In the standard model of cytokine-induced signal transducer and activator of transcription (STAT) protein family signaling to the cell nucleus, it is assumed that STAT3 is recruited to the cytoplasmic side of the cell surface receptor complex from within a cytosolic monomer pool. By using Superose-6 gel-filtration chromatography, we have discovered that there is little monomeric STAT3 (91 kDa) in the cytosol of liver cells (human hepatoma Hep3B cell line and rat liver). The bulk of STAT3 (and STAT1, STAT5a, and -b) was present in the cytosol as high molecular mass complexes in two broad distributions in the size range 200 -400 kDa ("statosome I") and 1-2 MDa ("statosome II"). Upon treatment of Hep3B cells with interleukin-6 (IL-6) for 30 min (i) cytosolic tyrosine-phosphorylated STAT3 was found to be in complexes of size ranging from 200 -400 kDa to 1-2 MDa; (ii) a small pool of monomeric STAT3 and tyrosinephosphorylated STAT3 eluting at 80 -100 kDa was observed, and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-called SIF-A "homodimer") coeluted with catalase at 230 kDa. In order to identify the protein components of the 200 -400-kDa statosome I cytosolic complexes, we used the novel technique of antibody-subtracted differential protein display using anti-STAT3 antibody. Eight polypeptides in the size range from 20 to 114 kDa co-shifted with STAT3; three of these (p60, p20a, and p20b) were co-shifted in an IL-6-dependent manner. In-gel tryptic fragmentation and mass spectroscopy identified the major IL-6-dependent STAT3-coshifted p60 protein as the chaperone GRP58/ER-60/ ERp57. Taken together, these data (i) emphasize the absence of a detectable STAT3 monomer pool in the cytosol of cytokine-free liver cells as posited by the standard model, and (ii) suggest an alternative model for STAT signaling in which STAT3 proteins function in the cytoplasm as heteromeric complexes with accessory scaffolding proteins, including the chaperone GRP58.

Signal transduction from the plasma membrane to the nucleus by STAT proteins is widely represented as exclusively a soluble cytosolic process. Using cell-fractionation methods, we observed that ϳ5% of cytoplasmic STAT3 was constitutively associated with the purified early endosome (EE) fraction in human Hep3B liver cells. By 15-30 min after interleukin-6 (IL-6) treatment, up to two-thirds of cytoplasmic Tyr-phosphorylated STAT3 can be associated with the purified early endosome fraction (Rab-5-, EEA1-, transferrin receptor-, and clathrin-positive fraction). Electron microscopy, immunofluorescence, and detergent dissection approaches confirmed the association of STAT3 and PY-STAT3 with early endosomes. STAT3 was constitutively associated with clathrin heavy chain in membrane and in the 1-to 2-MDa cytosolic complexes. The membrane association was dynamic in that, within 15 min of treatment with the vicinal-thiol cross-linker phenylarsine oxide, there was a dramatic increase in bulk STAT3 association with sedimentable membranes. The functional contribution of PY-STAT3 association with the endocytic pathway was evaluated in transient transfection assays using IL-6-inducible STAT3-reporter-luciferase constructs and selective regulators of this pathway. STAT3-transcriptional activation was inhibited by expression constructs for dominant negative dynamin K44A, epsin 2a, amphiphysin A1, and clathrin light chain but enhanced by that for the active dynamin species MxA. Taken together, these studies emphasize the contribution of the endocytic pathway to productive IL-6/STAT3 signaling.Diverse cytokines and growth factors, including various interleukins and interferons, signal to the cell nucleus by activating the JAK 2 /STAT (Janus kinase/signal transducers and activators of transcription) pathway at the plasma membrane at the level of raft microdomains (reviewed in Refs. 1-3). This signal transduction from the plasma membrane to the nucleus by Tyr-and/or Ser-phosphorylated STAT proteins is widely represented exclusively as a soluble cytosolic process.Although until recently it was considered that latent STAT proteins in the cytoplasm were monomeric, work from this and other laboratories showed that latent STATs exist in the cytosol already in the form of at least dimers and included higher order complexes (200 -400 kDa statosome I and 1-to 2-MDa statosome II complexes) (4 -8, reviewed in Refs. 9 and 10). The absence of free STAT monomers in the cytoplasm has now been extensively confirmed (11)(12)(13)(14). Moreover, recent fluorescence transfer and fluorescence correlation spectroscopy data confirm the existence of STAT3 dimers and higher order statosome complexes (200 -400 kDa and 1-2 MDa) in the cytoplasm of live cells (15)(16)(17).That different growth factor and cytokine receptors are associated with the endocytic pathway and can even maintain their ongoing signaling function from this membrane-bound compartment has been clearly delineated (18 -20). Nevertheless, today, the widely represented model of IL-6/STAT3 signa...

