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Phosphoinositide Messengers in Cellular Signaling and Trafficking

Tamás Balla, MD, PhD
  • Tamás Balla, MD, PhD, Head, Section on Molecular Signal Transduction
  • Yeun Ju Kim, PhD, Staff Scientist
  • Alejandro Alvarez-Prats, PhD, Postdoctoral Fellow
  • Maria-Luisa Guzman-Hernandez, PhD, Postdoctoral Fellow
  • Gerald Hammond, PhD, Postdoctoral Fellow
  • Marko Jovic, PhD, Postdoctoral Fellow
  • Mira Sohn, PhD, Postdoctoral Fellow
  • Eva Wisniewski, MD, Special Volunteer
  • Ljubisa Vitkovic, PhD, Special Volunteer

We investigate 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 signaling protein complexes that assemble on the surface of cellular membranes. Phosphoinositides regulate protein kinases and GTP–binding proteins as well as membrane transporters, including ion channels, thereby controlling many cellular processes such as proliferation, apoptosis, metabolism, cell migration, and differentiation. We focus on the phosphatidylinositol 4 (PtdIns4)–kinases (PI4Ks), a family of enzymes that catalyze the first committed step in polyphosphoinositide synthesis. Current work aims to: (i) understand the function and regulation of several PI4Ks involved in the control of cellular signaling and trafficking pathways; (ii) find specific inhibitors for the individual PI4Ks; (iii) define the molecular basis of PtdIns4P–regulated pathways through identification of PtdIns4P–interacting molecules; (iv) develop tools to analyze inositol lipid dynamics in live cells; and (v) determine the importance of the lipid-protein interactions in the activation of cellular responses by G protein–coupled receptors and receptor tyrosine kinases.

A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi.

Localization of inositol lipids in live cells is uniquely important to our understanding of the compartmentalized regulation of these lipids and the downstream biochemical pathways they control. Several inositol-lipid forms have been successfully imaged with protein domains that recognize specific isomers of the lipid. However, it has been problematic to image one particular member, phosphatidylinositol 4-phosphate (PtdIns4P), because in the past the probes used for this purpose had additional binding components that restricted their PtdIns4P recognition to only certain membrane compartments. For example, PtdIns4P was localized to Golgi membranes based on the distribution of lipid-binding modules from PtdIns4P effector proteins, but these probes were biased by additional interactions with other Golgi-specific determinants. Localizing PtdIns4P in all other membrane compartments is also highly desirable for studies in which the reorganization of host-cell membranes in virus-infected cells was shown to require PtdIns4P–rich membrane platforms. In our study, we derived a new PtdIns4P biosensor using the PtdIns4P binding of SidM (P4M) domain of the secreted effector protein SidM from the bacterial pathogen Legionella pneumophila. PtdIns4P was necessary and sufficient for localization of P4M, which revealed pools of the lipid associated not only with the Golgi but also with the plasma membrane and Rab7–positive late endosomes/lysosomes. PtdIns4P distribution was determined by the localization and activities of both its anabolic and catabolic enzymes. The probe also detected the enlarged ER compartment induced by the hepatitis C NS5A protein. Therefore, P4M reveals a wider cellular distribution of PtdIns4P than previous probes and will therefore be valuable for dissecting the biological functions of PtdIns4P in its assorted membrane compartments both in healthy cells and in during pathological conditions.

