Real‐time DNA binding measurements of the ETSl recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms

Abstract

The sequence‐specific DNA binding of recombinant p42 and p51 ETSl oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETSl or the phosphorylation of expressed Exon VII in p51 ETSl 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 ETSl displayed significant differences in kinetic behavior. p51 ETSl is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETSl exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETSl DNA binding states are characterized by rapid turnover, whereas the p42 ETSl DNA binding states are 4‐20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETSl with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETSl as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETSl by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETSl sequence‐specific DNA binding occurs through phosphorylation by a calcium‐dependent second messenger. The p42 ETSl lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.