Phosphorylation is involved in the activation of metal-regulatory transcription factor 1 in response to metal ions

We have studied the role of phosphorylation in the activation of metal-regu latory transcription factor-1 (MTF-1) and metallothionein (MT) gene express ion. We showed that MTF-1 is phosphorylated in vivo and that zinc stimulate s MTF-1 phosphorylation 2-4-fold. Several kinase inhibitors were used to ex amine the possible involvement of kinase cascades in the activation of MTF- 1. Metal-induced MT gene expression was abrogated by protein kinase C (PKC) , c-Jun N-terminal kinase (JN-K), phosphoinositide 3-kinase, and tyrosine-s pecific protein kinases inhibitors, as assayed by Northern analysis and by cotransfection experiments using a metal regulatory element-luciferase repo rter plasmid. The extracellular signal-activated protein kinase and the p38 kinase cascades did not appear to be essential for the activation Aff gene transcription by metals. By using dominant-negative mutants of PKC, JNK., mitogen-activated kinase kinase 4 (MKK4), and MKK7, we provide further evid ence supporting a role for PKC and JNK in the activation of MTF-1 in respon se to metals. Notably, increased MTF-1 DNA binding in response to zinc and MTF-1 nuclear localization was not inhibited in cells preincubated with the different kinase inhibitors despite strong inhibition of MTF-1-mediated ge ne expression. This suggests that phosphorylation is essential for MTF-1 tr ansactivation function. We hypothesize that metal-induced phosphorylation o f MTF-1 is one of the primary events leading to increased MTF-1 activity. T hus, metal ions such as cadmium could activate MTF-1 and induce MT gene exp ression by stimulating one or several kinases in the MTF-1 signal transduct ion pathway.