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J. Biol. Chem., Vol. 277, Issue 26, 23193-23207, June 28, 2002
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From the Center for Cell Signaling Research, Division of
Molecular Life Sciences and College of Pharmacy, Ewha Womans
University, Seoul 120-750, Korea
Heat shock (HS) induces a wide
variety of biological processes, including inhibition of protein
synthesis, elevated expression of heat shock proteins, induction of
thermotolerance, and apoptotic cell death in a
dose-dependent manner. We compared phosphorylated proteins
in heat-shocked and thermotolerant cells using proteome analysis. After
HS treatment of control RIF-1 and their thermotolerant derivatives,
TR-RIF-1 cells, cellular proteins were separated by two-dimensional gel
electrophoresis and the phosphorylated proteins were detected with the
anti-phosphotyrosine antibodies. We found that 93 proteins showed
significant changes in phosphorylation between control and
thermotolerant cells as a function of recovery time after HS; we
identified 81 of these proteins with peptide mass fingerprinting using
MALDI-TOF MS after in-gel trypsin digestion. These phosphorylated
proteins exhibit various cellular functions, including chaperones, ion
channels, signaling molecules, in transcription and translation
processes, in amino acid biosynthesis, oxidoreduction, energy
metabolism, and cell motility or structure, suggesting that HS turns on
the various signaling pathways by activating protein-tyrosine kinases
(PTKs). Of these, 20 proteins were previously identified phosphorylated
proteins and 64 were newly identified. These proteins can be grouped
into three families: 1) proteins highly phosphorylated in TR-RIF-1
cells at basal level and phosphorylated more significantly by HS in
RIF-1 than TR-RIF-1; 2) proteins highly phosphorylated in control RIF-1
cells at basal level and phosphorylated more easily by HS in TR-RIF-1
than in RIF-1 cells; and 3) proteins with a similar basal
phosphorylation level in both RIF-1 and TR-RIF-1 cells and responding
to HS similarly in both cells. Most of the phosphorylated proteins are
presumably involved in HS signaling in different ways, with the first
and second families of proteins influencing thermotolerance. The
possible tyrosine phosphorylation sites, the possible PTKs
phosphorylating these proteins, and the proteins binding to these
phosphorylated sites were predicted by the Netphos, ScanProsite, and
Scansite programs. These results suggest that HS can activate various
PTKs and HS responses can be regulated by phosphorylations of
proteins having various functions.
Proteomic Analysis of Protein Phosphorylations in Heat Shock
Response and Thermotolerance*
,, and
*
This work was supported in part by Korea Science and
Engineering Foundation through the Center for Cell Signaling Research at Ewha Womans University, by Korea Institute of Science & Technology Evaluation and Planning Research Fund for Women's Universities, and by
IMT2000 Project IMT2000-C5-2 for IT-BT.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.
Supported by the Brain Korea 21 Project. Both authors contributed
equally to this work.
§
To whom correspondence should be addressed: Division of Molecular
Life Sciences and College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea. Tel.: 82-2-3277-3038; Fax: 82-2-3277-3760; E-mail: kjl@mm.ewha.ac.kr.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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