REAL-TIME DNA-BINDING MEASUREMENTS OF THE ETS1 RECOMBINANT ONCOPROTEINS REVEAL SIGNIFICANT KINETIC DIFFERENCES BETWEEN THE P42 AND P51 ISOFORMS

The sequence-specific DNA binding of recombinant p42 and p51 ETS1 onco protein was examined quantitatively to determine whether the loss of t he Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activit y. The kinetics of sequence-specific DNA binding was measured using re al-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p 42 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 an d conversion to a stable complex. All of the p51 ETS1 DNA binding stat es are characterized by rapid turnover, whereas the p42 ETS1 DNA bindi ng states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ET S1 with spe specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by muta tional analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting t hat the regulation of p51 ETS1 sequence-specific DNA binding occurs th rough phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its spec ific DNA sequence is not sensitive to calcium signaling.