András Balla

A Pharmacological Map of the PI3-K Family Defines a Role for p110α in Insulin Signaling

Phosphoinositide 3-kinases (PI3-Ks) are an important emerging class of drug targets, but the unique roles of PI3-K isoforms remain poorly defined. We describe here an approach to pharmacologically interrogate the PI3-K family. A chemically diverse panel of PI3-K inhibitors was synthesized, and their target selectivity was biochemically enumerated, revealing cryptic homologies across targets and chemotypes. Crystal structures of three inhibitors bound to p110gamma identify a conformationally mobile region that is uniquely exploited by selective compounds. This chemical array was then used to define the PI3-K isoforms required for insulin signaling. We find that p110alpha is the primary insulin-responsive PI3-K in cultured cells, whereas p110beta is dispensable but sets a phenotypic threshold for p110alpha activity. Compounds targeting p110alpha block the acute effects of insulin treatment in vivo, whereas a p110beta inhibitor has no effect. These results illustrate systematic target validation using a matrix of inhibitors that span a protein family.

Dual Regulation of TRPV1 by Phosphoinositides

The membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PIP2] regulates many ion channels. There are conflicting reports on the effect of PtdIns(4,5)P2 on transient receptor potential vanilloid 1 (TRPV1) channels. We show that in excised patches PtdIns(4,5)P2 and other phosphoinositides activate and the PIP2 scavenger poly-Lys inhibits TRPV1. TRPV1 currents undergo desensitization on exposure to high concentrations of capsaicin in the presence of extracellular Ca2+. We show that in the presence of extracellular Ca2+, capsaicin activates phospholipase C (PLC) in TRPV1-expressing cells, inducing depletion of both PtdIns(4,5)P2 and its precursor PtdIns(4)P (PIP). The PLC inhibitor U73122 and dialysis of PtdIns(4,5)P2 or PtdIns(4)P through the patch pipette inhibited desensitization of TRPV1, indicating that Ca2+-induced activation of PLC contributes to desensitization of TRPV1 by depletion of PtdIns(4,5)P2 and PtdIns(4)P. Selective conversion of PtdIns(4,5)P2 to PtdIns(4)P by a rapamycin-inducible PIP2 5-phosphatase did not inhibit TRPV1 at high capsaicin concentrations, suggesting a significant role for PtdIns(4)P in maintaining channel activity. Currents induced by low concentrations of capsaicin and moderate heat, however, were potentiated by conversion of PtdIns(4,5)P2 to PtdIns(4)P. Increasing PtdIns(4,5)P2 levels by coexpressing phosphatidylinositol-4-phosphate 5-kinase inhibited TRPV1 at low but not at saturating capsaicin concentrations. These data show that at low capsaicin concentrations and other moderate stimuli, PtdIns(4,5)P2 partially inhibits TRPV1 in a cellular context, but this effect is likely to be indirect, because it is not detectable in excised patches. We conclude that phosphoinositides have both inhibitory and activating effects on TRPV1, resulting in complex and distinct regulation at various stimulation levels.

Phosphatidylinositol 4-Kinase IIIβ Regulates the Transport of Ceramide between the Endoplasmic Reticulum and Golgi

The recently identified ceramide transfer protein, CERT, is responsible for the bulk of ceramide transport from the endoplasmic reticulum (ER) to the Golgi. CERT has a C-terminal START domain for ceramide binding and an N-terminal pleck-strin homology domain that binds phosphatidylinositol 4-phosphate suggesting that phosphatidylinositol (PI) 4-kinases are involved in the regulation of CERT-mediated ceramide transport. In the present study fluorescent analogues were used to follow the ER to Golgi transport of ceramide to determine which of the four mammalian PI 4-kinases are involved in this process. Overexpression of pleckstrin homology domains that bind phosphatidylinositol 4-phosphate strongly inhibited the transport of C5-BODIPY-ceramide to the Golgi. A newly identified PI 3-kinase inhibitor, PIK93 that selectively inhibits the type III PI 4-kinase β enzyme, and small interfering RNA-mediated down-regulation of the individual PI 4-kinase enzymes, revealed that PI 4-kinase β has a dominant role in ceramide transport between the ER and Golgi. Accordingly, inhibition of PI 4-kinase III β either by wortmannin or PIK93 inhibited the conversion of [3H]serine-labeled endogenous ceramide to sphingomyelin. Therefore, PI 4-kinase β is a key enzyme in the control of spingomyelin synthesis by controlling the flow of ceramide from the ER to the Golgi compartment.

Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate; a comparative study

BackgroundPhosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)P2 also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)P2 levels in living cells is critical to understanding the biology of PtdIns(4,5)P2. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLCδ1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)P2 imaging tools.ResultsAll of the yeast PH domains that have been previously shown to bind PtdIns(4,5)P2 showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5)P2. None of these domains showed any advantage over the PLCδ1PH-GFP reporter and were compromised either in their expression levels, nuclear localization or by causing peculiar membrane structures. In contrast, the Tubby domain showed high membrane localization consistent with PtdIns(4,5)P2 binding and displayed no affinity for the soluble headgroup, Ins(1,4,5)P3. Detailed comparison of the Tubby and PLCδ1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)P2 and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation.ConclusionThese results showed that both the PLCδ1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)P2 changes in the plasma membrane, with distinct advantages and disadvantages. While the PLCδ1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P3 binding may compromise its accuracy to measure PtdIns(4,5)P2changes. The Tubby domain is more accurate to report on PtdIns(4,5)P2 but its higher affinity and lower sensitivity may limit its utility when phospholipase C activation is only moderate. These studies also demonstrated that similar changes in PtdIns(4,5)P2 levels in the plasma membrane can differentially regulate multiple effectors if they display different affinities to PtdIns(4,5)P2.

A membrane capture assay for lipid kinase activity

Phosphoinositide kinases such as PI3-kinase synthesize lipid second messengers that control diverse cellular processes. Recently, these enzymes have emerged as an important class of drug targets, and there is significant interest in discovering new lipid kinase inhibitors. We describe here a procedure for the high-throughput determination of lipid kinase inhibitor IC50 values. This assay exploits the fact that phosphoinositides, but not nucleotides such as ATP, bind irreversibly to nitrocellulose membranes. As a result, the radiolabeled lipids from a kinase assay can be isolated by spotting the crude reaction on a nitrocellulose membrane and then washing. We show that diverse phosphoinositide kinases can be assayed using this approach and outline how to perform the assay in 96-well plates. We also describe a MATLAB script that automates the data analysis. The complete procedure requires 3–4 h.

Acute depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate impairs specific steps in endocytosis of the G-protein-coupled receptor

Receptor endocytosis plays an important role in regulating the responsiveness of cells to specific ligands. Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been shown to be crucial for endocytosis of some cell surface receptors, such as EGF and transferrin receptors, but its role in G-protein-coupled receptor internalization has not been investigated. By using luciferase-labeled type 1 angiotensin II (AT1R), type 2C serotonin (5HT2CR) or β2 adrenergic (β2AR) receptors and fluorescently tagged proteins (β-arrestin-2, plasma-membrane-targeted Venus, Rab5) we were able to follow the sequence of molecular interactions along the endocytic route of the receptors in HEK293 cells using the highly sensitive method of bioluminescence resonance energy transfer and confocal microscopy. To study the role of plasma membrane PtdIns(4,5)P2 in receptor endocytosis, we used our previously developed rapamycin-inducible heterodimerization system, in which the recruitment of a 5-phosphatase domain to the plasma membrane degrades PtdIns(4,5)P2. Here we show that ligand-induced interaction of AT1, 5HT2C and β2A receptors with β-arrestin-2 was unaffected by PtdIns(4,5)P2 depletion. However, trafficking of the receptors to Rab5-positive early endosomes was completely abolished in the absence of PtdIns(4,5)P2. Remarkably, removal of the receptors from the plasma membrane was reduced but not eliminated after PtdIns(4,5)P2 depletion. Under these conditions, stimulated AT1 receptors clustered along the plasma membrane, but did not enter the cells. Our data suggest that in the absence of PtdIns(4,5)P2, these receptors move into clathrin-coated membrane structures, but these are not cleaved efficiently and hence cannot reach the early endosomal compartment.

Targeted expression of the inositol 1,4,5-triphosphate receptor (IP3R) ligand-binding domain releases Ca2+ via endogenous IP3R channels.

