We investigate signal transduction pathways that mediate the actions of hormones and growth factors 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 (if not all) signaling protein complexes that assemble on the surface of cellular membranes. For example, they regulate protein kinases and GTP-binding proteins as well as membrane transporters such as ion channels, thereby controlling many cellular processes such as proliferation, apoptosis, and metabolism. We focus on phosphatidylinositol 4–kinases (PI4Ks), a group of enzymes that catalyze the first committed step in phosphoinositide synthesis. Our current studies aim at understanding the function and regulation of several phosphatidylinositol (PI) 4–kinases in controlling the synthesis of hormone-sensitive phosphoinositide pools; characterizing the structural features that determine the catalytic specificity and inhibitor sensitivity of PI 3– and PI 4–kinases; defining the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology and other domains of selected regulatory proteins; developing tools to analyze inositol lipid dynamics in live cells; and 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 regulator of the plasma membrane pool of phosphoinositides
Balla A, Várnai, Tuymetova, Tóth
The cellular effects of many hormones and neurotransmitters are initiated by receptor-activated hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) in order to generate two important second messengers: Ins(1,4,5)P3 (InsP3) and diacylglycerol (DAG). InsP3 rapidly binds to its intracellular receptors and releases Ca2+ from non-mitochondrial Ca2+ stores to produce the cytoplasmic Ca2+ increase while DAG activates the various isoforms of PKC; both messengers trigger specific cellular responses of the target cell. We have shown previously that the sustained formation of these messengers depends on the activity of the type III PI 4-kinases (PI4Ks). None of the type-III PI4Ks are detectable by immunofluorescence in the plasma membrane, which is where the PI(4)P has to be generated for conversion to PI(4,5)P2. We have used small interfering RNA (siRNA) to downregulate the levels of the four cellular PI4Ks and applied the pleckstrin homology (PH) domains of the OSBP, FAPP1, and OSH2 proteins fused to GFP to monitor PI(4)P production in single COS-7 cells. The OSBP and FAPP1 PH domains could report on PI(4)P synthesis in the plasma membrane, but only after recovery from a robust PLC activation evoked by the addition of ionomycin, a Ca2+ ionophore, while the OSH2 PH domain can detect PI(4)P in the plasma membrane without stimulation of the PI turnover. After recovery from Ca2+ stimulation, the plasma membrane localization of PHOSBP and PHFAPP1 was inhibited by wortmannin, which inhibits both type-III PI4Ks, and by a concentration of phenylarsine-oxide (PAO) that specifically inhibits the type-IIIalpha, but not the type-IIIbeta enzyme. The localization of both PH domains was not affected by RNAi-mediated knock-down of either PI4KIIIbeta or PI4KIIalpha but was greatly inhibited by the knock-down of the PI4KIIIalpha enzyme. In HEK293 cells stably expressing the AT1a angiotensin receptor, angiotensin II (AngII) stimulation caused the transient translocation of the PHOSH2 tandem from the plasma membrane to the cytosol, indicating that PLC-mediated hydrolysis of PI(4)P parallels the changes observed in 32P-labeled PI(4)P. Whether monitored by the PHOSH2-tandem translocation or by metabolic labeling, wortmannin or PAO (but not a specific inhibitor of the PI4KIIIbeta enzyme) inhibited the resynthesis of PI(4)P (see below). We found similar inhibitor sensitivity when we analyzed the AngII-induced InsP3 and the cytoplasmic Ca2+ signal. Our observations suggest that the PI4KIIIalpha enzyme carries out synthesis of the agonist-regulated PI(4)P and PI(4,5)P2 pools. Further studies are in progress to determine how the PI4K isofom, which is primarily localized in the endoplasmic reticulum, generates PI(4)P in the plasma membrane.
Balla A, Tuymetova G, Tsiomenko A, Várnai P, Balla T. A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1. Mol Biol Cell 2005;16:1282-1295.
Várnai P, Balla A, Hunyady L, Balla T. Targeted expression of the all-helical segment of the IP3 receptor ligand binding domain is capable of inducing Ca2+ release via the endogenous IP3 receptor channels. Proc Natl Acad Sci USA 2005;102:7859-7864.
