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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
  • Hiroyuki Nakamura, PhD, Special Volunteer
  • Nathalie Reinhard, MS, 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.

Endosomal sorting of VAMP3 is regulated by PI4K2A.

We focused on the question of whether membrane fusion is controlled by inositol lipid kinases. Endocytic transport depends on membrane-specific fusion between cargo-containing vesicles and the membranes at target compartments. Fusion is mediated by SNARE proteins that form four-helix bundles between a single R-SNARE molecule on the donor/vesicle membrane and three cognate Q-SNAREs on the target membrane. In a proteomic analysis, we found that VAMP7 and VAMP3 associate with PI4K2A, one of the phosphatidylinositol kinase enzymes. Inositol lipids play important regulatory roles in controlling membrane dynamics and assembly of protein signaling complexes in cellular membranes. In experiments designed to understand the functional significance of the PI4K2A and VAMP3 association, we found that PI4K2A resides on VAMP3–positive tubulo-vesicular structures and that PI4K2A depletion resulted in accumulation of VAMP3 in endosomal compartments. PI4K2A knockdown also inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3–mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly reduced association of VAMP3 with Vti1a, VAMP3's cognate Q-SNARE. While the initial VAMP3 binding to PI4K2A did not require kinase activity, we found sorting of VAMP3 to be tightly linked to the endosomal levels of PtdIns4P because acute depletion of PtdIns4P on endosomes significantly delayed VAMP3 trafficking. Taken together, the results of this study identified PI4K2A as an endosomal regulator of an R-SNARE, providing mechanistic evidence in intact cells in support of previous in vitro data showing phospholipid modulation of SNARE function. The significance of the findings is that missorting of proteins is often the cause of a variety of diseases, neurodegenerative diseases in particular, and that better understanding the proteins and mechanism controlling the protein-sorting pathways will help us identify new targets to alter these processes and the course of such diseases.

The role of phosphatidylinositol 4-phosphate in Golgi organization

A critically important component of all eukaryotic cells, the Golgi is involved in the sorting and modification of protein and lipid cargoes destined for secretion or for degradation. The Golgi harbors a high concentration of phosphatidylinositol 4-phosphate (PI4P) made by four different PI 4-kinase enzymes (PI4Ks), which all can be found in the outer surface of the Golgi membranes. The importance of this high PI4P content is poorly understood. PI4P has been recognized as a docking point for numerous proteins, mostly responsible for non-vesicular transport of structural lipids such as cholesterol. In collaboration with the group of Gustavo Egea, we found that betaIII-spectrin, one of the important molecular components of the actin cytoskeleton, is recruited to the Golgi by PI4P. Using newly generated antibodies to specific peptide sequences of the human βIII-spectrin, the Egea group showed that the molecule is found in the Golgi complex, where it is enriched in the trans-Golgi and trans-Golgi network (TGN). We then used our recently developed drug-inducible enzymatic tool set to deplete the Golgi-associated pool of PI4P and showed that the manipulation displaced βIII-spectrin from the Golgi. Similarly, overexpression of a pleckstrin homology domain that specifically recognizes PI4P and competes with proteins that bind to the same lipid also reduced βIII-spectrin Golgi localization. Importantly, interference with actin dynamics using various toxins failed to affect the localization of βIII-spectrin to Golgi membranes. The functional importance of the presence of βIII-spectrin in the Golgi was demonstrated when depletion of βIII-spectrin, using siRNA technology or the microinjection of anti-βIII spectrin antibodies into the cytoplasm, led to the fragmentation of the Golgi. The Golgi fragments showed swollen distal Golgi cisternae and vesicular structures. Using a variety of protein transport assays, the Egea group also showed that the endoplasmic reticulum-to-Golgi and post-Golgi protein transports were impaired in βIII-spectrin–depleted cells, whereas the internalization of the Shiga toxin subunit B to the endoplasmic reticulum (ER) was unaffected. The studies showed that βIII-spectrin constitutes a major skeletal component of distal Golgi compartments, where it is necessary to maintain the structural integrity and secretory activity of the Golgi, and that PI4P appears to be highly relevant for coordinating this process.

