Phosphatidylinositol 4-Kinases and Cell Regulation

Tamás Balla, MD, PhD, 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
Andras Balla, PhD, Contractor
Peter Várnai, MD, PhD, Contractor

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 are a small fraction of the cellular phospholipids, but they play critical roles in regulating many 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 cellular processes such as proliferation, apoptosis, metabolism, and cell migration and differentiation. Our group focuses on the 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 phosphatidylinositol (PI) 4-kinases that control 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 the lipid-protein interactions in the activation of cellular responses by G protein–coupled receptors and receptor tyrosine kinases.

Identification of mammalian Sfk1 proteins as regulators of complex glyco-sphingolipid metabolism

Tóth, Kim, Balla A, Tuymetova,¹ Balla T

PI4Ks catalyze the first step in the synthesis of phosphoinositides, a small but uniquely important class of phospholipids that regulate almost every aspect of a cell’s life. These enzymes have remained highly conserved during evolution; two of them, Pik1 and Stt4, assume non-redundant and essential functions in S. cerevisiae. Pik1 is a Golgi-localized enzyme that regulates Golgi-to-cell surface secretion while Stt4 plays several roles in cell wall biogenesis, lysosomal functions, and lipid transport, apparently by maintaining the signaling pool of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in the plasma membrane. The mammalian homologues of the two enzymes, PI4KIIIbeta and PI4KIIIalpha (for Pik1 and Stt4, respectively), have been identified as type-III PI4Ks; however, progress in understanding their roles in mammalian cell physiology or finding their regulatory partners and mechanisms has been generally slow. The only known regulator of the Stt4 PI4K identified so far is the yeast Sfk1 protein (suppressor of four kinase), which rescues the temperature-sensitive allele of Stt4 in S. cerevisiae. Sfk1 is a non-essential gene encoding a protein with 6-membrane-spanning domains and of unknown function with no easily recognizable homologues in higher organisms.

Using multiple sequence alignments of Sfk1 homologues identified in various yeast and fungal genomes, we were able to determine the most conserved regions of these proteins and find their homologues in higher eukaryotes, including five genes in the mouse and human genomes. None of these proteins had any known or assigned functions or showed similarity to any proteins that could provide clues to their cellular roles. However, a recent report identified one of the proteins as the product of a gene sharply upregulated by p53 and as a lysosomal protein important for autophagy. A subsequent study recognized three additional human homologues, but neither study found the homology of the proteins to yeast Sfk1 or assigned any biochemical function to them. We characterized the five mammalian homologues of Sfk1 (designated as hSfk1–5) and found that the proteins are localized in distinct cellular compartments, with two of them located in lysosomes. RNAi-mediated knockdown of the individual proteins showed minor effects on phospholipid metabolism but a prominent effect on the level of certain complex glyco-sphingolipids, such as GM3. Overexpression as well as downregulation of hSfk1, one of the lysosome-associated forms, drastically changed lysosomal morphology. The data indicated that Sfk1 proteins play an important role in the metabolism of complex glyco-sphingolipids. We hypothesize that the proteins recognize and associate with glyco-sphingolipids within the membranes and regulate their trafficking and hence their metabolic fate within the cell. The functional connection between phosphoinositides and glycolipid metabolism rests in the ability of phosphoinositides to regulate the trafficking of these transmembrane domain proteins. Our studies reveal a novel connection between glyco-sphingolipid metabolism, lysosomal functions, and autophagy. Further studies will focus on identifying the molecular steps controlled by the Sfk proteins.

PI 4-kinase IIIalpha and FgF signaling in zebrafish development

Ma, Balla T; in collaboration with Blake, Liu

Previously, we identified four isoforms of PI4Ks and characterized their expression patterns during embryonic development in zebrafish. Even though these enzymes catalyze the same biochemical reaction, their intracellular localization and presumably their regulation are different. Studies in yeast show 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 these enzymes by injection of morpholinos targeting the splicing of exons that coding the catalytic domains of the individual enzymes.

Last year, we reported that downregulation of PI4KIIIalpha with morpholino oligonucleotides resulted in several developmental abnormalities, with the most severe effects observed in the hindbrain and the branchial arches in zebrafish. These changes were associated with highly increased 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 finbuds. To evaluate further the exact site of defect in the cascade of FgF signaling, we determined the expression pattern of otherFgf genes known to be upstream of Fgf8 induction in the pectoral fin bud. Our studies showed that the earliest detectable defects were in the movement of Fgf24-positive cells from the mesenchyme to the apical ectodermal ridge (AER) in the developing fin buds at the 32 hpf² stage, leading to the loss of Fgf10 induction in the underlying mesenchyme. We observed a similar defect after treatment of the embryos with LY294002, consistent with a role of PI 3-kinases in regulating the migration of the Fgf24-positive cells. The data suggest that PI4KIIIalpha is a part of the major pathway by which PI(4,5)P2 is synthesized in the plasma membrane to be used by PI 3-kinases for their signaling roles. The studies are the first to identify the role of a PI4K enzyme in the developmental process of a vertebrate organism and to reveal a functional connection between PI4KIIIalpha and the PI 3-kinases. Current studies aim to elucidate the role of PI4KIIIalpha enzyme in the production of PI(4,5)P2 in a zebrafish cell line.

