SCS | SHR | SME | SMR
| UMSPC | UMST | SSR
| Main Page
PHOSPHOINOSITIDE-CALCIUM SIGNALING IN CELL REGULATION
|
Tamás Balla, MD, PhD, Head, Unit on Molecular Signal Transduction Andras Balla, PhD, Postdoctoral Fellow |
|
|
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. While phosphoinositides constitute a small fraction of the cellular phospholipids, they play a critical role in the regulation of many (if not all) signaling protein complexes that assemble on the surface of cellular membranes. Phosphoinositides regulate protein kinases, GTP-binding proteins, and membrane transporters including ion channels, thereby controlling cellular processes such as proliferation, apoptosis, and metabolism. Our group focuses on the phosphatidylinositol 4-kinases (PI4Ks) that catalyze the first committed step in phosphoinositide synthesis. Current studies are aimed at (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. Inhibition of PI(3,4,5)P3-mediated cellular responses by pleckstrin homology domains Várnai, Bondeva,* A. Balla, Barshishat, T. Balla Pleckstrin-homology (PH) domains are protein modules of about 150 amino acids with a characteristic fold; they have attracted great interest because of their ability to bind to phosphoinositides. PH domains are present in a large variety of signaling molecules, including tyrosine- or serine-threonine kinases, guanine nucleotide exchange factors, GTPase-activating proteins, phospholipases, and a number of adaptor proteins. Given that several types of regulatory molecules possess PH domains that specifically bind to the same phosphoinositide [i.e., PI(3,4,5)P3], we wanted to investigate whether PH domains originating from various regulatory proteins and displaying identical lipid-binding specificity would interfere with PI(3,4,5)P3-regulated cellular responses in a similar manner. Therefore, several PH domains with the ability to bind to phosphatidylinositol 3,4,5,-trisphosphate [PI(3,4,5)P3] were expressed in NIH 3T3 and COS-7 cells as GFP fusion proteins to determine their effects on various cellular responses known to be activated by PI(3,4,5)P3. All these proteins, namely, the PH domains of Grp1, Akt, ARNO, and Btk, were found to show PDGF-stimulated and wortmannin-sensitive translocation from the cytosol to the plasma membrane, indicating their ability to recognize PI(3,4,5)P3. Remarkably, while overexpressed AktPH-GFP and BtkPH-GFP were quite potent in antagonizing the PI(3,4,5)P3-mediated activation of Akt, overexpression of the other PH domains showed no such inhibitory effect. In contrast, expression of the PH domains of Grp1 and ARNO, but not those of Akt or Btk, inhibited the attachment of freshly seeded cells to culture dishes. Activation of PLCgamma by EGF was also attenuated by the PH domains of Grp1, ARNO, and Akt but was significantly enhanced by the Btk PH domain. Followed over several days, the kinetics of expression of the various GFP-fused PH domains clearly showed a lipid-binding- dependent self-elimination of the PH-domain of Akt, consistent with its interference with the anti-apoptotic Akt signaling pathway. Moreover, NIH 3T3 cells expressing the various PH domains showed characteristic morphological changes: greatly elongated extensions in response to Grp1-PH and ARNO-PH and a larger foot-print size in response to the Akt PH. The data suggest that interaction with and sequestration of PI(3,4,5)P3 is not the sole mechanism by which PH domains interact with cellular membranes and that PH domains are likely to possess structural features that allow their more specific participation in the assembly and function of specific signaling pathways. Bondeva T, Balla A, Várnai P, BallaT. Structural determinants of Ras-Raf interaction analyzed in live cells. Mol Biol Cell 2002;13:2323-2333. Characterization of the pleckstrin homology domain of phospholipase C-delta4 Várnai, A. Balla, T. Balla; in collaboration with Jalink, Lee, Rhee Some of the most important regulators of phosphoinositide levels are the phospholipase C (PLC) enzymes that hydrolyze phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Of the various isoforms of PLCs, the delta forms are the most evolutionarily conserved and possess a unique regulatory feature, namely, of regulation by the lipid PI(4,5)P2 itself. Lipid regulation takes place via the pleckstrin homology (PH) domain of PLCdelta1, a protein module that is characterized by high-affinity binding to the lipid as well as to the water-soluble Ins(1,4,5)P3. Recent studies have identified a new isoform of PLCdelta, the delta4 enzyme, and indicated that its PH domain, unlike that of delta1, does not bind to Ins(1,4,5)P3. To understand the role of the PLCdelta4 PH domain in the localization and function of PLCdelta4 enzyme, we compared the inositol-lipid and inositol phosphate binding properties of the enzyme, and its isolated PH domain, with the similar features of the PLCdelta1 protein. Both PLCdelta enzymes showed binding to lipid micelles containing PI(4,5)P2, but the binding of PLCdelta4 was significantly less sensitive to inhibition by Ins(1,4,5)P3 than that of PLCdelta1. The isolated PH domain of PLCdelta4, expressed as a GFP fusion protein, also displayed PI(4,5)P2-dependent binding to lipid micelles as well as high-affinity binding to the soluble Ins(1,4,5)P3. A 10-fold higher concentration of InsP3 was required