Tamás Balla, MD, PhD, Head, Section on Molecular Signal Transduction
András Balla, PhD, Postdoctoral Fellow
Yeun Ju Kim, PhD, Postdoctoral Fellow
Hui Ma, PhD, Postdoctoral Fellow
Zsófia Szentpétery, PhD, Postdoctoral Fellow
Balázs Tóth, PhD, Postdoctoral Fellow
Peter Várnai, MD, PhD, Adjunct Investigator
Viviane Clement, Summer Student

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. Although phosphoinositides are but a small fraction of the cellular phospholipids, they play critical roles in the regulation of many (if not all) 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 phosphatidylinositol 4-kinases (PI4Ks), which catalyze the first committed step in phosphoinositide synthesis. Current studies focus on (1) understanding the function and regulation of several phosphatidylinositol (PI) 4-kinases in the control of the synthesis of hormone-sensitive phosphoinositide pools; (2) characterizing the structural features that determine the catalytic specificity and inhibitor sensitivity of PI 3- and PI 4-kinases; (3) defining the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology and other domains of selected regulatory proteins; (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.

Identification of PI4KIIIalpha as a key regulator of the transport of ceramide between the endoplasmic reticulum and the Golgi

Tóth, Balla A, Ma, Balla T; in collaboration with Shokat

Sphingomyelin (SM) is a critical lipid component of the plasma membrane, a lipid that together with cholesterol and glycolipids forms the special liquid-ordered microdomain of cellular membranes often referred to as rafts. Rafts concentrate many signaling proteins and contain inositol phospholipids and are therefore considered active zones in signal transduction. The regulation of cholesterol and sphingomyelin metabolism is intimately interrelated, but relatively little is known about the regulatory pathways that link them. Efficient synthesis of sphingomyelin in the Golgi has been shown to require a steady supply of ceramide from the ER by a process distinct from the vesicular transport between the two organelles. The mechanism of this transport was recently revealed by the identification of a lipid transport protein named CERT. CERT has a lipid-binding START domain at the C-terminus, a PH domain at the N-terminus, and a so-called FFAT (diphenylalanine in an acidic tract) domain that binds to the ER-localized protein VAP-A. The START domain is both necessary and sufficient for ceramide binding and transport, yet a mutation within the PH domain renders the molecule unable to fulfill its transport function, pointing to the PH domain as a critical component for docking/regulation of the molecule. The CERT PH domain shows a high degree of similarity to PH domains that specifically recognize phosphatidylinositol 4-phosphate (PtdIns4P), such as those of the OSBP, FAPP1, and FAPP2 proteins, and has been shown to localize to the Golgi. This finding indicates that PtdIns4P and PI4K enzymes are likely to regulate the transport function of CERT.

Given the presence of several PI4Ks in the Golgi and one in the ER, we were interested in determining which of these enzymes (if any) are important in supporting the transport of ceramide between the ER and the Golgi. Using a combination of pharmacological and genetic approaches, we followed the transport of exogenously added fluorescent ceramide analogues to the Golgi and tracked the fate of endogenous ceramide labeled with [ ³ H]-serine. When we added BODIPY® FL C5-ceramide (FL-Cer) to COS-7 cells at 4°C at very low concentration (to prevent its simple diffusion due to its partial water solubility), we noted that it accumulated in the plasma membrane (PM). Upon warming the cells to 35°C, we observed that FL-Cer rapidly appeared in the ER and subsequently in the Golgi, where it showed significant accumulation. The Golgi accumulation was completely prevented in cells expressing PH domains that bind to PtdIns4P in the Golgi (FAPP1- and OSBP-PH) and significantly but only partially inhibited by wortmannin (Wm) at concentrations that inhibit type III PI4Ks. The effect of Wm was mimicked by another PI4K inhibitor, PIK93, that inhibits PI4KIIIb but not PI4KIIIa and by siRNA-mediated knock-down of PI4KIIIb but not any other PI4Ks. We observed no additivity between knock-down of type-II PI4Ks and the inhibitors, suggesting that the partial inhibition of ceramide transport by interference with PI4KIIIb did not result from the participation of several PI4Ks in the process. Either Wm or PIK93 strongly but not completely inhibited the conversion of [ ³ H]-serine--labeled ceramide to sphingomyelin but not to glycosyl-ceramide, consistent with the inability of the cells to synthesize SM in the trans-Golgi network (TGN) when ceramide transport from the ER is impaired.

Our studies identified PtdIns4P as a critical regulatory lipid in the Golgi that facilitates ceramide transport and PI4KIIIb as the enzyme that controls the process. This finding is significant in underscoring the importance of inositide lipid--mediated regulation of the transport and metabolism of major phospholipids in mammalian cells, a hitherto unrecognized function of phosphoinositide kinases. Future research will address the question of whether PI4K enzymes also affect cholesterol metabolism.

Identification of PI 4-kinases in zebrafish

Ma, Balla T; in collaboration with Blake, Liu

Four PI4K enzymes have been identified in mammalian genomes; they fall into one of two groups. Type III PI4Ks are structural relatives of PI3Ks and are represented by the PI4KIIIa and PI4KIIIb enzymes, which are highly conserved from yeast to man. Their respective yeast orthologues, Stt4p and Pik1p, are essential genes with nonredundant functions. Type II PI4K enzymes (also in an --a and --b form) represent a completely distinct family of kinases, and their single yeast homologue, LSB6, is a nonessential gene. Pik1p and its mammalian homologue PI4KIIIb are Golgi-localized peripheral membrane proteins that have been implicated in Golgi-to-plasma membrane secretion in both yeast and mammalian cells. However, both the type IIa and IIb PI4K enzymes have also been described in the Golgi/TGN and shown to regulate vesicular trafficking from the TGN. The functions attributed to Stt4p and its orthologue PI4KIIIa have been linked to the plasma membrane and the ER but not to the Golgi.

