PHOSPHATIDYLINOSITOL 4-KINASES AND CELL REGULATION
, Head, Section on Molecular Signal Transduction
Yeun Ju Kim, PhD, Postdoctoral Fellow
Marek Korzeniowski, PhD, Postdoctoral Fellow
Hui Ma, PhD, Postdoctoral Fellow
Zsofia Szentpetery, PhD, Postdoctoral Fellow
Balázs Tóth, PhD, Postdoctoral Fellow
Peter Várnai, MD, PhD, Contractor
Ilia Kobrinsky, Summer Student
Our group investigates signal transduction pathways that mediate the actions of hormones, growth factors, and neurotransmitters in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Phosphoinositides constitute a small fraction of the cellular phospholipids but play critical roles in the regulation of many (if not all) signaling protein complexes that assemble on the surface of cell membranes. Phosphoinositides regulate protein kinases and GTP-binding proteins as well as membrane transporters and ion channels, thereby controlling many cellular processes, including proliferation, apoptosis, metabolism, cell migration, and differentiation. We focus on the group of enzymes termed phosphatidylinositol 4-kinases (PI4Ks), which catalyze the first committed step in phosphoinositide synthesis. Current studies aim to (1) understand the function and regulation of several PI4Ks as they relate to the synthesis of hormone-sensitive phosphoinositide pools; (2) characterize the structural features that determine the catalytic specificity and inhibitor sensitivity of PI 3- and PI 4-kinases; (3) define the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology and other domains of selected regulatory proteins; (4) develop tools to analyze inositol lipid dynamics in live cells; and (5) determine the importance of lipid-protein interactions in the activation of cellular responses by G protein–coupled receptors and receptor tyrosine kinases.
Visualization and manipulation of plasma membrane–adjacent endoplasmic reticulum structures and their relationship to STIM1 and Orai1 movements
The functional importance of the fraction of the endoplasmic reticulum (ER) positioned close to the plasma membrane (PM) has long been recognized, especially in phospholipid synthesis and transfer in both metazoan cells and yeast. Upon depletion of ER calcium stores, STIM1, a recently described ER protein, rapidly translocates to such a PM-adjacent ER compartment; here, it activates the newly identified calcium channel Orai1, forming the molecular basis of the so-called store-operated calcium entry (SOCE) phenomenon. The importance of this calcium entry pathway is underscored by the fact that mutations in Orai1 have been linked to severe inborn human immunodeficiencies. Moreover, this route of calcium entry is pivotal to the calcium-regulated activation of regulatory T cells that is mediated by the NFAT transcription factors. Studying both the molecular mechanism of STIM1 translocation to the PM-adjacent ER compartment and the molecular definition of this compartment could aid in identifying novel molecular targets for immunosuppression.
We used a chemically inducible molecular bridge formation between the PM and ER membranes to highlight the PM-adjacent ER compartment and to show that the compartment is the site at which STIM1 and its calcium channel partner Orai1 form a productive interaction upon ER calcium store depletion. By changing the length of the linkers connecting the plasma and ER membranes, we showed that Orai1 requires a larger space than STIM1 between the two membranes. Our finding suggests that Orai1 is part of a larger macromolecular cluster with an estimated 11to 14 nm protrusion to the cytoplasm while the cytoplasmic domain of STIM1 fits in a space calculated to be less than 6 nm. We also showed that agonist-induced translocation of STIM1 is rapidly reversible and only partially affects STIM1 in the juxtanuclear ER compartment. Our studies are the first to detect juxtaposed areas between the ER and the PM in living cells and reveal novel details of STIM1-Orai1 interactions.
Várnai P, Tóth B, Tóth D, Hunyady L, Balla T. Visualization and manipulation of plasma membrane endoplasmic reticulum contact sites indicates the presence of additional molecular components within the STIM1-Orai1 complex. J Biol Chem 2007;282:29678-90.
The role of PI 4-kinase IIIalpha in zebrafish development
Previously, we identified four isoforms of the phosphatidylinositol 4-kinase enzymes (PI4Ks) that are all expressed in zebrafish and then characterized their expression patterns during embryonic development. Even though the enzymes catalyze the same biochemical reaction, their intracellular localization and, presumably, their regulation vary. Based on studies in yeast, we determined that the enzymes assume non-redundant functions. To identify developmental processes and signal transduction pathways in which specific PI4Ks play pivotal roles, we downregulated the expression of the enzymes by injecting morpholinos that target the splicing of exons encoding the catalytic domains of the individual enzymes.