to displace the PLCdelta4 PH domain from the lipid vesicles compared with PLCdelta1-PH, with a lower Ins(1,4,5)P3 affinity reflected in the Ins(1,4,5)P3 binding assays. Importantly, the concentrations of InsP3 required to displace the full-length proteins from the lipid micelles were significantly higher than the isolated PH-domains with both isoforms of PLCdelta. When expressed in various cells, both PLCdelta1-PH-GFP and PLCdelta4-PH-GFP localized to the plasma membrane, but significantly higher concentrations of the latter were found in the cytosol. PLCdelta4-PH also showed prominent nuclear localization, with bright nucleolar dots that were never detected with the PLCdelta1-PH domain. However, the full-length PLCdelta4 was found to be associated primarily with the endoplasmic reticulum and showed neither a nuclear nor a prominent plasma membrane localization. The data suggest that the PH domain of PLCdelta4 is not responsible for the localization of the enzyme, probably because it is not fully exposed in the molecule. The differential effects of Ins(1,4,5)P3 on the membrane association and localization of PLCdelta1-PH and PLCdelta4-PH are partly attributable to differences in their PI(4,5)P2 and InsP3 affinities. Our data also suggest that intramolecular interactions between the PH domain and the rest of the molecule contribute to the different Ins(1,4,5)P3 sensitivities as well as to the differential localization of these proteins. Halet G, Tunwell R, Balla T, Swann K, Carroll J. The dynamics of plasma membrane PtdIns(4,5)P2 at fertilization of mouse eggs. J Cell Sci 2002;115:2139-2149. Varnai P, Lin X, Lee SB, Tuymetova G, Bondeva T, Spat A, Rhee SG, Hajnoczky G, Balla T. Inositol lipid binding and membrane localization of isolated pleckstrin homology domains. Studies on the PH-domains of PLCdelta 1 and p130. J Biol Chem 2002;277:27412-27422. Monitoring phosphatidylinositol 4-kinase activity in single cells using the PH domains of the oxysterol binding protein and the adaptor protein FAPP1 A. Balla, Tujmetova, Tsiomenko, Várnai, T. Balla Phosphatidylinositol 4-kinases catalyze the first reaction step in the synthesis of phosphoinositides. Four mammalian PI 4-kinases have been identified to date; the type-III enzymes (alpha and beta forms) have similarities to PI 3-kinases within their catalytic domains while the type-II forms (alpha and beta) belong to a new family of lipid kinases. We have previously described the differential cellular localization of these proteins and have been seeking methods by which their reaction product PI(4)P could be detected in single cells. Protein domains, including PH domains, fused to the green fluorescent protein (GFP) have been successfully used to follow localized inositol lipid changes in living cells, an area in which our group has been particularly active. Based on in vitro binding assays, we have shown that the PH domains of the oxysterol binding protein (OSBP) and the adaptor protein FAPP1 bind to PI(4)P very specifically. Therefore, we investigated the usefulness of the PH domains of the two proteins to detect PI(4)P in living cells. When expressed in COS-7 cells, OSBP-PH was associated with the Golgi and with small vesicles scattered around the cytoplasm, a small amount also being found in the plasma membrane. FAPP1-PH was more conned to the Golgi, especially in live cells. Expression of both domains at high levels caused fragmentation and tubulation of the Golgi that was reminiscent of the changes caused by brefeldin A. Although both PH domains showed co-localization with PI4K type-IIIbeta over the Golgi, inhibition of the enzyme by wortmannin did not change the distribution of either PH domains. In cells overexpressing the type-II PI 4-kinases, we observed no co-localization of either PH domains with the enzymes in the endocytic compartments and found only the Golgi-localized fraction of the type-IIbeta enzyme in co-localization with either of the PH domains. Activation of PLC by Ca2+ ionophores caused dissociation of OSBP-PH from all membrane sites, a condition that rapidly reversed after restoration of Ca2+ levels. The plasma membrane association of OSBP-PH was significantly higher after the Ca2+ transient and completely prevented by wortmannin. The data suggest that the localization of the OSBP and FAPP1 PH domains is only partially dependent on PI(4)P and that the Golgi localization of these constructs involves additional, probably protein-protein, interactions. However, the use of these molecular tools has allowed the identification of the type-III PI 4-kinases as the source of PI(4)P formation in the plasma membrane after Ca2+-induced PLC activation. Balla A, Tuymetova G, Barshishat M, Geiszt M, Balla T. Characterization of type-II phosphatidylinositol 4-kinase isoforms reveals association of the enzymes with endosomal vesicular compartments. J Biol Chem 2002;277:20041-20050. Balla T, Varnai P. Visualizing cellular phosphoinositide pools with GFP-fused protein-modules. Science STKE 2002;125:PL3. COLLABORATORS Kees Jalink, PhD, Division of Cell Biology, The Netherlands Cancer Institute, The Netherlands Sang Bong Lee, PhD, Laboratory of Cell Signaling, NHLBI, Bethesda MD Sue Goo Rhee, PhD, Laboratory of Cell Signaling, NHLBI, Bethesda MD
*Tzvetanka Bondeva, PhD, former Visiting Fellow
For further information, contact tambal@box-t.nih.gov |
|
