Isolation and characterization of STAT3 and PY‐STAT3 signaling endosomes

Abstract

Cytokine- and growth factor-initiated STAT signaling transits from the plasma membrane to the nucleus. Until recently, STAT signaling has been viewed exclusively as a soluble cytosolic process. We have previously presented evidence for membrane-associated STAT3 signaling through the cytoplasm [Shah et al (2005) The FASEB Journal 19, Abstract 209.9]. Using a detergent dissection approach to determine the relative magnitude of membrane-associated versus free cytosolic PY-STAT3 signaling, approximately 70 % of IL-6-activated cytoplasmic PY-STAT3 was found to be associated with cytoplasmic vesicular elements suggesting that the membrane-associated signaling is the major pathway. There was constitutive as well as an IL-6-enhanced association of cytoplasmic STAT3 and PY-STAT3 with vesicular components positive for markers of the endolysosomal pathways [clathrin heavy chain (CHC), Rab5, Rab7, LAMP1, LAMP2 and EEA1]. To test whether significant amounts of STAT3 and PY-STAT3 were associated with isolated endocytic organelles as predicted by the STAT3 signaling endosome model, early endosomes enriched for EEA1 and Rab5 were purified from IL-6-treated Hep3B cells [Aniento et al (1996) J. Cell. Biol. 133: 29-41]. STAT3 complexed with CHC constitutively associated with this early endosome fraction. At 15 min after IL-6, the majority of cytoplasmic PY-STAT3 was associated with early endosomes, with the remainder in the late endosome and low-density membrane fractions; there was little soluble PY-STAT3. These data provide direct evidence in support of the STAT3 signaling endosome pathway. Supported by a Susan G. Komen Foundation dissertation award (MS) and NIH-HL73301.