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Real-time DNA binding measurements of the ETS1 recombinant oncoproteins reveal significant kinetic differences
R J Fisher1, M Fivash, J Casas-Finet
1Laboratory of Cellular Biochemistry, PRI/DynCorp, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702.
Abstract:
The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.
Insights
The phosphorylation status of ETS1 oncoprotein significantly impacts its DNA binding kinetics and stability. While p51 ETS1 shows rapid binding and turnover, p42 ETS1 exhibits slower binding and more stable DNA interactions.
Area of Science:
- Molecular Biology
- Biochemistry
- Oncology
Background:
- ETS1 is an oncoprotein involved in gene regulation.
- Two isoforms, p42 ETS1 and p51 ETS1, exist with differing phosphorylation domains.
- Understanding their DNA binding is crucial for cancer research.
Purpose of the Study:
- To quantitatively assess the sequence-specific DNA binding of p42 and p51 ETS1.
- To determine the effect of Exon VII phosphorylation on ETS1 DNA binding activity.
- To elucidate the regulatory mechanisms of ETS1 DNA binding.
Main Methods:
- Quantitative analysis of DNA binding using surface plasmon resonance (BIAcore technology).
- Kinetic measurements of real-time binding and complex formation.
- Mutational analysis to determine DNA sequence specificity.
- In vitro phosphorylation assays using CAM kinase II.
Main Results:
- p51 ETS1 demonstrated fast initial binding and rapid complex turnover.
- p42 ETS1 exhibited slow initial binding with 4-20 times greater complex stability.
- Both isoforms formed 1:1 complexes with specific oligonucleotides.
- DNA sequence specificity was similar between p42 and p51 ETS1.
- Phosphorylation of p51 ETS1 by CAM kinase II abolished DNA binding.
- p42 ETS1 DNA binding was insensitive to calcium signaling.
Conclusions:
- ETS1 phosphorylation status critically modulates its DNA binding kinetics and stability.
- p51 ETS1 DNA binding is regulated by calcium-dependent phosphorylation.
- p42 ETS1, lacking the Exon VII domain, shows altered and unregulated DNA binding.
- These findings provide insights into ETS1 function in gene regulation and cancer.