Cellular Physiology of STAT3: Where's the Cytoplasmic Monomer?

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 tyrosine-phosphorylated STAT3 eluting at 80-100 kDa was observed, and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-called SIF-A "homodimer") co-eluted 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-co-shifted 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.