Evidence for the ability of nucleophosmin/B23 to bind ATP

Abstract

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 μM; 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 [α-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.

Role of Protein Kinase CK2 Phosphorylation in the Molecular Chaperone Activity of Nucleolar Protein B23

“…Although protein B23 is capable of binding ATP (32), extensive studies in our laboratory have never detected any significant ATPase activity in the protein itself under any conditions. 2 Thus, it is unlikely that the affinity of B23 for a substrate is regulated by direct nucleoside triphosphate hydrolysis.…”

“…Incorporation of 32 P from [␥-32 P]ATP into the complex catalyzed by CK2 was monitored in the linear phase of the reaction (Fig. 1A).…”

“…The N- 32 P was monitored by scintillation counting. The temperatures for the CK2 reactions were 37°C for rhodanese and citrate synthase and 30°C for HIV-1 Rev.…”

Nucleophosmin and its complex network: a possible therapeutic target in hematological diseases

“…In vitro functional studies suggest that NPM displays chaperone-like behaviors on a wide variety of denatured substrates: (i) NPM inhibits temperaturedependent and independent protein aggregation, due to thermal denaturation, protecting enzymes from loss of activity; (ii) NPM promotes renaturation of chemically denatured proteins and it appears to preferentially bind denatured substrates through the exposure of hydrophobic regions in both NPM and substrates (Szebeni and Olson, 1999). Although NPM binds ATP, at least in vitro (Chang et al, 1998), its binding to substrates is governed by association-dissociation thermodynamics, and release of the substrate does not require ATP hydrolysis (Szebeni and Olson, 1999), as seen with other molecular chaperones including Hsp70 and chaperonins (Ruddon and Bedows, 1997). Interestingly, it has been shown that phosphorylation of NPM by casein kinase II induces the release of denaturated substrates (Szebeni et al, 2003).…”

Nucleophosmin/B23, a multifunctional protein that can regulate apoptosis

“…Positively charged lysine residues in the C-terminus of B23 have been implicated in binding negatively charged ATP. 69 Mutation of lysine residues into asparagines disrupts B23 binding to ATP. Similarly, B23(K194N,K200N) and B23(K228N,K234N) mutants completely lost the binding activity to PIP3 compared to wild-type B23.…”