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Phosphoinositide Messengers in Cellular Signaling and Trafficking

Tamás Balla, MD, PhD, Head, Section on Molecular Signal Transduction
Yeun Ju Kim, PhD, Postdoctoral Fellow
Marek Korzeniowski, PhD, Postdoctoral Fellow
Naveen Bojjireddy, PhD, Postdoctoral Fellow
Marko Jovic, PhD, Postdoctoral Fellow
Maria-Luisa Guzman-Hernandez, 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.

Structural analysis of STM1 activation regulating 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 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 a protein resident in the endoplasmic reticulum (ER) that, upon depletion of the ER Ca²⁺ stores, rapidly translocates to the compartment of the ER adjacent to the plasma membrane (PM), 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 (Varnai et al.).

STIM1 molecules reside in the ER and consist of a luminal Ca²⁺-binding EF-hand domain and a SAM (sterile alpha motif) that mediates oligomerization. They have a single transmembrane segment and a cytoplasmic domain that is built from coiled-coil domains, acidic and basic domains, and proline-rich segments. It was shown earlier by several laboratories that the cytoplasmic tail of STIM1 is capable of activating the Orai1 Ca²⁺ channels, and a short segment named CAD/SOAR has been identified as the minimal segment capable of Orai1 activation (Varnai et al.).

We used a unique oligomerization strategy to show that the full cytoplasmic segment of STIM1 is a poor activator of Orai1 but becomes highly active upon clustering. In contrast, the CAD/SOAR domain is very active in opening Orai1 channels without oligomerization, suggesting that it must be kept inactive in the context of the whole cytosolic segment of STIM1. In search of an intramolecular silencing mechanism, we identified a short acidic segment within the first coiled-coiled domain of STIM1 that could form an intramolecular interaction with a basic sequence within CAD/SOAR recently identified as critical for Orai1 activation. We showed that mutations within the acidic stretch make STIM1 constitutively active and confirmed that the basic sequence within CAD/SOAR is essential for STIM1-mediated Orai1 activation. Intriguingly, the acidic stretch within STIM1 shows significant sequence homology with the C-terminus of Orai1 and could be used as a decoy to interfere with the ability of STIM1 to activate Orai1 channels—but only when expressed as part of the juxtamembrane first coiled-coil domain of STIM1. We suggest that the initial oligomerization of STIM1 unmasks the CAD/SOAR domain by breaking the intramolecular interaction that keeps the CAD/SOAR domain inactive in the quiescent STIM1 molecule (Korzeniowski et al.).

New approach to studying the role of Golgi phosphoinositides in trafficking and signaling

Phosphoinositides are essential lipid regulators of trafficking and signaling pathways of all eukaryotic cells. Phosphatidylinositol 4-phosphate (PtdIns4P) is an intermediate in the synthesis of several important phosphoinositide species but also serves as a regulatory molecule in its own right. PI4Ks are most abundant in the Golgi but are also found in the plasma membrane and in endocytic compartments. To investigate the role of Golgi PtdIns4P in orchestrating trafficking events, we used a novel drug-inducible molecular approach to rapidly deplete PtdIns4P from Golgi membranes by a recruitable Sac1 phosphatase enzyme. In this system the small (9 kDa) FRB fragment of the mTOR protein was fused with Tgn38, the Golgi/Tgn protein, and tagged with CFP, while the Sac1 phosphatase enzyme was trimmed of its C-terminal ER localization signal and fused to an mRFP-FKBP12 chimera. Expression of these constructs in HEK293 or COS-7 cells allowed rapid recruitment of the Sac1 phosphatase to the Golgi/Tgn, initiated by the rapamycin-induced heterodimerization of the FKBP12 protein with FRB. The utility of the system was shown by the rapid loss of Golgi localization of PH domains known to bind to PtdIns4P after Sac1 recruitment to the Golgi. Acute PtdIns4P depletion prevented the exit of cargo from the Golgi destined to both the plasma membrane and the late endosomes and led to the loss of some but not all clathrin adaptors from the Golgi membrane. Rapid PtdIns4P depletion in the Golgi also impaired, but did not eliminate, the replenishment of the plasma membrane PtdIns(4,5)P₂ during phospholipase C activation, revealing a hitherto unrecognized contribution of Golgi PtdIns4P to this process. This new approach will allow further studies on the role of phosphoinositides in endocytic compartments that have evaded detection using the conventional long-term manipulations of inositide kinase and phosphatase activities (Szentpetery et al.).

Imaging the ER-mitochondrial contact zones and measuring the local Ca²⁺ concentration in this compartment

A significant fraction of the ER makes close contacts with mitochondria (mito). ER-mito local coupling is essential for survival in yeast and is required for many aspects of cell function in mammalian cells, including lipid biosynthesis, protein trafficking, and calcium signaling. Regarding calcium, ER-mitochondrial junctions provide a site at which IP3R/RyR–mediated calcium oscillations are propagated locally to the mitochondria to control energy metabolism and mitochondrial apoptosis and to exert feedback effects on cytoplasmic calcium signaling. Indirect evidence supports the notion that, at the ER-mitochondrial junctions, the mitochondrial membrane can be exposed to a high-calcium microdomains generated by the open IP3Rs or ryanodine receptors (RyRs). The microdomain would attain activation of the low-affinity mitochondrial Ca²⁺ uniporter. Despite the broad significance of the ER-mito junctions and the emerging research efforts to further explore them, several fundamental aspects of the coupling remain elusive. Localization and dynamic monitoring of the ER-mito contacts in live cells have been prevented by the lack of a specific marker. Visualization and quantitation of the [Ca²⁺]_ER-mt changes were also precluded by the lack of an ER-mito interface–targeted probe.

