Membrane-associated STAT3 and PY-STAT3 in the Cytoplasm

Mehul Shah,Kirit Patel,Somshuvra Mukhopadhyay,Fang Xu,Gary Guo,Pravin B Sehgal

doi:10.1074/jbc.m508527200

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 approximately 5% 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.

Highlights

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)
We observed the association of STAT3 and IL-6-activated PY-STAT3 with cytoplasmic membrane fractions, which lacked detectable levels of the plasma membrane marker 5Ј-nucleotidase and of the cytosolic marker lactate dehydrogenase (Fig. 1A)
This association between STAT3 and CHC was observed in the soluble cytosol S100-derived 1- to 2-MDa statosome II complexes previously reported by us (Fig. 1D), reminiscent of the constitutively cycling membrane-associated and soluble pools of CHC in the endocytic pathway

Summary

Introduction

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). 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]. In cell fractionation experiments we reported the association of Western-blottable STAT3 and PY-STAT3 as well as DNA-binding competent PY-STAT3 with a large granular membrane fraction (the “P15 fraction”) as well as a P100 sedimentable cytoplasmic fraction [6, 7]. These data were at variance with the idea of IL-6/STAT3 signaling as exclusively a soluble cytosolic process. In this report we demonstrate the association of STAT3 and PY-STAT3 with the purified early endosome fraction and provide evidence for a significant role of this pathway in the transcriptional activation function of STAT3

Methods

Results

Conclusion