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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
  • Naveen Bojjireddy, PhD, Postdoctoral Fellow
  • Yeun Ju Kim, PhD, Postdoctoral Fellow
  • Marek Korzeniowski, PhD, Postdoctoral Fellow
  • Hui Ma, PhD, Postdoctoral Fellow
  • Zsofia Szentpetery, PhD, Postdoctoral Fellow

The Section on Molecular Signal Transduction 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 have 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. This group focuses on one family of enzymes, the phosphatidylinositol 4 (PtdIns4)–kinases (PI4Ks), that catalyze the first committed step in polyphosphoinositide synthesis. Current studies are aimed at (1) understanding the function and regulation of several PI4Ks in the control of cellular signaling and trafficking pathways; (2) finding specific inhibitors for the individual PI4Ks; (3) defining the molecular basis of PtdIns4P–regulated pathways through identification of PtdIns4P–interacting molecules; (4) developing tools to analyze inositol lipid dynamics in live cells; and (5) determining the importance of the lipid-protein interactions in the activation of cellular responses by G protein–coupled receptors and receptor tyrosine kinases.

Inositol lipid requirement for STM1/Orai1 mediated calcium entry

Calcium ions (Ca2+) are among the most ubiquitously used signaling molecules in eukaryotic cell regulation. They regulate a range of cellular processes such as muscle contraction, hormone secretion, and gene transcription. Ca2+ enters the cells via multiple Ca2+ entry routes formed by Ca2+ channels and transporters and is efficiently eliminated by Ca2+ pumps and exchangers. The tight control of the cells’ Ca2+ homeostasis is essential for cellular signaling and for maintenance of cellular integrity. Recent studies identified two protein families (STIM and Orai/CRACM) that mediate the specific form of Ca2+ entry termed store-operated calcium entry (SOCE). STIM1 is an endoplasmic reticulum– (ER) resident protein that rapidly translocates to the plasma membrane– (PM) adjacent compartment of the ER upon depletion of the ER Ca2+ stores, where the protein activates the calcium channel Orai1. The importance of this calcium entry pathway is underscored by the fact that mutations in Orai1 have been linked to severe inborn human immunodeficiencies and that this route of calcium entry is key to the calcium-regulated activation of T cells mediated by the NFAT transcription factors. Studying the molecular details of STIM1 translocation to the PM–adjacent ER compartment and identification of the domains responsible for STIM1/Orai1 interaction will help develop novel molecular approaches for immunosuppression.

Given that most ion channels and transporters are regulated by the phosphoinositides, a minor but critically important class of acidic phospholipids, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM–adjacent ER regions during Ca2+ store depletion. Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P2 levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, while PtdIns4P levels were reduced by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P2 depletion, substantially reduced endogenous or STIM1/Orai1–mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced ICRAC currents (CRAC=calcium release–activated calcium channel; the electrophysiological correlate of STIM1/Orai1–mediated SOCE). These results suggest an inositide requirement of Orai1 activation, but not STIM1 movements, and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai1 channel activity.

In another set of studies, we examined whether subplasmalemmal mitochondria are located proximal to the ER-PM regions close to the activated Orai1 channels. This question is important given that such a group of mitochondria could preferentially respond to Ca2+ influx occurring via the STIM1/Orai1 mechanism. For this purpose COS-7 cells were cotransfected with Orai1, STIM1 labeled with YFP or mRFP, and the mitochondrially targeted Ca2+-sensitive fluorescent protein inverse Pericam. Depletion of ER Ca2+ with ATP and thapsigargin (Tg) (in Ca2+-free medium) induced the appearance of STIM1 puncta in the 100 nm–wide subplasmalemmal space, as examined with total internal reflection fluorescence (TIRF) microscopy. In such cells, mitochondria were located either in the gaps between STIM1–tagged puncta or in remote, STIM1–free regions. After addition of Ca2+ to initiate Ca2+ influx via the activated Orai1 channels, mitochondrial (Ca2+m) increases were similar regardless of the mitochondrion–STIM1 distance. These observations indicate that specially positioned mitochondria are not likely to serve as uniquely sensitive sensors of Ca2+ influx occurring at the PM and the subplasmalemmal ER.

Phosphoinositides in enteropathogenic E. coli infection

Another research focus of the group was the analysis of the phosphoinositide changes upon invasion of mammalian cells by the enteropathogenic E. coli (EPEC) bacterium. EPEC is a major cause of severe infantile diarrhea in developing countries. Many bacterial pathogens use the cell’s own trafficking machinery to invade and move around within the cells and help spread the bacteria from one cell to another. Understanding the underlying molecular events will help us find new strategies to better fight bacterial infections.