Glucose-regulated protein 58 (GRP58/ER-60/ERp57), best known as a chaperone in the endoplasmic reticulum lumen, was previously identified by us as one of several accessory proteins in the S100 cytosol fraction of human hepatoma Hep3B cells that was differentially coshifted by anti-Stat3 antibody in an antibody-subtracted differential protein display assay. In the present study, the association between GRP58 and Stat3 in different cytoplasmic compartments was evaluated using cross-immunoprecipitation and cell-fractionation techniques. In the S100 cytosol fraction, three different anti-GRP58 polyclonal antibodies (pAb) cross-immunoprecipitated Stat3 (but not Stat1), and, conversely, anti-Stat3 pAb cross-immunoprecipitated GRP58. Both cytosolic Stat3 and GRP58 eluted during Superose-6 gel-filtration chromatography in complexes of size 200-400 kDa (statosome I), and anti-Stat3 pAb cross-immunoprecipitated GRp58 from these FPLC elution fractions. Using differential sedimentation and density equilibrium flotation methods, Stat3 and GRP58 were observed to be coassociated with cytoplasmic membranes enriched for the plasma membrane marker 5' nucleotidase but not with those containing the endoplasmic reticulum marker BiP/GRP78. The Stat3 and GRP58-containing plasma membrane fraction also contained Stat1, Stat5b, and gp130. Stat activation by orthovanadate caused the accumulation of PY-Stat3 in the GRP58-containing plasma membrane fraction. However, this PY-Stat3 was DNA-binding deficient. Likewise, excess exogenous recombinant human GRP58 prepared using a baculovirus expression system preferentially inhibited Stat3 DNA-binding activity in the S100 cytosol, suggesting that GRP58 may sequester activated Stat3. The new data confirm the association between GRP58 and Stat3 in cytosolic 200-400-kDa statosome I complexes and show that both GRP58 and Stat family members coassociate in the plasma membrane compartment. We suggest that the chaperone GRP58 may regulate signaling by sequestering inactive and activated Stat3.

STAT transcription factors signal from the plasma membrane to the nucleus in response to growth factors and cytokines. We have investigated whether plasma membrane "rafts" are involved in cytokine-activated STAT signaling. Cytokine-free human hepatoma Hep3B cells or cells treated with interleukin-6 (IL-6) or orthovanadate (a general activator of STATs) were fractionated, and plasma membrane raft fractions were obtained by equilibrium sedimentation or flotation through discontinuous sucrose gradients using either non-detergent or detergent-based (saponin or Triton X-100) methods. By Western blotting the plasma membrane raft fractions obtained using either non-detergent or detergent-based methods contained significant amounts of STAT1 and STAT3 (up to ϳ10% of the total cytoplasmic amount) as well as the integral raft proteins caveolin-1 and flotillin-1, the IL-6-receptor signal transducing chain gp130, the interferon-␥ receptor ␣ chain (IFN-␥R␣), and the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57). Upon activation of signaling by IL-6 or orthovanadate the respective Tyr-phosphorylated STAT species were now also observed in the membrane raft fraction but in a form deficient in DNA binding. The data show pre-association of STATs with plasma membrane rafts in flotation fractions, which also contained caveolin-1 and flotillin-1, and suggest that Tyr phosphorylation may not in itself be sufficient to cause the departure of PY-STATs from plasma membrane rafts. Methyl-␤-cyclodextrin, which sequesters cholesterol and disrupts plasma membrane rafts, markedly inhibited IL-6-and IFN-␥-induced STAT signaling. Signaling through specialized raft microdomains may be a general mechanism operating at the level of the plasma membrane through which cytokines and growth factors activate STAT species (the "raft-STAT signaling hypothesis").Signal transduction in mammalian cells is initiated by complex protein-protein interactions between ligands, receptors, and kinases at the level of the plasma membrane. It is now becoming clear that specialized microdomains at the cell surface, known as rafts and/or caveolae, are intimately involved in this process (1, 2). These lipid microdomains contain high concentrations of glycolipids, sphingomyelin, and cholesterol and represent platforms for conducting cellular functions such as vesicular trafficking and signal transduction (1, 2). Raft domains contain several integral raft proteins, which include caveolin and flotillin family members. Signaling processes shown to involve plasma membrane raft domains include immunoglobulin E signaling, T-cell antigen receptor signaling, B-cell receptor signaling, signaling involving epidermal growth factor, platelet-derived growth factor, insulin receptor, Ephrin B1 receptor, neurotrophin, Ha-Ras, nitric-oxide synthase and integrins (reviewed in Refs. 1-4). Caveolin-1 in rafts has been shown to modulate insulin-and Ha-Ras-mediated signaling (the "caveola signaling hypothesis") (3, 4). Despite these major advances in the understanding of rafts as...

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