Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion.
The high mobility group box 1 (HMGB1) protein can be secreted by activated monocytes and macrophages and functions as a late mediator of sepsis. HMGB1 contains two nuclear localization signals (NLSs) for controlled nuclear transport, and acetylation of both NLSs of HMGB1 is involved in nuclear transport toward secretion. However, phosphorylation of HMGB1 and its relation to nuclear transport have not been shown. We show here that HMGB1 is phosphorylated and dynamically shuttled between cytoplasmic and nuclear compartments according to its phosphorylation state. Phosphorylation of HMGB1 was detected by metabolic labeling and Western blot analysis after treatments with TNF-alpha and okadaic acid, a phosphatase inhibitor. Hyperphosphorylated HMGB1 in RAW 264.7 and human monocytes was relocated to the cytoplasm. In a nuclear import assay, phosphorylated HMGB1 in the cytoplasm did not enter the nucleus. We mutated serine residues of either or both NLSs of HMGB1 to glutamic acid to simulate a phosphorylated state and examined the binding of HMGB1 to karyopherin-alpha1, which was identified as the nuclear import protein for HMGB1 in this study. Substitution to glutamic acid in either NLSs decreased the binding with karyopherin-alpha1 by approximately 50%; however, substitution of both NLSs showed no binding, and HMGB1 was relocated to the cytoplasm and subsequently secreted. These data support the hypothesis that HMGB1 could be phosphorylated and that the direction of transport is regulated by phosphorylation of both NLS regions.
Youn
Ju Ho
JH
Department of Microbiology, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemoon-gu, Seoul 120-752, Korea.
Shin
Jeon-Soo
JS
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D024243:HMGB1 Protein / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D051379:Mice; D019913:Nuclear Localization Signals / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport / Q000502:physiology
D024243:HMGB1 Protein; D019913:Nuclear Localization Signals