We first investigated the role of PI4KIIIalpha. Downregulation of PI4KIIIalpha resulted in several developmental abnormalities, most severely affecting the zebrafish’s hindbrain and branchial arches. The changes were associated with highly elevated apoptotic activity and were partially mimicked by treatment of the embryos with the PI 3-kinase inhibitor LY294002. The most striking phenotype of PI4KIIIalpha downregulation was the lack of pectoral fin development. Downstream targets of the FGF8 signaling pathway, such as MKP3 (a MAP kinase phosphatase) and Sef, were strongly inhibited in the morphant embryos, especially in the branchial arches and pectoral fin buds, whereas genes mediating hedgehog (Hh) signaling were only marginally affected. In HEK293T cells, downregulation of PI4KIIIalpha by RNAi-mediated gene silencing inhibited constitutive Akt activation and potentiated the FGF- but not EGF-stimulated MAPK response. The data suggest that PI4KIIIalpha is critically important for supplying phosphoinositides in order for PI 3-kinases to activate anti-apoptotic pathways; in addition, PI4KIIIalpha’s downregulation changes the balance between the MAPK and PI3K signaling pathways in FGF signaling, causing a defect in pectoral fin development. To define the molecular details of how this PI4K isoform contributes to signaling from FGF receptors, we have undertaken further studies.
Balla A, Balla T. Phosphatidylinositol 4-kinases: old enzymes with emerging functions. Trends Cell Biol 2006;16:351-61.
Role of plasma membrane phosphatidylinositol 4,5-bisphosphate in regulating ion channels and clathrin-mediated receptor endocytosis
Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is the major phosphoinositide species associated with numerous molecular events critical for cellular signaling in mammalian cells. PtdIns(4,5)P2 is hydrolyzed by phospholipase C enzymes to generate diacylglycerol and Ins(1,4,5)P3, two pivotal second messengers. PtdIns(4,5)P2 is also converted by Class I PI 3-kinases to PtdIns(3,4,5)P3, another important membrane-bound messenger molecule. PtdIns(4,5)P2 directly interacts with several ion channels, transporters, and actin-binding proteins and regulates enzymes such as PLC and PLD. Several molecules that are part of the receptor internalization machinery have also been shown to contain inositide-binding domains, but the lipid species that regulates them in the cell has not been firmly established. It is a major challenge to understand how a single type of molecule is able to regulate so many processes simultaneously and perhaps independently within the PM.
Last year, we reported the development of a new approach for quickly regulating membrane PtdIns(4,5)P2 levels through application of a drug-inducible membrane-targeting strategy based on the heterodimerization of the FRB domain of mTOR and FKBP12. In this approach, we fused the enzyme of interest—in this case, a type-IV phosphoinositide 5-phosphatase (5-ptase)—to the FKBP12 protein; upon addition of rapamycin (Rapa), the enzyme rapidly translocated to the membrane where its binding partner, the FRB domain, is targeted. We showed that the manipulation rapidly eliminates PtdIns(4,5)P2 from the PM. We applied our newly developed strategy to studying the PtdIns(4,5)P2 regulation of a number of ion channels and transporters. In collaboration with Tibor Rohacs, we compared the phosphoinositide regulation of the TrpV1 and TrpM8 channels (the calcium channel responsible for hot and cold sensations, respectively). Intriguingly, while both channels are regulated by phosphoinositides, the regulation of TrpM8 is relatively simple; it requires PtdIns(4,5)P2 for activity, and PtdIns4P cannot substitute for the former lipid. In contrast, TrpV1 channels are inhibited as well as stimulated by PtdIns(4,5)P2 depending on the dose of capsaicin while PtdIns4P is able to support TrpVI’s calcium channel activity. The molecular basis for this regulatory difference between channels is currently under investigation in Tibor Rohacs’s laboratory. In similar experiments, we also showed that the Orai1 channel, which supports capacitative calcium entry, does not require PtdIns(4,5)P2 for its activity.
In collaboration with Wouter Mooolenaar’s group, we revealed the regulation of the GAP junction protein connexin43 by PtdIns(4,5)P2 and showed that transport of small molecules from cell to cell via GAP junctions formed by connexin43 requires PtdIns(4,5)P2 in the PM; decreasing the level of the lipid rapidly shuts down this form of intercellular communication. Moolenaar’s group has also shown that connexin43 regulation by PtdIns(4,5)P2 occurs via receptor activation of PLCbeta3, which associates indirectly with the connexin43 protein.
Last year, we also reported that PtdIns(4,5)P2 is required for the internalization of transferrin receptors and, following up on our observation and in collaboration with Pietro De Camilli’s group, we investigated the role of PtdIns(4,5)P2 in PM recruitment of several clathrin adaptor proteins. PtdIns(4,5)P2 depletion resulted in a rapid loss of clathrin puncta from the PM, which correlated with a massive dissociation of endocytic adaptors. The remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which are concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the PM. These changes correlated with an arrest of motility at the cell edge. Our findings demonstrate the critical importance of PtdIns(4,5)P2 in clathrin coat dynamics and Arp2/3-dependent actin regulation.
