Membrane-associated STAT3 and PY-STAT3 in the Cytoplasm

Abstract

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...

Cytoplasmic provenance of STAT3 and PY-STAT3 in the endolysosomal compartments in pulmonary arterial endothelial and smooth muscle cells: implications in pulmonary arterial hypertension

“…The following constitutive expression constructs were used in our studies: 1) N1-EGFP and STAT3-EGFP from Dr. Taddaki Miyazaka, Hokkaido University, Sapporo, Japan (54); 2) dominantnegative (DN) dynamin II K44A (Dyn II K44A) from Dr. Lois Greene, National Institutes of Health (NIH), Bethesda, MD (55); 3) murine cav-1 wild-type (WT) from Dr. Ferruccio Galbiati, University of Pittsburgh, Pittsburgh, PA (52); 4) myc-tagged canine cav-1-WT and cav-1-S80A-myc and cav-1-S80E-myc, which are single point mutations in the scaffolding domain of cav-1 from Dr. Jeffery Pessin, SUNY Stony Brook, Stony Brook, NY (48); 5) a myc-tagged double point mutation in the scaffolding domain of cav-1 (canine species) cav-1-F92A/V94A and the corresponding myc-tagged cav-1-WT from Dr. Michael Quon, NIH (30); 6) human GRP58-WT from Drs. Mohammed Bourdi and Lace Pohl, NIH (8); and 7) p950M4-luciferase, a well-characterized STAT3 responsive luciferase construct, which contains four copies of STAT3 DNA-binding element from the human angiotensin promoter from Dr. Ashok Kumar, New York Medical College, Valhalla, NY (26,46).…”

“…Recombinant human IL-6 (R&D Systems, Minneapolis, MN) was used at 10 ng/ml for 30 min unless otherwise specified (24,25,29,39). Phenylarsine oxide (PAO) was from Sigma-Aldrich (St. Louis, MO) and was used at 5 M for 30 min (46).…”

“…Luciferase reporter assays were carried out essentially as described by us earlier (26,46). PAEC or Hep3B cells in six-well plates were transfected with the p950M4-luciferase reporter together with the constitutive ␤-galactosidase expression plasmid pCH110 and either murine cav-1-WT, myc-tagged canine cav-1-WT, cav-1-S80A-myc, cav-1-S80E-myc, cav-1-F92A/V94A, or GRP58-WT using PolyFect.…”

“…5,39,46). In these cell types, components of the endocytic pathway (such as dynamin, epsin, Eps15, and amphiphysin A1) were essential for optimal transcriptional activity of PY-STAT3 (5,46). Additionally, other groups have established that that STAT3 constitutively shuttled between the nucleus and the cytoplasm (35) and that phosphorylation of STAT3 was not a prerequisite for transcriptional activity (57).…”

“…Moreover, we and others have established that the transcytoplasmic trafficking of PY-STAT3 was not a free cytosolic process but was initiated at plasma membrane raft microdomains (with or without cav-1) and proceeded along the cytoplasmic endocytic pathway at least in human hepatocytes (Hep3B cells) and in murine NIH/3T3 and BALB/c 3T3 fibroblast cells ("signaling endosome hypothesis"; Refs. 5,39,46). In these cell types, components of the endocytic pathway (such as dynamin, epsin, Eps15, and amphiphysin A1) were essential for optimal transcriptional activity of PY-STAT3 (5,46).…”