efflux of DA. Furthermore, we have shown that phosphatidylinositol (4, 5)‐bisphosphate (PIP2) directly interacts with the DAT and facilitates AMPH‐induced DA efflux, but is not required for DA uptake. Specially, PIP2 binds DAT through electrostatic interactions with positively charged DAT N‐terminal residues. Disrupting the interactions between DAT and PIP2 or depleting PIP2 diminishes reverse transport (efflux) of DA. Previous studies on the human serotonin transporter show that non‐N‐terminal PIP2 binding sites (within the fourth intracellular loop) are essential for AMPH‐induced reverse transport of serotonin, but not substrate uptake. Together, these data led us to hypothesize that PIP2 coordinates functional interactions between the N‐terminus and the fourth intracellular loop to promote reverse transport of DA. Here, we demonstrate that depletion of PIP2 after DAT N‐terminus phosphorylation does not affect AMPH‐induced DA efflux, suggesting that N‐terminus phosphorylation is the limiting step of reverse transport of DA. Notably, we show that residue R443, which resides in the fourth intracellular loop, also modulates AMPH‐induced DA efflux. Thus, we focus on the role of the interaction between the DAT N‐terminus and R443 in mediating the reverse transport of DA. Furthermore, we translate our molecular discoveries in vivo by using a coordinated genetic and pharmacological approach to study AMPH‐induced behaviors in Drosophila melanogasterSupport or Funding Information1F31MH114316 (JIA), AHA Predoctoral Award (AS), DA035263(AG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A PIP2 Binding Site on a Human TRP Channel: Simulation Studies of PKD2

Polycystin-2 (PKD2) is a member of the transient receptor potential (TRP) superfamily of non-selective cation channels. Mutations in this channel and homologous channels lead to a common genetic disease, autosomal dominant polycystic kidney disease (ADPKD). Phosphatidylinositol (4,5) bisphosphate (PIP2) suppresses epidermal growth factor induced PKD2 activation, and hydrolysis of PIP2 can release this inhibition1, suggesting PIP2 may be a modulator of PKD2 channels. Using multi-scale molecular dynamics simulations based on a recent electron cryo-microscopy (cryo-EM) structure of PKD2 (PDB ID: 5K47) we revealed a potential PIP2 binding site between S3, S4 and S5 transmembrane helices. This binding site is close to the vanilloid/lipid-binding site observed in the TRPV1 channel (PDB ID: 5RIZ). Free energy profiles for the interaction of PIP2, phosphatidylserine (PS) and of phosphatidylcholine (PC) with PKD2 support the existence of a discrete binding site, which has a strong selectivity for PIP2 binding. Close examination of the protein-lipid interaction at the lowest-energy binding distance showed that the head group of PIP2 is coordinated by a cluster of basic residues, which are all highly conserved. This suggests that the binding site may play a key role in lipid-mediated modulation of PKD2 channel activity. (1) Ma, R., Li, W., Rundle, D., Kong, J., Akbarali, H. I., and Tsiokas, L. (2005) Mol. Cell. Biol.25, 8285–8298.

PIP2 epigenetically represses rRNA genes transcription interacting with PHF8

Phosphoinositides are present in the plasma membrane, cytoplasm and inside the cell nucleus. Here we identify phosphatidylinositol-4,5-bisphosphate (PIP2) as a regulator of rRNA genes transcription at the epigenetic level. We show that PIP2 directly interacts with histone lysine demethylase PHF8 (PHD finger protein 8) and represses demethylation of H3K9me2 through this interaction. We identify the C-terminal K/R-rich motif as PIP2-binding site within PHF8, and address the function of this PIP2-PHF8 complex. PIP2-binding mutant of PHF8 has increased the activity of rDNA promoter (20%) and expression of pre-rRNA genes (47S-100%; 45S-66%). Furthermore, trypsin digestion reveals a potential conformational change of PHF8 upon PIP2 binding. These observations identify the function of nuclear PIP2, and suggest that PIP2 contributes to the fine-tuning of rDNA transcription.

