Interactions of STAT3 with Caveolin-1 and Heat Shock Protein 90 in Plasma Membrane Raft and Cytosolic Complexes

Abstract

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-␤-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°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.Fever ("calor") is a common response of the body to infection and injury (reviewed in Refs. 1 and 2). Interleukin-6 (IL-6) 1 is a major systemic mediator of this "acute-phase" response that includes stimulation of the liver to synthesize and secrete a large number of protective plasma proteins such as anti-proteinases, clotting factors, complement factors, and scavenger proteins that help to limit the site of injury or infection (1, 2). Furthermore, IL-6 is itself a centrally acting pyrogen (3-5).How is the major signaling pathway used by IL-6 to enhance acute-phase plasma protein gene expression in hepatocytes, the JAK-STAT3 pathway (Janus kinase-signal transducer and activator of transcription 3) (6 -11), maintained during an increase in body temperature? We report the identification of caveolin-1 (cav-1) and heat shock protein 90 (HSP90) as STAT3-associated proteins at the level of plasma membrane rafts and in high molecular mass cytoplasmic complexes, and we explore their role in cytokine signaling at normal and elevated temperatures.In previous cell fractionation studies of human hepatom...

Pyrrolidine Dithiocarbamate Inhibits Interleukin-6 Signaling through Impaired STAT3 Activation and Association with Transcriptional Coactivators in Hepatocytes

“…Treatment of HepG2 cells with PDTC resulted in a reduction in the extent of Hsp90 interaction with endogenous STAT3 and STAT3-ER. As was shown previously by others (31,32), the inhibitor geldanamycin, known to bind tightly to Hsp90, has been found to disrupt STAT3-Hsp90 interactions. Prompted by these observations, we examined the redistribution of STAT3 by IL-6 in HepG2 cells subjected to geldanamycin.…”

“…Recent studies have shown the association of Hsp90 with STAT3 in cytosol and plasma membrane complexes where the gp130/JAK signaling module is known to reside (31,49). Therefore, we examined whether cell treatment with PDTC can cause release of STAT3 from Hsp90.…”

“…A recent study has shown that the chaperone Hsp90 is a key component of the multimeric protein complex encompassing STAT3 (31). We investigated whether Hsp90 was indeed associated with the endogenous STAT3.…”

Hsp90 Inhibition Accelerates Cell Lysis

“…Geldanamycin treatment efficiently dissociated several Hsp90-related protein complexes in lipid rafts (56,57). Since lipid rafts are usually isolated after a nonionic detergent extraction, the similar experimental condi- FIG.…”

Signal transduction of erbB receptors in trastuzumab (Herceptin) sensitive and resistant cell lines: Local stimulation using magnetic microspheres as assessed by quantitative digital microscopy

“…Immunomagnetic microsphere separation and microparticle based flow cytometry play an important role in experimental protocols and analytical procedures (19,20). Recently, microspheres have also served as powerful tools for studying complex cell biological phenomena such as collagen phagocytosis in fibroblasts (21,22), viscoelastic responses of endothelial cells upon thrombin stimulation measured by magnetic microsphere-based microrheology (23), and re-organization of lipid rafts and signalling complexes upon various stimuli (24)(25)(26)(27)(28)(29)(30).…”