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Originally published In Press as doi:10.1074/jbc.M112153200 on February 27, 2002

J. Biol. Chem., Vol. 277, Issue 18, 15621-15628, May 3, 2002
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Rapid Phosphorylation of Heterogeneous Nuclear Ribonucleoprotein C1/C2 in Response to Physiologic Levels of Hydrogen Peroxide in Human Endothelial Cells*

James R. StoneDagger and Tucker Collins§

From the Departments of Pathology, Children's Hospital and Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Hydrogen peroxide (H2O2) has been implicated as a signaling agent in numerous signal transduction pathways in mammalian cells. However, to date, no sensor for low concentrations (<10 µM) of H2O2 has been identified. Using a functional proteomic approach, nuclear extracts from human umbilical vein endothelial cells were analyzed by two-dimensional PAGE with or without prior treatment with a low concentration of H2O2. A protein doublet with a molecular mass of 39-41 kDa and a pI of ~5.0 was observed to be consistently altered by the treatment. Using proteolytic peptide mass fingerprinting, the protein was identified as heterogeneous nuclear ribonucleoprotein C1/C2, a nuclear restricted, pre-mRNA-binding protein. Upon two-dimensional PAGE, each heterogeneous nuclear ribonucleoprotein-C splice form was present as multiple spots because of differing levels of phosphorylation. Upon treatment with H2O2, there was an increase in phosphorylation at 10-20 min, which partially reversed by 30 min. Subsequently, at 60 min after treatment, a population of unphosphorylated protein was transiently present. The effects were observed with as little as 1 µM H2O2 and were maximal with 5-8 µM H2O2. The H2O2-stimulated phosphorylation was inhibited by catalase, but not by the transcriptional inhibitor actinomycin D.

* This work was supported in part by National Institutes of Health Grant R37 HL35716.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Grant T32 HL07627.

§ To whom correspondence should be addressed: Dept. of Pathology, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-5806; Fax: 617-734-4721; E-mail: tcollins@rics.bwh.harvard.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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