No studies have been published on the importance of these enzymes for whole organisms other than yeast. PI4KIIIb mutations result in impaired male spermatogenesis in Drosophila owing to a cytokinesis defect; in the plant Arabidopsis, the lack of PI4KIIIb enzyme greatly impairs root hair growth owing to a defect in Golgi-to-plasma membrane vesicular transport. Given that elimination of these enzymes is likely to yield developmental abnormalities in vertebrates, we decided to investigate the role of the enzymes in zebrafish development. As a first step, we cloned all PI4K enzymes from cDNAs prepared from 24-hour embryos. All four PI4Ks were present in the fish and highly homologous to their human counterparts. The fish type-II enzymes possessed PI 4-kinase activity and showed the same intracellular localization in COS-7 cells as their human counterparts.

We assessed the expression pattern of all four PI4Ks by whole-mount in situ hybridization and observed moderate and ubiquitous expression of the enzymes throughout embryonic development, with notable differences in the stages and areas of greatest expression. At 48 hours post-fertilization, both the type IIIa and IIa enzymes showed highest expression in the brain, with expression also detected in the branchial arches and pectoral fin buds. We are now injecting antisense morpholinos directed against the individual PI4K isoforms to determine the functional significance of the presence of four distinct enzymes during zebrafish development.

Inducible localized changes in membrane phosphoinositides by a novel targeting strategy

Várnai, Balla T; in collaboration with Rohács

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is the major phosphoinositide species in mammalian cells and has been associated with numerous molecular events critical for cellular signaling. PtdIns(4,5)P2 is hydrolyzed by phospholipase C enzymes to generate DAG and Ins(1,4,5)P3, two pivotal second messengers. PtdIns(4,5)P2 is also converted by Class I PI 3-kinases to PtdIns(3,4,5)P3, another important membrane-bound messenger molecule. PtdIns(4,5)P2 directly interacts with several ion channels, transporters, and actin-binding proteins and regulates enzymes such as PLC and PLD. Several molecules that are part of the receptor internalization machinery have also been shown to contain inositide-binding domains, but the exact lipid species that regulates them in the cell has not been firmly established. It is a major challenge to understand how a single type of molecule is able to regulate so many processes simultaneously, and perhaps independently, within the plasma membrane (PM).

Part of the difficulty in studying the several functions of PtdIns(4,5)P2 is that it is hard to manipulate phosphoinositide levels within cells. For example, most data on channel regulation rely on the addition of phospholipids to excised patches and on the use of inhibitors, such as high concentrations of Wm, to inhibit PtdIns(4,5)P2 formation. Several attempts have been made to alter the level of PtdIns(4,5)P2 in intact cells by overexpressing either PIP 5-kinase or 5-phosphatase enzymes that generate and eliminate the lipid, respectively. However, given that sustained changes in the level of PtdIns(4,5)P2 initiate a number of trafficking and signaling events, prolonged exposure to such changes will alter the disposition of the cells by the time the effects are analyzed, making it difficult to draw firm conclusions about a direct effect of the lipid on any single process.

To overcome this problem, we developed a strategy for promptly regulating membrane PtdIns(4,5)P2 levels by a drug-inducible membrane-targeting strategy based on the heterodimerization of the FRB domain of mTOR and FKBP12. In this approach, the enzyme of interest (in this case, a type-IV phosphoinositide 5-phosphatase) is fused to the FKBP12 protein and, upon addition of rapamycin (Rapa) or an analogue that does not interact with endogenous proteins, the enzyme rapidly translocates to the membrane where its binding partner, the FRB-domain, is targeted. This method has been used successfully to manipulate small GTP binding proteins at the PM or to study the effects of β-arrestin membrane recruitment.

Rapa-induced PM recruitment of a truncated type-IV 5-phosphatase containing only the 5-phosphatase domain fused to FKBP12 rapidly lowered PM PtdIns(4,5)P2, as monitored by the PLCd1PH-GFP fusion construct. The decrease was paralleled by rapid termination of the ATP-induced Ca2+ signal and prompt inactivation of menthol-activated TRPM8 channels. Depletion of PM PtdIns(4,5)P2 was associated with a complete blockade of transferrin uptake and inhibition of EGF internalization. None of these changes were observed upon Rapa-induced translocation of an mRFP-FKBP12 fusion protein that was used as a control. The data demonstrate that rapid, inducible depletion of PM PtdIns(4,5)P2 is a powerful tool for studying the various regulatory roles of this phospholipid and the differential sensitivities of various processes to PtdIns(4,5)P2 depletion.

COLLABORATORS

Trevor Blake, MS, Genetics and Molecular Biology Branch, NHGRI, Bethesda, MD
Gyorgy Hajnóczky, MD, PhD, Thomas Jefferson University, Philadelphia, PA
Paul Liu, MD, PhD, Genetics and Molecular Biology Branch, NHGRI, Bethesda, MD
Tibor Rohács, MD, PhD, University of Medicine and Dentistry of New Jersey, Newark, NJ
Kevan Shokat, PhD, University of California San Francisco, San Francisco, and University of California Berkeley, Berkeley, CA

For further information, contact tambal@mail.nih.gov.