Nucleophosmin (B23) Targets ARF to Nucleoli and Inhibits Its Function

Abstract

The ARF tumor suppressor is a nucleolar protein that activates p53-dependent checkpoints by binding Mdm2, a p53 antagonist. Despite persuasive evidence that ARF can bind and inactivate Mdm2 in the nucleoplasm, the prevailing view is that ARF exerts its growth-inhibitory activities from within the nucleolus. We suggest ARF primarily functions outside the nucleolus and provide evidence that it is sequestered and held inactive in that compartment by a nucleolar phosphoprotein, nucleophosmin (NPM). Most cellular ARF is bound to NPM regardless of whether cells are proliferating or growth arrested, indicating that ARF-NPM association does not correlate with growth suppression. Notably, ARF binds NPM through the same domains that mediate nucleolar localization and Mdm2 binding, suggesting that NPM could control ARF localization and compete with Mdm2 for ARF association. Indeed, NPM knockdown markedly enhanced ARF-Mdm2 association and diminished ARF nucleolar localization. Those events correlated with greater ARF-mediated growth suppression and p53 activation. Conversely, NPM overexpression antagonized ARF function while increasing its nucleolar localization. These data suggest that NPM inhibits ARF's p53-dependent activity by targeting it to nucleoli and impairing ARF-Mdm2 association.

ACKNOWLEDGMENTS

We thank Pier Giuseppe Pelicci, Chuck Sherr, Martine Roussel, Gerry Zambetti, and Gordon Peters for reagents. We are also grateful to Jason Weber, Emanuela Colombo, Pier Giuseppe Pelicci, Martine Roussel, and Chuck Sherr for sharing findings prior to publication. These studies were performed with assistance from the University of Iowa Flow Cytometry Facility, the Holden Comprehensive Cancer Center, and core facilities of the Diabetes and Endocrinology Research Center at the University of Iowa.

This work was supported by grants to F.W.Q. (RO1-CA79889) and D.E.Q. (RO1-CA90367).