Cellular physiology of STAT3: Where's the cytoplasmic monomer?

In the standard model of cytokine-induced signal transducer and activator o f transcription (STAT) protein family signaling to the cell nucleus, it is assumed that STATE 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 mon omeric STATE (91 kDa) in the cytosol of liver cells (human hepatoma Hep3B c ell line and rat liver). The bulb: of STAT3 (and STAT1, STAT5a, and -b) was present in the cytosol as high molecular mass complexes in two broad distr ibutions in the size range 200-400 kDa ("statosome I") and 1-2 MDa ("statos ome II"). Upon treatment of Hep3B cells with interleukin-6 (IL-6) for 30 mi n (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 STATE and tyrosine-phosphorylated STATE eluting at 80-100 kDa was observed , and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-cal led SIE-A "homodimer") coeluted with catalase at 230 kDa. In order to ident ify the protein components of the 200-400-kDa statosome I cytosolic complex es, we used the novel technique of antibody-subtracted differential protein display using anti-STAT3 antibody. Eight polypeptides in the size range fr om 20 to 114 kDa co-shifted with STATE; 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 cytoso l of cytokine-free liver cells as posited by the standard model, and (ii) s uggest an alternative model for STAT signaling in which STAT3 proteins func tion in the cytoplasm as heteromeric complexes with accessory scaffolding p roteins, including the chaperone GRP58.