Metal ions and phosphatidylinositol 4,5-bisphosphate as interacting effectors of α-type plant phospholipase D

Plant phospholipases D (PLD) are typically characterized by a C2 domain with at least two Ca2+ binding sites. In vitro, the predominantly expressed α-type PLDs need 20–100 mM CaCl2 for optimum activity, whereas the essential activator of β- or γ-type PLDs, phosphatidylinositol 4,5-bisphosphate (PIP2), plays a secondary role. In the present paper, we have studied the interplay between PIP2 and metal ion activation of the well-known α-type PLD from cabbage (PLDα). With mixed micelles containing phosphatidyl-p-nitrophenol as substrate, PIP2-concentrations in the nanomolar range are able to activate the enzyme in addition to the essential Ca2+ activation. Mg2+ ions are able to replace Ca2+ ions but they do not activate PLDα. Rather, they abolish the activation of the enzyme by Ca2+ ions in the absence, but not in the presence, of PIP2. The presence of PIP2 causes a shift in the pH optimum of PLDα activity to the acidic range. Employing fluorescence measurements and replacing Ca2+ by Tb3+ ions, confirmed the presence of two metal ion-binding sites, in which the one of lower affinity proved crucial for PLD activation.Moreover, we have generated a homology model of the C2 domain of this enzyme, which was used for Molecular Dynamics (MD) simulations and docking studies. As is common for C2 domains, it shows two antiparallel β-sheets consisting of four β-strands each and loop regions that harbor two Ca2+ binding sites. Based on the findings of the MD simulation, one of the bound Ca2+ ions is coordinated by five amino acid residues. The second Ca2+ ion induces a loop movement upon its binding to three amino acid residues. Docking studies with PIP2 reveal, in addition to the previously postulated PIP2-binding site in the middle of the β-sheet structure, another PIP2-binding site near the two Ca2+ ions, which is in accordance with the experimental interplay of PIP2, Ca2+ and Mg2+ ions.

Phospholipid binding to the FAK catalytic domain impacts function

Focal adhesion kinase is an essential nonreceptor tyrosine kinase that plays an important role in development, in homeostasis and in the progression of human disease. Multiple stimuli activate FAK, which requires a change in structure from an autoinhibited to activated conformation. In the autoinhibited conformation the FERM domain associates with the catalytic domain of FAK and PI(4,5)P2 binding to the FERM domain plays a role in the release of autoinhibition, activating the enzyme. An in silico model of FAK/PI(4,5)P2 interaction suggests that residues on the catalytic domain interact with PI(4,5)P2, in addition to the known FERM domain PI(4,5)P2 binding site. This study was undertaken to test the significance of this in silico observation. Mutations designed to disrupt the putative PI(4,5)P2 binding site were engineered into FAK. These mutants exhibited defects in phosphorylation and failed to completely rescue the phenotype associated with fak -/- phenotype fibroblasts demonstrating the importance of these residues in FAK function. The catalytic domain of FAK exhibited PI(4,5)P2 binding in vitro and binding activity was lost upon mutation of putative PI(4,5)P2 binding site basic residues. However, binding was not selective for PI(4,5)P2, and the catalytic domain bound to several phosphatidylinositol phosphorylation variants. The mutant exhibiting the most severe biological defect was defective for phosphatidylinositol phosphate binding, supporting the model that catalytic domain phospholipid binding is important for biochemical and biological function.

CP1 Opens IKs Channels by Substituting PIP2

The voltage-gated potassium channel KCNQ1 co-assembles with the auxiliary subunit KCNE1 to form the IKs channel in the heart. The IKs current, a slow delayed rectifier K+ current, plays a key role in repolarization of cardiac action potentials. Mutations in the KCNQ1 gene are associated with long QT (LQT) syndrome, atrial fibrillation and short QT syndrome, which lead to cardiac arrhythmias. It was shown that KCNQ1 and IKs channels require PIP2 (Phosphatidylinositol 4,5-bisphosphate) for activation. This lipid acts as a cofactor of the channel for the movements of the voltage sensor domain to trigger the pore gate domain to open. Using the previously identified PIP2 binding site in a homology structural model of KCNQ1 as the target, an in-silico screening of chemical compounds in the Available Chemical Database (ACD, Molecular Design, Ltd.) and a subsequent experminetal testing on the IKs channels expressed in Xenopus oocytes identified CP1 as a novel IKs opener. CP1 increases IKs currents by two-fold and alters voltage dependence and kinetics of activation and deactivation of IKs channels. The potentiation effect of CP1 on IKs currents has an EC50 of 7.4 µM. Consistent with targeting the PIP2 binding site,CP1 rescues the IKs current with the coexpression of CiVSP, which depletes PIP2 and inhibits the currents. The KCNQ1/IKs specific blocker, Chromanol 293B inhibits the enhanced current due to CP1 application, suggesting that CP1 substitutes for PIP2in activating IKs channels. In preliminary experimetns with Guinea-pig ventricular myocytes, CP1 caused similar changes in chromanol sensitive IKs while also shortening action potential duration. CP1 as an opener of the IKs channels may provide a novel therapy to treat congential and acquired LQT syndromes.

Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating.

KATP channels are metabolic sensors that couple cell energetics to membrane excitability. In pancreatic β-cells, channels formed by SUR1 and Kir6.2 regulate insulin secretion and are the targets of antidiabetic sulfonylureas. Here, we used cryo-EM to elucidate structural basis of channel assembly and gating. The structure, determined in the presence of ATP and the sulfonylurea glibenclamide, at ~6 Å resolution reveals a closed Kir6.2 tetrameric core with four peripheral SUR1s each anchored to a Kir6.2 by its N-terminal transmembrane domain (TMD0). Intricate interactions between TMD0, the loop following TMD0, and Kir6.2 near the proposed PIP2 binding site, and where ATP density is observed, suggest SUR1 may contribute to ATP and PIP2 binding to enhance Kir6.2 sensitivity to both. The SUR1-ABC core is found in an unusual inward-facing conformation whereby the two nucleotide binding domains are misaligned along a two-fold symmetry axis, revealing a possible mechanism by which glibenclamide inhibits channel activity.

Differential lipid binding of vinculin isoforms promotes quasi-equivalent dimerization

The main cause of death globally remains debilitating heart conditions, such as dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), which are often due to mutations of specific components of adhesion complexes. Vinculin regulates these complexes and plays essential roles in intercalated discs that are necessary for muscle cell function and coordinated movement and in the development and function of the heart. Humans bearing familial or sporadic mutations in vinculin suffer from chronic, progressively debilitating DCM that ultimately leads to cardiac failure and death, whereas autosomal dominant mutations in vinculin can also provoke HCM, causing acute cardiac failure. The DCM/HCM-associated mutants of vinculin occur in the 68-residue insert unique to the muscle-specific, alternatively spliced isoform of vinculin, termed metavinculin (MV). Contrary to studies that suggested that phosphoinositol-4,5-bisphosphate (PIP2) only induces vinculin homodimers, which are asymmetric, we show that phospholipid binding results in a domain-swapped symmetric MV dimer via a quasi-equivalent interface compared with vinculin involving R975. Although one of the two PIP2 binding sites is preserved, the symmetric MV dimer that bridges two PIP2 molecules differs from the asymmetric vinculin dimer that bridges only one PIP2 Unlike vinculin, wild-type MV and the DCM/HCM-associated R975W mutant bind PIP2 in their inactive conformations, and R975W MV fails to dimerize. Mutating selective vinculin residues to their corresponding MV residues, or vice versa, switches the isoform's dimeric constellation and lipid binding site. Collectively, our data suggest that MV homodimerization modulates microfilament attachment at muscular adhesion sites and furthers our understanding of MV-mediated cardiac remodeling.

Improved Side Chain Dynamics in MARTINI Simulations of Protein-Lipid Interfaces.

Specific interactions of protein side chains and lipid membranes regulate the localization, orientation, and activity of many peripheral proteins. Here, we introduce a modification of the coarse-grained MARTINI protein model, called 'side chain fix' (scFix), that was necessary and sufficient to correctly sample the side chain dynamics of β-strands in several globular proteins. When compared to μs long atomistic simulations or previous experimental findings, scFix MARTINI simulations reproduced all key interactions between the well-studied PLC-δ1 pleckstrin homology domain and a phosphatidylinositol-4,5-bisphosphate (PIP2) containing lipid membrane. Moreover, the extended runtime and higher sampling speed enabled the systematic mapping of the protein's rolling motion at the membrane, the identification of short-lived and stable binding orientations, as well as the verification and prediction of already known and of novel transient PIP2 binding sites. scFix also showed promise to maintain proper side chain orientation in other secondary structural motifs of the α-spectrin SH3 domain, the B1 domain of protein G, and the villin headpiece. This suggests that scFix improves on the predictive power of MARTINI simulations regarding protein-lipid and protein-ligand interactions.

