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Generation of recombinant affinity reagents against a two-phosphosite epitope of ATF2
Jennifer E McGinnis1, Brian K Kay1
1Department of Biological Sciences, University of Illinois at Chicago, 900 S. Ashland Ave., MBRB 4318, MC 567, Chicago, IL 60607 USA.
Abstract:
Activating Transcription Factor 2 (ATF2) plays an important role in mammalian cell proliferation, apoptosis and DNA repair. Its activation is dependent on the sequential phosphorylation of residue threonine 71 (T71) followed by threonine 69 (T69) in its transactivation domain. While these modifications can be directed by a variety of kinases, the time to reach full phosphorylation is dependent on which signaling pathway has been activated, which is thought to be important for proper temporal regulation. To explore this phenomenon further, there have been ongoing efforts to generate affinity reagents for monitoring phosphorylation events in cellular assays. While phospho-specific antibodies have been valuable tools for monitoring cell signaling events, those raised against a peptide containing two or more adjacent phosphosites tend to cross-react with that peptide's various phospho-states, rendering such reagents unusable for studying sequential phosphorylation. As an alternative, we have employed the N-terminal Forkhead-associated 1 (FHA1) domain of yeast Rad53p as a scaffold to generate recombinant affinity reagents via phage display and were successful in generating a set of reagents that can distinguish between the dual-phosphorylated epitope, 63-IVADQpTPpTPTRFLK-77, and the mono-phosphorylated epitope, 63-IVADQpTPTPTRFLK-77, in the human ATF2 transactivation domain.
Insights
Researchers developed new reagents to study sequential phosphorylation of Activating Transcription Factor 2 (ATF2). These tools can distinguish between mono- and dual-phosphorylated states, crucial for understanding cell signaling and temporal regulation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Activating Transcription Factor 2 (ATF2) is vital for cell proliferation, apoptosis, and DNA repair.
- ATF2 activation requires sequential phosphorylation at threonine 71 (T71) and threonine 69 (T69).
- Existing phospho-specific antibodies struggle to differentiate between sequential phosphorylation states.
Purpose of the Study:
- To develop novel affinity reagents for monitoring sequential phosphorylation of ATF2.
- To overcome limitations of current antibodies in distinguishing mono- vs. dual-phosphorylated ATF2.
- To enable precise temporal regulation studies of ATF2 signaling pathways.
Main Methods:
- Utilized the N-terminal Forkhead-associated 1 (FHA1) domain of yeast Rad53p as a scaffold.
- Employed phage display technology to generate recombinant affinity reagents.
- Designed reagents to specifically recognize distinct phosphorylation states of the ATF2 transactivation domain.
Main Results:
- Successfully generated affinity reagents capable of distinguishing between dual-phosphorylated and mono-phosphorylated ATF2 epitopes.
- Demonstrated the ability to differentiate between the 63-IVADQpTPpTPTRFLK-77 (dual) and 63-IVADQpTPTPTRFLK-77 (mono) states.
- Provided tools for accurate monitoring of sequential phosphorylation events in cellular assays.
Conclusions:
- Developed novel, highly specific affinity reagents for studying ATF2 phosphorylation.
- These reagents overcome the cross-reactivity issues of traditional antibodies for sequential phosphosites.
- Facilitates deeper understanding of ATF2's role in cellular processes through precise temporal signaling analysis.
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