u-0126 and 1-7-phenanthroline

The membrane-permeable intracellular heavy metal chelator, 1,10-phenanthroline, which prevents progesterone-induced germinal vesicle breakdown (GVBD), would be expected to regulate phosphorylation (activation) of the MAP kinase (MAPK) cascade in Xenopus oocytes. Here, our experiments show that 1,10-phenanthroline itself results in the phosphorylation of MAPK in both oocytes and a cell-free system. In contrast, 1,7-phenanthroline, the nonchelating analogue, had no effect. A supplement of zinc (as a heavy metal) given to 1,10-phenanthroline-loaded oocytes suppressed the stimulatory effects of 1,10-phenanthroline, while 1,10-phenanthroline withdrawal caused dephosphorylation of activated MAPK. Further, treatment with a MEK (a MAPK kinase) inhibitor, PD 098059 or U0126, suppressed 1,10-phenanthroline-stimulated MAPK phosphorylation, indicating that 1,10-phenanthroline can phosphorylate MAPK in a MEK-dependent fashion. Our results suggest that phosphorylation of MAPK by 1,10-phenanthroline depends on the interaction of MEK. Thus, the intracellular heavy metal (zinc) regulates MAPK phosphorylation and 1,10-phenanthroline can serve as a unique tool for investigating MAPK phosphorylation mechanism.

Topics: Animals; Blotting, Western; Butadienes; Cell-Free System; Chromones; Colforsin; Cycloheximide; Enzyme Inhibitors; Female; Flavonoids; Indoles; Kinetics; Maleimides; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Morpholines; Nitriles; Oocytes; Pentetic Acid; Phenanthrolines; Phosphorylation; Progesterone; Threonine; Tyrosine; Xenopus laevis; Zinc Sulfate