A Novel Site of Competitive PIP2 and Calmodulin Interaction to KCNQ1 C-Terminus Helix B is Crucial for IKs Channel Activity

KCNQ1 and KCNE1 co-assembly generates the IKS potassium current, which is crucial to the cardiac action potential repolarization. Mutations in their corresponding genes cause cardiac arrhythmias. In the KCNQ1 C-terminus (CT), proximal helices A and B form sites for calmodulin (CaM) binding, whereas distal coiled-coil helices C and D are necessary for subunit tetramerization. Studies identified basic residues in KCNQ1 at S2-S3 and S4-S5 intracellular linkers and proximal CT as PIP2 binding sites. Our recent crystallographic data showed that CaM embraces the two proximal anti-parallel helices B and A with its calcified N-lobe and apo C-lobe, respectively. Here we identified a novel site of competitive PIP2 and calmodulin interaction to helix B that is crucial for IKS channel activity. PIP2 competes with CaM binding to purified His-tagged KCNQ1 CT in the presence of Ca2+ only (IC50 = 39 µM). Conversely, recombinant WT Ca2+-CaM or CaM3,4 (IC50 = 1 µM) but neither WT apoCaM nor CaM1,2 compete with PIP2 binding to purified His-tagged KCNQ1 CT. Recombinant WT Ca2+-CaM or CaM3,4 but not WT apoCaM or CaM1,2 in the patch pipet significantly attenuate the decrease in IKS current resulting from PIP2 breakdown by Dr-VSP. Molecular docking, dynamic simulation and subsequent biochemical and electrophysiological validation experiments indicate that K526 and K527 in helix B form a novel and crucial site for competitive PIP2 and calmodulin interaction. Our data suggest that upon PIP2 breakdown (e.g. following GPCR-Gq signaling), PIP2 unbinds from helix B and allows the calcified CaM N-lobe to replace it, which guaranties the maintenance of the channel open state in front of stressful PIP2 depletion events.

Self-Organization of Actomyosin Networks Attached to Artificial Membranes

The shape and mechanical stability of cells are highly dependent on their cytoskeleton. Actin is one of the main proteins which contributes to this biological network. The mechanical movements of the cell rely on the interaction of F-actin with several other proteins. One of these proteins is myosin II, a molecular motor protein. By hydrolysing ATP myosin is able to walk along F-actin or to induce tension on these filaments.In our model system F-actin networks are attached to pore spanning lipid bilayers (PSLBs) via electrostatical interactions or the linker protein ezrin which mimics the biological situation. Ezrin has a PIP2 binding site located at the N-terminus and a F-actin binding site at the C-terminus and is responsible for the linkage of F-actin to PIP2 present in the PSLB. Several actin binding proteins and cross-linkers are introduced during the polymerization of actin filaments.Besides the visual self-organization the mechanical properties of different F-actin networks are examined. Atomic force microscopy is used to determine the lateral membrane tension of the PSLB dependent on different actomyosin networks. The viscoelastic properties of the network will be recorded by passive microrheology using the mean square displacement of a polymer beads Brownian movement.

Mechanism of Focal Adhesion Kinase Mechanosensing.

Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP2 binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP2 concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP2-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments.

Characterization of the part of N-terminal PIP2 binding site of the TRPM1 channel

Transient receptor potential melastatin-1 (TRPM1) is a calcium channel that is essential for the depolarization of photo-responsive retinal bipolar cells, but most of the physiological functions and cellular roles of this channel are still poorly understood. Most transient receptor potential (TRP) channels are typically regulated by intracellular proteins and other signaling molecules. Phosphatidylinositol-4,5 bisphosphate (PIP2), a minor phospholipid component of cell membranes, has previously been shown to directly bind TRP channels and to play a unique role in modulating receptor function. To characterize the binding of PIP2 as a potential regulator of TRPM1, we utilized biophysical methods and molecular modeling to study the interactions of PIP2 with an N-terminal fragment of TRPM1 (residues A451–N566). The basic N-terminal residue K464 of TRPM1 suggests that it is part of putative pleckstrin homology (PH) domain and is involved in the interactions with PIP2. This is the first report detailing the binding of PIP2 at the N-terminus of the TRPM1 receptor.

