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

Olivier Larochelle,Jae-Won Soh,Valéry Gagné,Carl Séguin,Jean Charron

doi:10.1074/jbc.m108313200

Abstract

We have studied the role of phosphorylation in the activation of metal-regulatory transcription factor-1 (MTF-1) and metallothionein (MT) gene expression. We showed that MTF-1 is phosphorylated in vivo and that zinc stimulates MTF-1 phosphorylation 2-4-fold. Several kinase inhibitors were used to examine 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 (JNK), phosphoinositide 3-kinase, and tyrosine-specific protein kinases inhibitors, as assayed by Northern analysis and by cotransfection experiments using a metal regulatory element-luciferase reporter plasmid. The extracellular signal-activated protein kinase and the p38 kinase cascades did not appear to be essential for the activation of MT gene transcription by metals. By using dominant-negative mutants of PKC, JNK, mitogen-activated kinase kinase 4 (MKK4), and MKK7, we provide further evidence supporting a role for PKC and JNK in the activation of MTF-1 in response 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 gene expression. This suggests that phosphorylation is essential for MTF-1 transactivation function. We hypothesize that metal-induced phosphorylation of MTF-1 is one of the primary events leading to increased MTF-1 activity. Thus, metal ions such as cadmium could activate MTF-1 and induce MT gene expression by stimulating one or several kinases in the MTF-1 signal transduction pathway.

Highlights

¶ Supported by Predoctoral Fellowships from the Fonds pour la Formation des Chercheurs et l’Aide ala Recherche du Quebec and the CRSNG
By using several kinase inhibitors, mitogen extracellular signal-regulated kinase kinase 1 (MEK1)-null mutant cells and dominant negative mutants of protein kinase C (PKC), Jun N-terminal kinase (JNK), and its upstream activators MAP kinase kinase 4 (MKK4) and MKK7, we provide evidence supporting a role for PKC, phosphoinositide 3-kinase (PI3K), and JNK, but not for extracellular signal-activated protein kinase (ERK) or p38, in the activation of metal-regulatory transcription factor-1 (MTF-1) and the induction of MT gene expression in response to metal ions
MTF-1 Is Involved in the Action of PKC, PI3K, JNK, and a Tyrosine Protein Kinase on Metal Induction of MT—To ascertain that the observed inhibition of MT mRNA accumulation in cells treated with the protein kinase inhibitors reflects an inhibition at the transcriptional level and that the intracellular signaling pathway(s) involved in this inhibition converge on MTF-1, transfection experiments were performed in HepG2 cells with the reporter plasmids, 1843MT1-LUC and (MREa)6-LUC

Summary

EXPERIMENTAL PROCEDURES

Materials—PD98059, SB202190, and LY294002 inhibitors were purchased from Calbiochem. The PKC inhibitors GF109203X, Ro-31-8220, and H7 and the JNK inhibitor dicoumarol were obtained from ICN Pharmaceuticals (Costa Mesa, CA). Cells were exposed to the DNACaPO4 precipitate for 3 h, shocked for 3 min at 37 °C with 15% glycerol in HEPES-buffered saline, incubated for 12 h in growth medium, and treated or not with protein kinase inhibitors and metals. Center de Recherche, l’Hotel-Dieu de Quebec) or anti-MAPK-activated protein-K2 (MAPKAPK2) antibodies Some cells were transfected with the expression plasmid pCMV-HA-LacZ (Invitrogen), which encodes for an HA-␤-galactosidase fusion protein with a molecular mass of ϳ115 kDa. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS for 48 h, serumand phosphate-starved for 1 h by incubation in phosphate-free Dulbecco’s modified Eagle’s medium, and incubated with 3 mCi of 32Pi/ml in the same medium for 90 min. The clarified cell lysates were preabsorbed on protein A-Sepharose beads for 30 min at 4 °C and centrifuged, and His-HA-MTF-1 or HA-LacZ was immunoprecipitated with an anti-HA monoclonal antibody (HA.; Babco, Richmond, CA).

RESULTS

H7–25 H7–50 H7–100

DISCUSSION