JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 22:555–563 (2002) © Mary Ann Liebert, Inc. Association of the Chaperone Glucose-Regulated Protein 58 (GRP58/ER-60/ERp57) with Stat3 in Cytosol and Plasma Membrane Complexes GARY G. GUO, 1 KIRIT PATEL, 1 VINITA KUMAR, 1 MEHUL SHAH, 1 VICTOR A. FRIED, 1 JOSEPH D. ETLINGER, 1 and PRAVIN B. SEHGAL 1,2 ABSTRACT Glucose-regulated protein 58 (GRP58/ER-60/ERp57), best known as a chaperone in the endoplasmic reticu- lum 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-sub- tracted 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-im- munoprecipitated 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 59 nu- cleotidase 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 orthovana- date 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 us- ing 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 mem- bers coassociate in the plasma membrane compartment. We suggest that the chaperone GRP58 may regulate signaling by sequestering inactive and activated Stat3. 555 INTRODUCTION I N THE STANDARD MODEL for cytokine-inducedsignaling through the cytoplasmiccompartmentby the Stat pathway, it has been proposed that Stat family proteins exist in the cytoplasmic com- partment as monomers that are recruited to the plasma mem- brane by binding to the cytoplasmic portion of the respective ligand-activated cytokine receptor polypeptide. (1–3) At the plasma membrane, the Stat proteins are then activated by Tyr- phosphorylation by receptor-associated Janus family kinases (Jak), which are themselves activated by Tyr phosphorylation in trans as a result of binding of cytokine ligand to its recep- tor. The Tyr-phosphorylated Stat proteins depart the receptor complex and dimerize, and then the Stat dimers translocate to the nucleus. (1–3) Recent data from this and several other labo- ratories (4–8) have suggested modifications of aspects of this standard model. Yeung et al., (4) using gel-filtration sieving chromatography, detected multimeric cytosolic complexes of signalingproteinsand cytoskeletalcomponentsin macrophages stimulated with colony-stimulatingfactor-1 (CSF-1). Tyr-phos- phorylated Stat3, Stat5a, and Stat5b were detected by Western blotting in two broad distributions of sizes 1–5 MDa and 300–400 kDa, which size estimates were far larger than those expected for simple Stat dimers (approximately 180 kDa). Lackmann et al. (5) reported that Stat1 in detergent-free cytoso- lic extracts of HeLa cells was in high molecular weight com- Departments of 1 Cell Biology & Anatomy and 2 Medicine, New York Medical College, Valhalla, NY 10595.