In a separate set of studies in collaboration with the group of Vivek Malhotra, we studied the role of PI4P in generating membrane curvature in the Golgi membrane. BAR (Bin/Amphiphysin/Rvs) domains are protein modules found in many membrane-associated molecules that are able to bend biological membranes and generate the curvature essential for the budding process. The Malhotra group showed that the BAR domain–containing proteins arfaptin1 and arfaptin2 are localized to the TGN and, by virtue of their ability to sense and/or generate membrane curvature, could play an important role in the biogenesis of transport carriers. The group found that arfaptins contain an amphipathic helix (AH) preceding the BAR domain, which is essential for their binding to PI4P–containing liposomes and the TGN of mammalian cells. The binding of arfaptin1, but not arfaptin2, to PI4P was found to be regulated by protein kinase D (PKD)–mediated phosphorylation at Ser100 within the AH. It is of importance that PKD also phosphorylates and activates one of the PI4K enzymes, PI4KB, in the Golgi, generating most of the PI4P in this compartment. Using our drug-inducible enzymatic tool set to deplete the Golgi-associated pool of PI4P, we showed that arfaptins require PI4P to re-associate with the Golgi once they are released by brefeldin A treatment. Notably, we found that only arfaptin1 was required for the PKD–dependent trafficking of chromogranin A by the regulated secretory pathway. Taken together, the findings revealed the importance of PI4P and PKD in the recruitment of arfaptins at the TGN and their requirement in the events leading to the biogenesis of secretory storage granules.

Pharmacological and genetic targeting of PI4KA reveals its important role in maintaining plasma membrane PtdIns4P and PtdIns(4,5)P2 levels and cellular responsiveness to Gq-coupled receptors.

The host factor PI4KA is essential for Hepatitis C virus (HCV) replication and hence a target for drug development. PI4KA has also been linked to ER exit sites and generation of plasma membrane phosphoinositides. In a collaboration with GlaxoSmithKline (GSK), we investigated whether targeting of PI4KA is a feasible approach to fight HCV infection. GSK has developed highly specific and potent inhibitors of PI4KA and a conditional knockout mouse for PI4KA to study the importance of this enzyme in vitro and in vivo. We used the inhibitors in cultured cells and showed that PI4KA is essential for the maintenance of plasma-membrane PtdIns(4,5)P2 pools during strong stimulation of receptors coupled to PLC activation. Intriguingly, cells were able to maintain their plasma membrane PtdIns(4,5)P2 levels when treated with the PI4KA inhibitors but not when challenged with Gq-coupled receptor stimulation. In studies with whole animals, pharmacological blockade of PI4KA in adult mice led to sudden death closely correlating with PtdIns(4,5)P2 inhibition. Genetic inactivation of PI4KA also led to death; however, the pathology only partially overlapped with that elicited by acute pharmacological blockade. Genetic inactivation of PI4KA led to severe gastrointestinal abnormalities, while acute pharmacological blockade led to sudden death with symptoms of acute cardiovascular shock. The studies highlighted the risks of targeting PI4KA as an anti–HCV strategy and also pointed to important distinctions between genetic and pharmacological studies when selecting host targets as putative therapeutics.

Publications

  1. Altan-Bonnet N, Balla T. Phosphatidylinositol 4-kinases: hostages harnessed to build panviral replication platforms. Trends Biochem Sci 2012;37:293-302.
  2. Salcedo-Sicilia L, Granell S, Jovic M, Sicart A, Mato E, Johannes L, Balla T, Egea G. βIII spectrin regulates the structural integrity and the secretory protein transport of the Golgi complex. J Biol Chem 2013;288:2157-2166.
  3. Kim YJ, Hernandez ML, Balla T. Inositol lipid regulation of lipid transfer in specialized membrane domains. Trends Cell Biol 2013;23:270-278.
  4. Cruz-Garcia D, Ortega-Bellido M, Scarpa M, Villeneuve J, Jovic M, Porzner M, Balla T, Seufferlein T, Malhotra V. Recruitment of arfaptins to the trans-Golgi network by PI(4)P and their involvement in cargo export. EMBO J 2013;32:1717-1729.
  5. Balla T. Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev 2013;93:1019-1137.

Collaborators

  • Nihal Altan-Bonnet, PhD, Rutgers, The State University of New Jersey, Newak, NJ
  • Julie A. Brill, PhD, Hospital for Sick Children, Toronto, Canada
  • Gustavo Egea, PhD, School of Medicine, Universitat de Barcelona, Barcelona, Spain
  • László Hunyady, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary
  • Vivek Malhotra, DPhil, Centre for Genomic Regulation, Barcelona, Spain
  • Christopher Moore, PhD, GlaxoSmithKline, Infectious Diseases Discovery Performance Unit, Hillsborough, NC
  • Péter Várnai, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary

Contact

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

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