Pharmacological discrimination between the functionsof PI 4-kinases
Balla A, Tuymetova; in collaboration with Shokat
Downregulation by siRNA has been the only tool available for studying the specific involvement of PI4K isoforms in various cellular functions. However, even low residual enzyme activity is often sufficient to maintain function, and the prolonged depletion of the enzymes needed for effective downregulation changes the entire trafficking pattern and lipid transport of the cells, making it difficult to pinpoint the primary process that requires PI4K activity. Therefore, identifying and developing specific inhibitors for the individual PI4Ks is highly desirable. To that end, we have been testing selected PI3K inhibitors under development that are active against the type-III PI4Ks in Kevan Shokat’s laboratory. One of the tested inhibitors was two orders of magnitude more potent against the PI4KIIIbeta than the PI4KIIIalpha and showed no activity against the type-II PI4K enzymes. In addition, myricetin was more potent against PI4KIIIbeta than the PI4KIIIalpha enzyme. Of the inhibitors subjected to testing we found phenylarsine-oxide (PAO) to be most active against the PI4KIIIalpha enzyme, but at higher concentrations PAO also inhibited the other PI4Ks. Based on molecular modeling of the PI4K type-III enzymes, we generated mutant enzymes with altered inhibitor sensitivity. One of the mutants of PI4KIIIbeta (Y583M) was 300 times less sensitive to wortmannin than the wild-type and retained about 90 percent catalytic activity. Similarly, we have generated a mutant PI4KIIIalpha enzyme (C1843S) that shows 20-fold reduced PAO sensitivity. We are using the inhibitors and mutant enzyme in combination with RNAi to analyze the cellular functions of the PI4K enzyme.
Balla A, Tuymetova G, Tsiomenko A, Várnai P, Balla T. A plasma membrane pool of phosphatidylinositol 4-phosphate is generated by phosphatidylinositol 4-kinase type-III alpha: studies with the PH domains of the oxysterol binding protein and FAPP1. Mol Biol Cell 2005;16:1282-1295.
Identification of residues in pleckstrin homology domains that affect their signaling function
Várnai, Tóth; in collaboration with Buday, Hunyady
Pleckstrin homology domains are small protein modules found in many signaling proteins. They mediate the interaction of proteins with cellular membranes and confer regulation by inositol phospholipids. Many PH domains are able to interact with specific phosphoinositides even when isolated from their parent proteins, a feature that makes them popular for visualizing phosphoinositides in living cells. Overexpression of PH domains also interferes with cellular responses, and we showed previously that distinct PH domains capable of binding to the lipid PI(3,4,5)P3 have selective inhibitory effects on certain PI(3,4,5)P3-mediated cellular responses, but not on others. This finding raised the possibility that the inhibitory effects of PH domains are attributable to the domains’ sequestering not only the inositol lipid in question but also protein binding partners, resulting in more pathway-specific inhibition. To validate this assumption, we generated mutations on the surface of the PH domains of Akt and Grp1 that should not influence the domains’ lipid binding and membrane interaction but should alter their protein-protein interaction. The studies yielded mutant PH domains indistinguishable from their wild-type counterparts with respect to inositol-lipid or inositol-phosphate binding but showed greatly reduced inhibition of Akt activation or of cell spreading (for AktPH and Grp1PH, respectively) when expressed in COS-7 cells. The data point to the importance of protein-protein interactions of PH domains in addition to the domains’ interaction with phosphoinositides. Further studies are in progress to determine the importance of the domains in the context of the whole molecule and, over time, to identify the protein binding partner of the selected PH domains.
Balla T. Inositol-lipid binding motifs: signal integrators through protein-lipid and protein-protein interactions. J Cell Sci2005;118:2093-2104.
Várnai P, Bondeva T, Tamas P, Buday L, Hunyady L, Balla T. Selective cellular effects of overexpressed pleckstrin homology domains that recognize PI(3,4,5)P3 suggest their interaction with protein binding partners. J Cell Sci 2005;118:4879-4888.
COLLABORATORS
László Buday, MD, PhD, DSc, Semmelweis University, Faculty of Medicine, Budapest, Hungary
László Hunyady, MD, PhD, DSc, Semmelweis University, Faculty of Medicine, Budapest, Hungary
Kevan Shokat, PhD, University of California San Francisco, San Francisco, and University of California Berkeley, Berkeley, CA
Peter Steinbach, PhD, Center for Information Technology, NIH, Bethesda, MD
For further information, contact tambal@mail.nih.gov.