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* Department of Microbiology, Brain Korea 21 Project for Medical Science,
Institute for Immunology and Immunological Diseases, and
National Core Research Center for Nanomedical Technology, Yonsei University College of Medicine, Seoul, Korea
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-
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-1, 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-1 by 
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.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Korea Science and Engineering Foundation through the National Core Research Center for Nanomedical Technology Grant R15-2004-024-00000-0, National Research and Development Program for Cancer Control Grant 0320250-2, and Korean Health 21 Research and Development Program Project 01-PJ10-PG6-01GM03-0002 of the Ministry of Health and Welfare, Yonsei University Biomolecular Secretion Research Center, and Brain Korea 21 Project for Medical Sciences, Republic of Korea.
2 Address correspondence and reprint requests to Dr. Jeon-Soo Shin, Department of Microbiology, Yonsei University College of Medicine, 134 Shinchon-dong Seodaemoon-gu, Seoul 120-752, South Korea. E-mail address: jsshin6203{at}yumc.yonsei.ac.kr
3 Abbreviations used in this paper: HMGB1, high mobility group box 1 protein; CHX, cycloheximide; CRM1, chromosome region maintenance 1; DC, dendritic cell; EGFP, enhanced GFP; KAP, karyopherin; NLS, nuclear localization signal sequence; OA, okadaic acid; PBMo, peripheral blood monocyte; TB, transport buffer; TSA, trichostatin A; WCL, whole cell lysate.
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