Molecular characterization of the ARF tumor suppressor protein

It is generally accepted that the ARF tumor suppressor induces p53-dependent growth arrest by sequestering the p53 antagonist Mdm2 in the nucleolus. Previous mutagenic studies of murine ARF suggested that residues 1 through 14 and 26 through 37 were critical for Mdm2 binding. We show that mouse ARF residues 6 to 10 and 21 to 25 are required for ARF-induced growth arrest whereas residues 1 to 5 and 29 to 34 are dispensable. Surprisingly, unlike wild-type ARF, the growth-inhibitory mutants D1–5 and D29–34 failed to stabilize p53 yet induced its transcriptional activation. Conversely, mutants lacking conserved residues 6 to 10 and 21 to 25 were unable to suppress growth in p53-positive cells despite nucleolar localization and the ability to import Mdm2. Those observations contrasted with the ability of wild-type ARF to block growth in some cells without importing endogenous Mdm2 to nucleoli, revealing a lack of correlation between ARF activity and Mdm2 relocalization. Together, our findings show that ARF can inhibit growth independent of p53 stabilization and Mdm2 relocalization.; The above findings, as well as accumulating evidence in the literature, suggest that other factors besides p53 and Mdm2 contribute to ARF signaling. For instance, recent studies demonstrate that ARF can block growth in p53/Mdm2-null cells and that it associates with numerous cellular proteins. Here, we identify a novel ARF-interacting protein, nucleophosmin (NPM/B23). Complexes between wild-type ARF and endogenous NPM, a small nucleolar phosphoprotein, were observed in both growing and growth-arrested fibroblasts regardless of the p53 status. Thus, ARF-NPM association is not sufficient to cause growth arrest and occurs independently of p53. Importantly, amino-terminal domains of ARF that are essential for Mdm2 binding and growth inhibitory activity (residues 1 to 14 and 26 to 37) were also required for efficient in vitro and in vivo association with NPM. In keeping with that finding, distinct ARF-NPM and ARF-Mdm2 complexes were observed within cells. These data imply that NPM may regulate ARF function, the possibilities of which are discussed.