Spatial analysis of key signaling proteins by high-content solid-phase cytometry in Hep3B cells treated with an inhibitor of Cdc25 dual-specificity phosphatases.
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 kappaB. 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.
Departments of Pharmacology and Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
A
A
Vogt
T
T
Adachi
A P
AP
Ducruet
J
J
Chesebrough
K
K
Nemoto
B I
BI
Carr
J S
JS
Lazo
D016475:3T3 Cells; D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051379:Mice; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D015398:Signal Transduction; D013379:Substrate Specificity; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D002070:Butadienes; D004791:Enzyme Inhibitors; D009570:Nitriles; C113580:U 0126; D020687:cdc25 Phosphatases