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Differential effects of monoclonal antibodies on activating transcription factor-1 and cAMP response element binding
D J Orten1, J M Strawhecker, S D Sanderson
1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198.
Abstract:
Activating transcription factor-1 (ATF1) and cAMP response element binding protein (CREB) have been implicated in cAMP-, calcium-, and virus-induced transcriptional alterations. Although CREB and ATF1 share extensive homology, they appear to mediate distinct cellular functions. We investigated the effect on DNA binding and in vitro transcription of four monoclonal antibodies (mAb) that bound to domains in either the regulatory region (mAb 1 and 3) or unique regions near the DNA-binding domains (mAb 4 and 5) of ATF1.mAb 1 and 3 supershifted both ATF1 and CREB in a DNA binding assay but did not affect in vitro transcription. mAb 4 prevented ATF1-DNA binding while supershifting CREB.DNA complexes and inhibited in vitro transcription by 95% from the CRE-containing murine proliferating cell nuclear antigen promoter. mAb 5 reacted specifically with ATF1 and did not prevent DNA binding or affect in vitro transcription. The mAb 4 epitope was located within ATF1 amino acid residues 205-219, including the first 3 basic residues in the putative DNA-binding domain. Secondary structural analysis predicted that this region comprises a transition site from alpha-helix to a turn-like conformation in ATF1. The transition to turn-like motifs is predicted to occur in CREB after 5 additional residues, with a correspondingly longer alpha-helical domain. Although regulatory domains distinct from DNA binding regions are thought to account for most of the differences in activity of members of the CREB subfamily, our results suggest that small structural variations adjacent to DNA binding regions may also contribute to the distinct functional activities of ATF1 and CREB.
Insights
Monoclonal antibodies targeting Activating Transcription Factor-1 (ATF1) reveal distinct DNA binding and transcription roles for ATF1 and CREB. Structural variations near DNA-binding domains influence their unique cellular functions.
Area of Science:
- Molecular Biology
- Transcription Factors
- Protein Structure-Function Relationships
Background:
- Activating transcription factor-1 (ATF1) and cAMP response element binding protein (CREB) are homologous transcription factors involved in cellular responses.
- Despite structural similarities, ATF1 and CREB mediate distinct cellular functions, the basis of which is not fully understood.
Purpose of the Study:
- To investigate the functional differences between ATF1 and CREB by examining the effects of specific monoclonal antibodies (mAbs) on their DNA binding and transcriptional activity.
- To identify the structural regions responsible for the distinct activities of ATF1 and CREB.
Main Methods:
- Utilized four monoclonal antibodies (mAbs) targeting different domains of ATF1.
- Performed DNA binding assays and in vitro transcription assays using a CRE-containing promoter.
- Analyzed antibody binding sites and predicted secondary protein structures.
Main Results:
- mAbs 1 and 3 recognized regulatory domains, affecting both ATF1 and CREB DNA binding but not transcription.
- mAb 4, targeting a region near the DNA-binding domain, inhibited ATF1 DNA binding and transcription by 95% while affecting CREB.
- mAb 5 specifically bound ATF1 without altering DNA binding or transcription.
Conclusions:
- Structural variations adjacent to the DNA-binding domain, specifically within residues 205-219 of ATF1, contribute to the distinct functional activities of ATF1 and CREB.
- These findings suggest that differences in alpha-helix to turn-like transitions near the DNA-binding domain play a role in the functional divergence of ATF1 and CREB, beyond differences in regulatory domains.