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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 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 one family of enzymes, the phosphatidylinositol 4 (PtdIns4)–kinases (PI4Ks), that catalyze the first committed step in polyphosphoinositide synthesis. Current studies aim to (i) understand the function and regulation of several PI4Ks 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 PtdIns4 P–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.
Identification of a new ER-derived phosphatidylinositol-synthesizing organelle
Polyphosphoinositides (PPIs) are phosphorylated forms of phosphatidylinositol (PtdIns) formed by a variety of PI and PIP kinases in eukaryotic cells. Although present in minute amounts, these phospholipids are of enormous interest because of their pivotal roles in regulating virtually every cellular process within eukaryotic cells. These lipids first rose to prominence as precursors of important second messengers, generated upon stimulation of certain groups of cell-surface receptors. However, PPIs have proven to be more versatile in that they also regulate ion channels and transporters and control membrane fusion and fission events and hence are master regulators of vesicular transport, secretion, and endocytosis; they also play key roles in lipid transport and disposition.
While the distribution and dynamics of PPIs have been mapped with various lipid-binding domains, our knowledge of the localization of PtdIns itself and the mechanism of its distribution within cells has been limited. We found that the PtdIns–synthesizing enzyme PIS associates with a rapidly moving compartment of endoplasmic reticulum (ER) origin that makes ample contacts with various organelles, including the plasma membrane. These PIS–positive objects originated from the ER, and their generation was abolished by expression of a GTP–locked form of the small GTPase Sar1, a regulator of ER exit sites. Mutations that rendered PIS catalytically incompetent also prevented the objects' movement to the mobile fraction. Density-gradient fractionation showed that PIS activity was associated primarily with light membrane fractions that were separable from the bulk of the ER. Importantly, the two CDP–diacylglycerol (DAG) synthase enzymes that provide PIS with its substrate, CDP-DAG, were found in the tubular ER but not in the PIS–positive organelle. To determine the site(s) of PtdIns localization, we also expressed a PtdIns–specific bacterial PLC (PI-PLC) enzyme to generate DAG and detected the latter with a high-affinity DAG sensor. Surprisingly, after PI–PLC expression, DAG was detected in rapidly moving cytoplasmic objects, confirming the presence of PtdIns primarily in cytoplasmic structures. Using targeted PI-PLC enzymes, we also depleted PtdIns in various membrane compartments and showed that PtdIns has to be depleted in the plasma membrane in order to have an impact on PtdIns(4,5)P2 levels. We propose a model in which PtdIns is synthesized in a highly mobile lipid distribution platform and is delivered to other membranes during multiple contacts by yet-to-be-defined lipid transfer mechanisms.
Genomic organization of PI4KIIIalpha and characterization of its isoforms
Phosphatidylinositol 4-kinases (PI4Ks) catalyze the first committed step in phosphoinositide synthesis. In the mammalian genome, four PI4Ks belong to two distinct families. Type III PI4Ks are relatives of the PI 3-kinases and exist in alpha and beta forms while type II PI4Ks (also with alpha and beta forms) are smaller proteins forming a distinct family. Each of these kinases appears to be dedicated to specific regulatory processes that are under investogation.
Two isoforms of the mammalian PI4KIIIα have been described and annotated in GenBank—a larger isoform of about 230kDa (isoform 2) and a shorter splice variant containing only the C-terminus of approximately 97kDa that includes the catalytic domain (isoform 1). However, Northern analysis of human tissues and cancer cells showed only a single transcript of about 7.5 kb, except for the proerythroleukemia line K562, which contains a significantly higher level of the 7.5 kb transcript along with smaller ones of 2.4 kb, 3.5 kb, and 4.2 kb. Bioinformatic analysis confirmed the high copy number of PI4KIIIα transcript in K562 cells along with several genes located in the same region in Chr22, including two pseudogenes that cover most exons coding for isoform 1, consistent with chromosome amplification. A panel of polyclonal antibodies, raised against peptides within the C-terminal half of PI4KIIIα, failed to detect the shorter isoform 1 in either COS-7 cells or K562 cells, even though the antibodies detected the larger isoform 2. Moreover, expression of a cDNA encoding isoform 1 yielded a protein of about 97 kDa that showed no catalytic activity and failed to rescue hepatitis C virus replication. These data drew attention to PI4KIIIα as one of the genes found in Chr22q11, a region affected by chromosomal instability, but did not substantiate the existence of a functionally relevant short form of PI4KIIIα.
The role of PI4KIIIalpha in HCV replication
Viruses use several of the host cell's signaling machinery for infection, viability, and replication. During infection, the virus initiates a sequence of highly organized subcellular events that change the cellular architecture and physiology to favor the viral replication process. 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, which include many human pathogens such as picornaviruses (e.g., the enteroviral genus members poliovirus [PV] and coxsackie-virus B3 [CVB3], rhinovirus, and hepatitis A virus), 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. Recent studies identified PI4KIIIa as an essential host factor for the replication of HCV in the liver. Our group has been directly involved in research addressing the role of PI4Ks in viral replication; this year, we collaborated with Ralf Bartenschlager's group to address the role(s) of PI4KIIIa in HCV–infected cells. The studies showed that PI4KIIIa is associated with the non-structural viral protein NS5A and that the association increases the enzyme's catalytic activity. Accordingly, HCV–infected hepatocytes showed elevated levels of PtdIns4P, and interference with PI4KIIIa activity prevented the characteristic structural change induced in the membrane architecture of HCV–infected cells. The studies identified PI4KIIIa as a major mediator of the process by which HCV re-organizes the intracellular membranes to generate its replication platform and the NS5A protein as an important link to alter the activity of this lipid kinase. Targeting PI4KIIIa, and possibly other PI4Ks, may help fight several forms of viral infections.
Development of a non-radioactive PI 4-kinase assay adaptable for high-throughput screening
Recent studies have shown that several small RNA viruses require PI4Ks for their replication. Among these, HCV requires PI4KIIIa while some enteroviruses require PI4KIIIb. The studies identified type III PI4Ks as potential targets for antiviral therapy and made screening for PI4K inhibitors highly desirable. Unfortunately, the current methods for detecting in vitro PI4K kinase activity are based on radioactive incorporation of phosphate from 32P-labeled ATP to PtdIns and involve a lipid extraction step with organic solvents. To overcome this limitation, we collaborated with Andrew Tai to develop a non-radioactive fluorescent assay based on ADP detection. When the inhibitor sensitivities of the various PI4K enzymes were tested and compared, the method proved highly sensitive and yielded results identical to those of the conventional radioactive kinase assay . The assay was adaptable regardless of type of PI4K used. The new method will be highly suitable in screens for PI4K–inhibitory compounds.