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 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.
Fisher
R J
RJ
Laboratory of Cellular Biochemistry, PRI/DynCorp, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702.
Fivash
M
M
Casas-Finet
J
J
Erickson
J W
JW
Kondoh
A
A
Bladen
S V
SV
Fisher
C
C
Watson
D K
DK
Papas
T
T
D001483:Base Sequence; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004586:Electrophoresis; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017354:Point Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D011994:Recombinant Proteins; D015682:Retroviridae Proteins, Oncogenic; C050379:oncogene proteins v-ets; D004247:DNA; D017871:Calcium-Calmodulin-Dependent Protein Kinases