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J. Biol. Chem., Vol. 276, Issue 23, 20544-20550, June 8, 2001
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From the Departments of Protein phosphorylation frequently results in the
subcellular redistribution of key signaling molecules, and this spatial change is critical for their activity. Here we have probed the effects
of a Cdc25 inhibitor,
2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, or Compound 5, on
the spatial regulation and activation kinetics of tyrosine
phosphorylation-dependent signaling events using two methods: (i) high-content, automated, fluorescence-based, solid-phase cytometry and (ii) a novel cellular assay for Cdc25A activity in intact
cells. Immunofluorescence studies demonstrated that Compound 5 produced
a concentration-dependent nuclear accumulation of
phospho-Erk and phospho-p38, but not nuclear factor
Spatial Analysis of Key Signaling Proteins by
High-content Solid-phase Cytometry in Hep3B Cells Treated with an
Inhibitor of Cdc25 Dual-specificity Phosphatases*
,,,,¶
Pharmacology and
§ Surgery, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261
B. Immunoblot analysis confirmed Erk phosphorylation and nuclear accumulation, and
in vitro kinase assays showed that Compound 5-activated Erk was competent to phosphorylate its physiological substrate, the transcription factor Elk-1. Pretreatment of cells with the MEK inhibitor U-0126 prevented the induction by Compound 5 of phospho-Erk (but not phospho-p38) nuclear accumulation and protected cells from the
antiproliferative effects of Compound 5. Overexpression of Cdc25A in
whole cells caused dephosphorylation of Erk that was reversed by
Compound 5. The data show that an inhibitor of Cdc25 increases Erk
phosphorylation and nuclear accumulation and support the hypothesis
that Cdc25A regulates Erk phosphorylation status.
*
This work was supported by National Institutes of Health
Grants CA 78039, CA 52995, and CA 82723; the Fiske Drug Discovery Fund;
and a seed grant from the Pittsburgh Tissue Engineering Initiative.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.
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