Virtually all functions of a cell are influenced by cytoplasmic [Ca2+] increases. Inositol 1,4,5-trisphosphate receptor (IP3R) channels, located in the endoplasmic reticulum (ER), release Ca2+ in response to binding of the second messenger, IP3.IP3Rs thus are part of the information chain interpreting external signals and transforming them into cytoplasmic Ca2+ transients. IP3Rs function as tetramers, each unit comprising an N-terminal ligand-binding domain (LBD) and a C-terminal channel domain linked by a long regulatory region. It is not yet understood how the binding of IP3 to the LBD regulates the gating properties of the channel. Here, we use the expression of IP3 binding protein domains tethered to the surface of the endoplasmic reticulum (ER) to show that the all-helical domain of the IP3R LBD is capable of depleting the ER Ca2+ pools by opening the endogenous IP3Rs, even without IP3 binding. This effect requires the domain to be within 50 Å of the ER membrane and is impaired by the presence of the N-terminal inhibitory segment on the LBD. These findings raise the possibility that the helical domain of the LBD functions as an effector module possibly interacting with the channel domain, thereby being part of the gating mechanisms by which the IP3-induced conformational change within the LBD regulates Ca2+ release.

Design of drug-resistant alleles of type-III phosphatidylinositol 4-kinases using mutagenesis and molecular modeling.

Molecular modeling and site directed mutagenesis were used to analyze the structural features determining the unique inhibitor sensitivities of type-III phosphatidylinositol 4-kinase enzymes (PI4Ks). Mutation of a highly conserved Tyr residue that provides the bottom of the hydrophobic pocket for ATP yielded a PI4KIIIbeta enzyme that showed greatly reduced wortmannin sensitivity and was catalytically still active. Similar substitutions were not tolerated in the type-IIIalpha enzyme rendering it catalytically inactive. Two conserved Cys residues located in the active site of PI4KIIIalpha were found responsible for the high sensitivity of this enzyme to the oxidizing agent, phenylarsine oxide. Mutation of one of these Cys residues reduced the phenylarsine oxide sensitivity of the enzyme to the same level observed with the PI4KIIIbeta protein. In search of inhibitors that would discriminate between the closely related PI4KIIIalpha and -IIIbeta enzymes, the PI3Kgamma inhibitor, PIK93, was found to inhibit PI4KIIIbeta with significantly greater potency than PI4KIIIalpha. These studies should aid development of subtype-specific inhibitors of type-III PI4Ks and help to better understand the significance of localized PtdIns4P production by the various PI4Ks isoforms in specific cellular compartments.

Nucleolar localization of phosphatidylinositol 4-kinase PI4K230 in various mammalian cells

Previous immunohistochemical investigations could not detect PI4K230, an isoform of mammalian phosphatidylinositol 4‐kinases (also called type III α), in the nucleus and nucleolus of cells in spite of its predicted nuclear localization signals.

Molecular targets for pharmacological cytoprotection.

Cell death is common to many pathological conditions. In the past two decades, research into the mechanism of cell death has characterized the cardinal features of apoptosis and necrosis, the two distinct forms of cell death. Studies using in vivo disease models have provided evidence that apoptosis is induced by an array of pathological stimuli. Thus, molecular components of the machinery of apoptosis are potential pharmacological targets. The mechanism of apoptosis can be dissected into: (i) the initiation and signaling phase, (ii) the signal amplification phase, and (iii) the execution phase. Reflecting on the diversity of apoptotic stimuli, the initiation and signaling phase utilizes a variety of molecules: free radicals, ions, plasma membrane receptors, members of the signaling kinase cascades, transcription factors, and signaling caspases. In most of the apoptotic scenarios, impairment of mitochondrial function is an early event. Dysfunctioning mitochondria release more free radicals and hydrolytic enzymes (proteases and nucleases), amplifying the primary death signal. In the final phase of apoptosis, executioner caspases are activated. Substrates of the executioner caspases include nucleases, members of the cellular repair apparatus, and cytoskeletal proteins. Partial proteolysis of these substrates leads to distinctive morphological and biochemical changes, the hallmarks of apoptosis. The first steps toward pharmacological utilization of specific modifiers of apoptosis have been promising. However, since the potential molecular targets of cytoprotective therapy play important roles in the maintenance of cellular homeostasis, specificity (diseased versus healthy tissue) of pharmacological modulation is the key to success.