In collaboration with the Hajnoczky group, we used the FKBP12-FRB drug-inducible heterodimerization strategy to visualize the ER-mito contact zones and to drive a genetically encoded Ca²⁺ indicator into this compartment for measurements of local Ca²⁺ elevations. For this, the FKBP12 domain fused to mRFP was targeted to the mito outer membrane by a short mito-targeting sequence from AKAP1, and the FRB domain tagged with CFP was targeted to the ER using the short C-terminal targeting sequence of Sac1. These constructs co-expressed in mammalian cells were localized in the mito and ER membranes, respectively. However, after addition of rapamycin, they rapidly concentrated in areas where the two organelles were juxtaposed, highlighting these compartments in confocal microscopy. In subsequent experiments the genetically encoded Ca²⁺ indicator Pericam, or its mutated version that has lower Ca²⁺ affinity, was fused with the mito-targeted FKBP12, allowing measurements of Ca²⁺ concentrations either on the surface of mitochondria or, after rapamycin addition, within the ER-mito contact zones. The experiments showed for the first time that Ca²⁺ concentrations in the contact zones can reach as high as 20-25 µM during InsP3-induced Ca²⁺ release from the ER. By changing the linker length between the two organelles, it was possible to show that there is an optimal distance between these organelles for Ca²⁺ transfer—too narrow a space probably excludes large molecules such as the InsP3R, making the Ca²⁺ transfer suboptimal (Csordás et al.).

Phosphatidylinositol 4-kinases are important host factors required for viral replication of several RNA viruses.

Viruses critically rely on their host cells for viability and replication. During infection, the virus and host cell become engaged in a dynamic duet, during which the virus initiates a sequence of subcellular events that are spatio-temporally highly organized and dramatically change the cellular architecture and physiology. Many RNA viruses and even some DNA viruses such as the poxviruses rely on host intracellular membranes for replication. The plus-strand RNA virus families that include many human pathogens like picornaviruses (such as the enteroviral genus members poliovirus [PV] and coxsackievirus B3 [CVB3], rhinovirus, and hepatitis A), coronaviruses (SARS) and flaviviruses (hepatitis C virus [HCV], yellow fever virus, dengue fever virus, and West Nile virus) are particularly dependent on organizing their replication and assembly on intracellular membranes. The properties of the replication membranes that are able to support viral RNA replication and the general question of what makes a membrane suitable for replication are poorly understood.

In collaboration with the Altan-Bonnet laboratory, we studied the in situ properties of the viral RNA replication membranes in cells infected with plus-strand RNA viruses. These studies showed how remodeling of the host secretory pathway by enteroviral replication proteins generates organelles with unique protein and lipid composition geared for replicating viral RNA. They also revealed that enteroviral proteins modulate effector recruitment by GBF1 and Arf1, promoting recruitment of the phosphatidylinositol 4-kinase PI4KIIIbeta to secretory organelle membranes over coat proteins, leading to disassembly of conventional secretory organelles and assembly of "replication organelles" that are juxtaposed to ER exit sites. PI4KIIIbeta at these organelle membranes produces a lipid microenvironment rich in PtdIns4P, which facilitates enteroviral RNA synthesis. Furthermore, the studies showed that the enteroviral RNA-dependent RNA polymerase exhibits specific affinity for PtdIns4P lipids over other cellular lipids, thus providing a mechanism for membrane recruitment of this protein in the host. Most importantly, inhibitors of PI4KIIIbeta were found to inhibit replication of several RNA viruses, offering a new potential target protein to fight infection by these viral pathogens (Hsu et al.).

Publications

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.
Korzeniowski M, Manjares, IM, Varnai P, Balla T. Activation of STIM1-ORAI1 involves and intramolecular switching mechanism. Science Signaling 2010; in press.
Szentpetery Z, Varnai P, Balla T. Acute manipulation of Golgi phosphoinositides to assess their importance in cellular trafficking and signaling. Proc Natl Acad Sci U S A 2010 107:8225-8230.
Csordás G, Várnai P, Golenár T, Roy S, Purkins G, Schneider TG, Balla T, Hajnóczky G. Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface. Mol Cell 2010 39:121-132.
Hsu N-Y, Ilnytska O, Belov G, Santiana M, Chen Y-H, Pau C, Takvorian P, Schaar H, Kaushik-Basu N, Balla T, Cameron C, Ehrenfeld E, van Kuppeveld FJM and Altan-Bonnet N. Viruses reorganize secretory pathway to form organelles with specific lipid microenvironment for RNA replication. Cell 2010 141:799-811.

Collaborators

Laszlo Hunyady, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary
Peter Varnai, MD, PhD, Semmelweis University, Faculty of Medicine, Budapest, Hungary
Nihal Altan-Bonnet, PhD, Department of Biological Sciences, Rutgers University, New Jersey, USA
Gyorgy Hajnoczky, MD, PhD, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, USA

Contact

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

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