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Dominant-negative mutants of cJun inhibit AP-1 activity through multiple mechanisms and with different potencies
1Biomarkers and Prevention Research Branch, Division of Clinical Sciences, National Cancer Institute, Rockville, Maryland 20850, USA.
Abstract:
We have previously described a dominant-negative mutant of cJun that lacks the transactivation domain (TAD) of cJun and prevents AP-1-mediated transcriptional activation by quenching endogenous Jun or Fos proteins. We now report the development of a panel of cJun mutants that have inactivating mutations in the TAD, DNA-binding domain (DBD), or leucine zipper domain. These mutants are all unable to activate transcription, but only TAD and DBD mutants function in a dominant-negative fashion by inhibiting both cJun-induced transcriptional activation and transformation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in ras-transfected rat embryo cells. Although the TAD and DBD mutants both function as transdominant inhibitors, they work through different mechanisms and with different inhibitory potencies. The DBD mutants, which function by inhibiting DNA binding, are relatively weak inhibitors, whereas the TAD mutants inhibit by quenching and are much more potent. Dimerization assays demonstrate that mutations in the DBD decrease the dimerization affinity of these mutants with cJun. These results demonstrate that the most potent dominant-negative mutants of cJun are proteins that have intact DBDs and quench the activity of the endogenous transcription factors.
Insights
Researchers developed new cJun mutants to study AP-1 transcriptional activation. TAD mutants, which quench transcription factor activity, proved more potent dominant-negative inhibitors than DBD mutants.
Area of Science:
- Molecular Biology
- Cellular Biology
- Cancer Research
Background:
- cJun is a transcription factor involved in AP-1 mediated gene expression.
- Dominant-negative mutants of cJun can inhibit AP-1 transcriptional activity.
- Previous work established a cJun mutant lacking the transactivation domain (TAD).
Purpose of the Study:
- To develop and characterize a panel of cJun mutants with inactivating mutations in different domains.
- To investigate the dominant-negative mechanisms and potencies of these cJun mutants.
- To identify the most effective cJun mutants for inhibiting AP-1 activity.
Main Methods:
- Site-directed mutagenesis to create cJun mutants in the TAD, DNA-binding domain (DBD), and leucine zipper domain.
- Assays for transcriptional activation and inhibition.
- Cell transformation assays using ras-transfected rat embryo cells treated with 12-O-tetradecanoylphorbol-13-acetate.
- Dimerization assays to assess binding affinity.
Main Results:
- All generated cJun mutants were transcriptionally inactive.
- TAD and DBD mutants exhibited dominant-negative effects, inhibiting cJun-induced transcription and cell transformation.
- TAD mutants inhibited by quenching and were more potent than DBD mutants, which inhibited DNA binding.
- Mutations in the DBD reduced dimerization affinity with cJun.
Conclusions:
- Potent dominant-negative cJun mutants possess intact DNA-binding domains.
- Quenching of endogenous transcription factor activity is a highly effective dominant-negative mechanism.
- TAD mutants represent the most potent dominant-negative inhibitors of cJun activity.