Evidence for the ability of nucleophosmin/B23 to bind ATP.
By taking advantage of its ability to be retained by ATP-agarose, we have demonstrated that nucleophosmin/B23 is capable of binding ATP. The specificity of the binding was confirmed by the absence of significant binding to AMP-agarose and by its loss when nucleophosmin/B23 in nuclear extracts was preincubated with ATP. Preincubation of the nuclear extracts with other ribonucleotide triphosphates (GTP, CTP, UTP) did not compete for the binding of nucleophosmin/B23 to ATP-agarose. The purified recombinant nucleophosmin/B23 was also able to be retained by ATP-agarose. The Kd for binding of ATP to the purified recombinant nucleophosmin/B23, on the basis of retention on a nitrocellulose membrane, was 86.5+/-8.3 microM; the number of binding sites was 0.68 per nucleophosmin/B23 protein molecule. To determine the possible ATP-binding site of nucleophosmin/B23, various deletion clones including the two mutants in which the putative ATP-binding sequence had been deleted were constructed. Deletion of the portions of the molecule (residues 83-152 and 185-240) had little effect on the ATP binding. The C-terminal deleted mutant (residue 242 to the C-terminus deleted) lost most of its ability to be retained by ATP-agarose and to bind [alpha-32P]ATP on a nitrocellulose membrane. The results indicate that the C-terminal portion (residues 242-294) contains the essential ATP-binding site of nucleophosmin/B23.
Chang
J H
JH
Cancer Biochemistry Laboratory, Department of Pharmacology, Chang Gung University, Taiwan, Republic of China.
Lin
J Y
JY
Wu
M H
MH
Yung
B Y
BY
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002466:Cell Nucleolus / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000090243:Nucleophosmin; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion
D002352:Carrier Proteins; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D000090243:Nucleophosmin; D000255:Adenosine Triphosphate