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Isolation and characterization of STAT3 and PY‐STAT3 signaling endosomes
Author(s) -
Shah Mehul,
Xu Fang,
Sehgal Pravin
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a935-d
Subject(s) - endosome , microbiology and biotechnology , cytoplasm , endocytic cycle , biology , cytosol , chemistry , endocytosis , biochemistry , cell , intracellular , enzyme
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.