The phosphoinositides (PIs) phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate PtdIns(3,4,5)P3 are present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells and modulate the activity of proteins involved in EPEC infection. However, the role of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in EPEC pathogenesis remains obscure. In a set of experiments using the fluorescent phosphoinositide probes developed in our laboratory, we collaborated with Benjamin Aroeti's group to show that EPEC induces transient PtdIns(4,5)P2 accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, was also observed at these sites. Acute PtdIns(4,5)P2 depletion partially diminished EPEC adherence to the cell surface and actin pedestal formation. These findings were consistent with a bimodal role, whereby PtdIns(4,5)P2 contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. We also showed that EPEC induces PtdIns(3,4,5)P3 clustering at bacterial infection sites, in a translocated intimin receptor– (Tir) dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, formed complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PtdIns(3,4,5)P3 beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity.

G protein-coupled receptor-promoted trafficking of Gbeta1gamma2 leads to AKT activation at endosomes via a mechanism mediated by Gbeta1gamma2-Rab11a interaction

G protein–coupled receptors activate heterotrimeric G proteins at the plasma membrane, where most of their effectors are intrinsically located or to which they are transiently associated as the external signal is being transduced. This paradigm has been extended to the intracellular compartments by studies in yeast showing that trafficking of G-alpha activates phosphatidylinositol 3-kinase (PI3K) at endosomal compartments, suggesting that vesicle trafficking regulates potential actions of G-alpha and possibly G-beta-gamma at the level of endosomes. In collaboration with Guadalupe Reyes-Cruz, we showed that G-beta-gamma interacts with Rab11a and that the two proteins colocalize at early and recycling endosomes in response to activation of lysophosphatidic acid (LPA) receptors. This agonist-dependent association of G-beta-gamma with Rab11a–positive endosomes contributes to the recruitment of PI3K and phosphorylation of AKT at this intracellular compartment. These events are sensitive to the expression of a dominant negative Rab11a mutant or treatment with Wortmannin, suggesting that Rab11a–dependent G-beta-gamma trafficking promotes the activation of the PI3K/AKT signaling pathway associated with endosomal compartments. In addition, RNA interference–mediated Rab11a depletion, or expression of a dominant negative Rab11a mutant attenuated LPA–dependent cell survival and proliferation, suggesting that endosomal activation of the PI3K/AKT signaling pathway in response to G-beta-gamma trafficking, via its interaction with Rab11, is a relevant step in the mechanism controlling these fundamental events.

Live cell imaging with protein domains capable of recognizing phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 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 PLCd1 fused to GFP. We performed 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. All the yeast PH domains that have been previously shown to bind to 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 PLCd1PH-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 PLCd1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)P2 and therefore displays a lower sensitivity as a reporter on changes of this lipid during phospholipase C activation. These results showed that both the PLCd1PH-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 PLCd1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P3 binding may compromise its accuracy to measure PtdIns(4,5)P2 changes. The Tubby domain is a more accurate reporter 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 could differentially regulate multiple effectors if the latter displayed different affinities to PtdIns(4,5)P2.

Publications

  • Korzeniowski MK, Popovic MA, Szentpetery Z, Varnai P, Stojilkovic SS, Balla T. Dependence of STIM1/Orai1-mediated calcium entry on plasma membrane phosphoinositides. J Biol Chem 2009 284:21027-21035.
  • Korzeniowski MK, Szanda G, Balla T, Spat A. Store-operated Ca2+ influx and subplasmalemmal mitochondria. Cell Calcium 2009 46:49-55.
  • Varnai P, Hunyady L, Balla T. STIM and Orai: the long-awaited constituents of store-operated calcium entry. Trends Cell Biol 2009 30:118-128.
  • Balla T, Szentpetery Z, Kim YJ. Phosphoinositide signaling: new tools and insights. Physiology 2009 24:231-244.
  • Sason H, Milgrom M, Weiss AM, Melamed-Book N, Balla T, Grinstein S, Backert S, Rosenshine I, Aroeti B. Enteropathogenic Escherichia coli subverts phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon epithelial cell infection. Mol Biol Cell 2009 20:544-555.

Collaborators

  • Benjamin Aroeti, PhD, The Hebrew University, Jerusalem, Israel
  • Laszlo Hunyady, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary
  • Guadalupe Reyes-Cruz, PhD, Centro de Investigación y de Estudios Avanzados-Instituto Politécnico Nacional, Mexico City, Mexico
  • Andras Spat, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary
  • Peter Varnai, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary

Contact

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

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