betadex and geldanamycin

The heterotrimeric G proteins, G(12) and G(13), are closely related in their sequences, signaling partners, and cellular effects such as oncogenic transformation and cytoskeletal reorganization. Yet G(12) and G(13) can act through different pathways, bind different proteins, and show opposing actions on some effectors. We investigated the compartmentalization of G(12) and G(13) at the membrane because other G proteins reside in lipid rafts, membrane microdomains enriched in cholesterol and sphingolipids. Lipid rafts were isolated after cold, nonionic detergent extraction of cells and gradient centrifugation. Galpha(12) was in the lipid raft fractions, whereas Galpha(13) was not associated with lipid rafts. Mutation of Cys-11 on Galpha(12), which prevents its palmitoylation, partially shifted Galpha(12) from the lipid rafts. Geldanamycin treatment, which specifically inhibits Hsp90, caused a partial loss of wild-type Galpha(12) and a complete loss of the Cys-11 mutant from the lipid rafts and the appearance of a higher molecular weight form of Galpha(12) in the soluble fractions. These results indicate that acylation and Hsp90 interactions localized Galpha(12) to lipid rafts. Hsp90 may act as both a scaffold and chaperone to maintain a functional Galpha(12) only in discrete membrane domains and thereby explain some of the nonoverlapping functions of G(12) and G(13) and control of these potent cell regulators.

Topics: 3T3 Cells; Acylation; Animals; Benzoquinones; beta-Cyclodextrins; Cell Line; COS Cells; Cyclodextrins; Cysteine; Detergents; Dimerization; DNA-Binding Proteins; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, G12-G13; Heterotrimeric GTP-Binding Proteins; HSP90 Heat-Shock Proteins; Humans; Immunoblotting; Lactams, Macrocyclic; Membrane Microdomains; Mice; Octoxynol; Protein Binding; Protein Structure, Tertiary; Protein Transport; Quinones; Signal Transduction; Subcellular Fractions; Transfection

Interleukin-6 (IL-6) initiates STAT3 signaling in plasma membrane rafts with the subsequent transit of Tyr-phosphorylated STAT3 (PY-STAT3) through the cytoplasmic compartment to the nucleus in association with accessory proteins. We initially identified caveolin-1 (cav-1) as a candidate STAT3-associated accessory protein due to its co-localization with STAT3 and PY-STAT3 in flotation raft fractions, and heat shock protein 90 (HSP90) due to its inclusion in cytosolic STAT3-containing 200-400-kDa complexes. Subsequent immunomagnetic bead pullout assays showed that STAT3, PY-STAT3, cav-1, and HSP90 interacted in plasma membrane and cytoplasmic complexes derived from uninduced and stimulated Hep3B cells. This was a general property of STAT3 in that these interactions were also observed in alveolar epithelial type II-like cells, lung fibroblasts, and pulmonary arterial endothelial cells. Exposure of Hep3B cells to the raft disrupter methyl-beta-cyclodextrin for 1-10 min followed by IL-6 stimulation for 15 min preferentially inhibited the appearance of PY-STAT3 in the cav-1-enriched sedimentable cytoplasmic fraction, suggesting that these complexes may represent a trafficking intermediate immediately downstream from the raft. Because IL-6 is known to function in the body in the context of fever, the possibility that HSP90 may help preserve IL-6-induced STAT3 signaling at elevated temperature was investigated. Geldanamycin, an HSP90 inhibitor, markedly inhibited IL-6-stimulated STAT3 signaling in Hep3B hepatocytes cultured overnight at 39.5 degrees C as evaluated by DNA-shift assays, trafficking of PY-STAT3 to the nucleus, cross-precipitation of HSP90 by anti-STAT3 polyclonal antibody, and reporter/luciferase construct experiments. Taken together, the data show that IL-6/raft/STAT3 signaling is a chaperoned pathway that involves cav-1 and HSP90 as accessory proteins and suggest a mechanism for the preservation of this signaling during fever.

Topics: Animals; Benzoquinones; beta-Cyclodextrins; Cattle; Caveolin 1; Caveolins; Cell Fractionation; Cell Membrane; Cell Nucleus; Cells, Cultured; Cyclodextrins; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Endothelium, Vascular; Filipin; Genes, Reporter; Heat-Shock Proteins; Hepatocytes; Hot Temperature; HSP90 Heat-Shock Proteins; Humans; Interferon-gamma; Interleukin-6; Isomerases; Lactams, Macrocyclic; Macromolecular Substances; Membrane Microdomains; Molecular Chaperones; Phosphorylation; Protein Binding; Protein Disulfide-Isomerases; Protein Transport; Quinones; Respiratory Mucosa; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Tyrosine