Determination of the crystal structure of phosphatidylinositol 4-kinase IIα

There are four distinct phosphatidylinositol 4 kinases in metazoan eukaryotic cells. These enzymes were recently identified as essential host factors for RNA virus replication in mammalian cells, and it is likely that they play a role in the life cycle of other pathogens. Recently, the PI4K enzyme of the malaria parasite, Plasmodium falciparum, was described as a promising drug target to combat malaria infection. Therefore, the kinases have attracted significant attention not only because of their physiological roles in normal cells, but also because of their potential as drug targets to fight various forms of infections. Structural information about these proteins is enormously useful to understand their mode of catalysis and for rational drug design. Several efforts have been geared toward solving the structures of PI4Ks. Recently, the structure of the human PI4K type III beta was solved in Roger Williams's laboratory at the MRC, Cambridge. The enzyme belongs to the PI 3-kinase family of enzymes, and the structure of its catalytic domain shows high degree of similarity to already known structures of PI3Ks. In contrast, the primary sequence of the type II PI4Ks has already suggested that it belongs to a different and unique enzyme family. In collaboration with Evžen Bouřa's laboratory, the atomic structure of phosphatidylinositol 4-kinase type IIα (PI4K IIα) was solved in complex with ATP by X-ray crystallography at 2.8 Å resolution. The structure revealed a non-typical kinase fold that could be divided into N- and C-lobes, with the ATP–binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C-lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, the structural results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane. Together with an ongoing screen to find PI4K type II–specific inhibitors, the structural studies will be of high value to understand the role of the enzyme in various intracellular processes as well as in cellular pathologies.

Secretion of VEGF-165 has unique characteristics including shedding from the plasma membrane.

Vascular endothelial growth factor (VEGF) is a critical regulator of endothelial cell differentiation and vasculogenesis during both development and tumor vascularization. A very large amount of literature has accumulated on the biological effects of the various VEGF isoforms and the signal transduction pathways activated by VEGF receptors. In contrast, surprisingly little is known about the way VEGF-A is produced and secreted by cells. Given that VEGF-A contains a signal sequence, it is generally believed that the factor is co-translationally translocated to the ER lumen, where it undergoes proteolytic cleavage of its signal sequence. VEGF-165 is a major form, which is secreted from the cells via a poorly characterized pathway. We used GFP– and epitope-tagged VEGF-165 and found that its early trafficking between the ER and the Golgi requires the small GTP–binding proteins Sar1 and Arf1 and that its glycosylation in the Golgi compartment is necessary for efficient post-Golgi transport and secretion from the cells. The relative temperature insensitivity of VEGF secretion and its Sar1 and Arf1 inhibitory profiles distinguished it from other cargoes using the “constitutive” secretory pathway. Prominent features of VEGF secretion were the retention of the protein on the outer surface of the plasma membrane and the stimulation of its secretion by Ca2+ and PKC. Importantly, shedding of VEGF-165 from the cell surface together with other membrane components appears to be a unique feature by which VEGF is delivered to the surroundings to exert its known biological actions. Understanding VEGF trafficking can reveal additional means by which tumor vascularization can be inhibited by pharmacological interventions.

Publications

  1. Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev 2013;93:1019-1137.
  2. Kim YJ, Hernandez ML, Balla T. Inositol lipid regulation of lipid transfer in specialized membrane domains. Trends Cell Biol 2013;23:270-278.
  3. Hammond GR, Machner MP, Balla T. A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi. J Cell Biol 2014;205:113-126.
  4. Baumlova A, Chalupska D, Róźycki B, Jovic M, Wisniewski E, Klima M, Dubankova A, Kloer DP, Nencka R, Balla T, Boura E. The crystal structure of the phosphatidylinositol 4-kinase IIα. EMBO Rep 2014;15:1085-1092.
  5. Guzmán-Hernández ML, Potter G, Egervári K, Kiss JZ, Balla T. Secretion of VEGF-165 has unique characteristics, including shedding from the plasma membrane. Mol Biol Cell 2014;25:1062-1071.

Collaborators

  • Nihal Altan-Bonnet, PhD, Laboratory of Host-Pathogen Dynamics, NHLBI, Bethesda, MD
  • Evžen Bouřa, PhD, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
  • Julie A. Brill, PhD, Hospital for Sick Children, Toronto, Canada
  • László Hunyady, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary
  • Jozef Z. Kiss, MD, PhD, Université de Genève, Geneva, Switzerland
  • Péter Várnai, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary

Contact

For more information, email ballat@mail.nih.gov.

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