Fra-2-positive autoregulatory loop triggered by mitogen-activated protein kinase (MAPK) and Fra-2 phosphorylation sites by MAPK.
We reported previously that activation of endogenous activator protein 1 (AP-1) in chicken embryo fibroblasts is essential for the cellular transformation induced by v-src, and we further showed that the activation of AP-1 is accompanied by elevation of Fra-2 and c-Jun expression and also high-level phosphorylation of Fra-2 by activated endogenous extracellular signal-regulated kinase [mitogen-activated protein kinase (MAPK)]. Here, we report that the transcriptional activity of Fra-2/c-Jun heterodimer was greatly enhanced by cotransfecting a constitutively active mutant of MEK1 gene (MEK-DD) into F9 cells, indicating that Fra-2 was converted into an active transactivator after phosphorylation by MAPK. High-level expression of MEK-DD alone was sufficient to induce clear cellular transformation of chicken embryo fibroblasts, which caused constitutive activation of endogenous MAPK, hyperphosphorylation of Fra-2, and elevation of fra-2 and c-jun gene expression. These results indicate that phosphorylation of Fra-2 by MAPK plays an important role in stimulating endogenous AP-1 activity in a positive autoregulation mechanism, in which phosphorylated Fra-2 induces fra-2 expression through AP-1 binding sites present in its promoter. We also localized the Fra-2 phosphorylation sites by MAPK to three threonine and three serine residues in the COOH-terminal region by means of site-directed mutagenesis and showed that the threonine residues were more susceptible to MAPK.
Murakami
M
M
Department of Gene Regulation, Institute of Medical Science, University of Tokyo, Japan.
Ui
M
M
Iba
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002642:Chick Embryo; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D051921:Fos-Related Antigen-2; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012967:Sodium Dodecyl Sulfate; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D051921:Fos-Related Antigen-2; C497873:Fosl2 protein, mouse; D014157:Transcription Factors; D013912:Threonine; D012967:Sodium Dodecyl Sulfate; D017871:Calcium-Calmodulin-Dependent Protein Kinases