Abstract PR02: Targeting PI3K: The PIP2 binding site

Abstract Structural biology has yielded many significant insights into inhibitor binding and isoform selectivity, directing the design of many important clinical candidates. To date, structural studies have focused on the ATP-binding site and ATP-competitive inhibitors. We have recently determined the structure of PI3Kα; in complex with the soluble lipid substrate mimetic, diC4-PIP2. The substrate binds in a positively charged pocket, defined by the activation and P-loops of the kinase domain, and the iSH2 domain of p85. The positively charged residues responsible for binding PIP2 that are present on the activation loop of Class I PI3K are missing in Class II and III PI3K. This explains why these two classes have lower affinity for PIP2. The complex structure provides insights into the catalysis and regulation of PI3K. Key interactions between the activation loop and nSH2 domain may modulate active and inactive conformations of the enzyme upon the binding of phosphorylated receptor tyrosine kinases. In addition, a second lipid substrate was identified in the structure. It binds in a hydrophobic pocket between the Adaptor Binding Domain (ABD), kinase domain and iSH2 domain of p85. Fluorescence quenching studies confirm the ability of PI3K to bind an additional PIP2 molecule. This abstract is also being presented as Poster B17. Citation Format: Michelle Miller, Oleg Schmidt-Kittler, David M. Bolduc, Evan T. Brower, Daniele Chaves-Moreira, Marc Allaire, Kenneth W. Kinzler, Ian G. Jennings, Philip E. Thompson, Philip A. Cole, L. Mario Amzel, Bert Vogelstein, Sandra B. Gabelli. Targeting PI3K: The PIP2 binding site. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR02.

Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels

All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81-104; Fürst, O., Mondou, B., and D'Avanzo, N. (2014) Front. Physiol. 4, 404-404). Structural components of a PIP2-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP2 regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP2 in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP2 resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP2, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP2. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP2-binding site, both forms of PIP2 strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature's mutations conferred a high affinity activation of vertebrate Kir channels by PIP2, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification.

The Na+/H+ Exchanger-3 (NHE3) Activity Requires Ezrin Binding to Phosphoinositide and Its Phosphorylation.

Na+/H+ exchanger-3 (NHE3) plays an essential role in maintaining sodium and fluid homeostasis in the intestine and kidney epithelium. Thus, NHE3 is highly regulated and its function depends on binding to multiple regulatory proteins. Ezrin complexed with NHE3 affects its activity via not well-defined mechanisms. This study investigates mechanisms by which ezrin regulates NHE3 activity in epithelial Opossum Kidney cells. Ezrin is activated sequentially by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and phosphorylation of threonine 567. Expression of ezrin lacking PIP2 binding sites inhibited NHE3 activity (-40%) indicating that ezrin binding to PIP2 is required for preserving NHE3 activity. Expression of a phosphomimetic ezrin mutated at the PIP2 binding region was sufficient not only to reverse NHE3 activity to control levels but also to increase its activity (+80%) similar to that of the expression of ezrin carrying the phosphomimetic mutation alone. Calcineurin Homologous Protein-1 (CHP1) is part, with ezrin, of the NHE3 regulatory complex. CHP1-mediated activation of NHE3 activity was blocked by expression of an ezrin variant that could not be phosphorylated but not by an ezrin variant unable to bind PIP2. Thus, for NHE3 activity under baseline conditions not only ezrin phosphorylation, but also ezrin spatial-temporal targeting on the plasma membrane via PIP2 binding is required; however, phosphorylation of ezrin appears to overcome the control of NHE3 transport. CHP1 action on NHE3 activity is not contingent on ezrin binding to PIP2 but rather on ezrin phosphorylation. These findings are important in understanding the interrelation and dynamics of a CHP1-ezrin-NHE3 regulatory complex.

Allosteric Regulation of Focal Adhesion Kinase by PIP2 and ATP

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that regulates cell signaling, proliferation, migration, and development. A major mechanism of regulation of FAK activity is an intramolecular autoinhibitory interaction between two of its domains—the catalytic and FERM domains. Upon cell adhesion to the extracellular matrix, FAK is being translocated toward focal adhesion sites and activated. Interactions of FAK with phosphoinositide phosphatidylinsositol-4,5-bis-phosphate (PIP2) are required to activate FAK. However, the molecular mechanism of the activation remains poorly understood. Recent fluorescence resonance energy transfer experiments revealed a closure of the FERM-kinase interface upon ATP binding, which is reversed upon additional binding of PIP2. Here, we addressed the allosteric regulation of FAK by performing all-atom molecular-dynamics simulations of a FAK fragment containing the catalytic and FERM domains, and comparing the dynamics in the absence or presence of ATP and PIP2. As a major conformational change, we observe a closing and opening motion upon ATP and additional PIP2 binding, respectively, in good agreement with the fluorescence resonance energy transfer experiments. To reveal how the binding of the regulatory PIP2 to the FERM F2 lobe is transduced to the very distant F1/N-lobe interface, we employed force distribution analysis. We identified a network of mainly charged residue-residue interactions spanning from the PIP2 binding site to the distant interface between the kinase and FERM domains, comprising candidate residues for mutagenesis to validate the predicted mechanism of FAK activation.