Balla A, Kim YJ, Varnai P, Szentpetery Z, Knight Z, Shokat KM, Balla T. Maintenance of hormone sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase III alpha. Mol Biol Cell 2007;19:711-721.
Knight ZA, Feldman ME, Balla A, Balla T, Shokat KM. A membrane capture assay for lipid kinase activity. Nat Protocols 2007;2:2459-2488.
Lukacs V, Thyagarajan B, Varnai P, Balla A, Balla T, Rohacs T. Dual regulation of TRPV1 by phosphoinositides. J Neurosci 2007;27:7070-7080.
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;177:881-891.
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;104:3793-3798.

Cell biology of the STIM1 and Orai1 proteins regulating store-operated calcium entry

Korzeniowski, Várnai, Szentpetery, Balla T; in collaboration with Hunyady

STIM1, a recently described endoplasmic reticulum (ER) protein, rapidly translocates to the ER compartment adjacent to the plasma membrane (PM) upon depletion of the ER calcium stores. At that location, it activates the newly identified calcium channel Orai1, forming the molecular basis of the so-called store-operated calcium entry (SOCE) phenomenon. This calcium entry pathway is significant for two reasons. First, mutations in Orai1 have been linked to severe inborn human immunodeficiencies, and, second, this route of calcium entry is pivotal to the calcium-regulated activation of regulatory T-cells 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 expedite the identification of novel molecular targets for immunosuppression.

We tagged STIM1 and Orai1 with fluorescent proteins to follow their movements in live cells. Given that the mechanism of Ca²⁺-regulated formation of the ER-PM junctional contact sites between the two proteins is not understood, we generated new research tools for the visualization and manipulation of these contact sites. We based our approach on the inducible heterodimerization of the FRB fragment of mTOR and the FKBP12 protein targeted to the cytoplasmic leaflet of the ER and PM, respectively. In cells expressing these constructs, addition of rapamycin generates contact areas between the PM and ER that are highly reminiscent of those generated by expressed STIM1 after store depletion, yet the contact zones do not activate Ca²⁺ influx even though STIM1 is also present within them. Curiously, increasing STIM1 expression alone also produces contact zones between the PM and ER that coalesce into large sheets of membranes where the ER and PM are juxtaposed, again without activation of Orai1 channels. Our most recent data suggest that the binding partner of the ER-localized STIM1 in the PM at the large sheets is neither Orai1 nor the PM-localized endogenous STIM1 protein and that the structural requirements for ER-PM sheet formation differ from those that trigger STIM1-Orai1 interaction in the Ca²⁺-depleted state. The carboxy-terminal polybasic tail of STIM1 is required for generation of both the constitutive contact zones and the large ER sheets as well as for microtubule association. However, this segment is dispensable for the store-operated calcium influx process. The data point to additional molecular players in the STIM1-Orai1 partnership. As a result, our current studies aim to identify these putative plasma membrane components.

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.

Translocation of C-met to the nucleus is necessary for induction of a Ca²⁺ signal

Várnai, Balla T; in collaboration with Nathanson

Hepatocyte growth factor (HGF) is important for cell proliferation, differentiation, and related activities. HGF acts through its receptor c-Met, which activates downstream signaling pathways. HGF binds to c-Met at the plasma membrane, where c-Met signaling is generally believed to be initiated. In collaboration with Michael Nathanson’s laboratory, we found that c-Met rapidly translocates to the nucleus upon stimulation with HGF. Intriguingly, the calcium signals induced by HGF result from PI(4,5)P2 hydrolysis and inositol 1,4,5-trisphosphate (InsP3) formation within the nucleus rather than within the cytoplasm. We based this conclusion on the observation that an InsP3-binding domain targeted to the nucleus was able to inhibit HGF- but not vasopressin-induced Ca²⁺ signals while the same domain expressed in the cytosol and excluded from the nucleus had a reverse effect in that it was inhibitory on vasopressin- but not HGF-induced Ca²⁺ signaling. Therefore, while vasopressin generated InsP3 at the plasma membrane, c-Met receptor stimulation appears to bypass the cytoplasm and generate the messenger in the nucleus. We found that translocation of c-Met to the nucleus depends on the adaptor protein Gab1 and importin 1 and that formation of Ca²⁺ signals in turn depends upon this translocation. Our studies suggest a unique mechanism of Ca²⁺ signaling well suited to affecting primarily nuclear processes.

Gomes DA, Rodrigues MA, Leite MF, Gomez MV, Varnai P, Balla T, Bennett AM, Nathanson MH. c-met must translocate to the nucleus to initiate calcium signals. J Biol Chem 2008;283:4344-4351.
Várnai P, Balla T. Visualization and manipulation of phosphoinositide dynamics in live cells using engineered protein domains. Pflügers Arch 2007;455:69-82.

¹ Galina Tuymetova, PhD, former Postdoctoral Fellow
² hours post-fertilization

Collaborators

Trevor Blake, MS, Genetics and Molecular Biology Branch, NHGRI, Bethesda, MD
Pietro De Camilli, PhD, Yale University Medical School, New Haven, CT
Laszlo Hunyady, MD, PhD, 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
Michael Nathanson, MD, PhD, Yale University Medical School, New Haven, CT
Tibor Rohacs, MD, PhD, University of Medicine and Dentistry of New Jersey, Newark, NJ