Lukacs V, Thyagarajan B, Varnai P, Balla A, Balla T, Rohacs T. Dual regulation of TRPV1 by phosphoinositides. J Neurosci 2007;27:7070-80.
van Zeijl L, Ponsioen B, Giepmans BN, Ariaens A, Postma FR, Várnai P, Balla T, Divecha N, Jalink K, Moolenaar WH. Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate. J Cell Biol 2007;177:881-91.
Várnai P, Balla T. Visualization and manipulation of phosphoinositide dynamics in live cells using engineered protein domains. Pflügers Arch 2007;455:69-82.
Várnai P, Thyagarajan B, Rohacs T, Balla T. Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels to study multiple regulatory functions of the lipid in intact living cells. J Cell Biol 2006;175:377-82.
Zoncu R, Perera RM, Sebastian R, Nakatsu F, Chen H, Balla T, Ayala G, Toomre D, De Camilli PV. Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci USA 2007;104:3793-98.
Roles of PI 4-kinases and PI transfer proteins in supplying phosphatdylinositol to the plasma membrane
Compartmentalization of phospholipid synthesis is a major factor in the unique lipid composition of organellar membranes. As with most other phospholipids, PtdIns is primarily synthesized in the ER and transported to other membranes either by vesicular transport or, as now increasingly recognized, by lipid transfer proteins. The central signaling role of PtdIns(4,5)P2 in the PM relies on the steady supply of PtdIn4P formed by phosphatidylinositol 4-kinase (PI4K) enzymes from PtdIns. Most of the PtdIn4P is produced in the Golgi, as most of the PI4Ks are also localized to compartments spanning the ER to the trans-Golgi network. At present, it is not clear how PtdIns4P of the PM is generated, specifically whether it is produced from PtdIns by a PI4K in the PM—a pathway clearly defined by earlier studies—or whether some yet unknown mechanism makes a contribution from the ER-Golgi compartment. This question recently resurfaced when we showed that the production of PtdIns4P in the PM requires the activity of a PI4K (PI4KIIIalpha) that is localized in the ER/Golgi interface.
PtdIns transfer proteins (PITPs) have been identified as responsible for the transfer of PtdIns (and, in some cases, the transfer of phosphatidylcholine or sphingomyelin) from the ER to the Golgi or the PM. Given that PI4Ks require PtdIns as substrate, the connection between PITPs and PI4Ks has long been established, mainly based on yeast studies. At the same time, more and more data suggest that PI4Ks regulate the transport of lipids (cholesterol, ceramide, and glucosylceramide) by lipid-transfer proteins within the cell. In view of the ER/Golgi localization of PI4KIIIalpha that regulates the PM supply of PtdIns4P, it would be particularly interesting to know whether the enzyme regulates the transfer of PtdIns between the ER/Golgi and PM or whether PITPs contribute to the supply of PtdIns for this enzyme. In our initial studies, we set up the RNAi-mediated gene silencing of PITPalpha and PITPbeta, two closely related members of the mammalian PITP proteins. We were able to knock down the individual PITPs by about 90 percent with great specificity in HEK293 cells and are now evaluating the effects of the downregulation of the PITP isoforms on calcium signaling and inositol lipid kinetics during angiotensin II stimulation. We will combine these studies with those in which individual PI4Ks are downregulated and the effects of PITP and PI4K knockdown on the PM and Golgi PtdIns4P generation are evaluated.
Balla T. Phosphoinositide-derived messengers in endocrine signaling. J Endocrinol 2006;188:135-53.
Várnai P, Balla T. Live cell imaging of phosphoinositide dynamics with fluorescent protein domains. Biochim Biophys Acta 2006;1761:957-67.
COLLABORATOR
Trevor Blake, MS, Genetics and Molecular Biology Branch, NHGRI, Bethesda, MD
Shamshad Cockcroft, PhD, University College London, London, UK
Pietro De Camilli, MD, Department of Cell Biology, Yale University School, of Medicine, New Haven, CT
Laszlo Hunyady, MD, PhD, DCs, Semmelweis University Faculty of Medicine, Budapest, Hungary
Paul Liu, MD, PhD, Genetics and Molecular Biology Branch, NHGRI, Bethesda, MD
Wouter Moolenaar, PhD, The Netherlands Cancer Institute, Amsterdam, The Netherlands
Tibor Rohacs, MD, PhD, University of Medicine and Dentistry of New Jersey, Newark, NJ
For further information, contact ballat@mail.nih.gov.
