Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2

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Abstract
The INK4a-ARF locus encodes two proteins, p16INK4a and p19ARF, that restrain cell growth by affecting the functions of the retinoblastoma protein and p53, respectively. Disruption of this locus by deletions or point mutations is a common event in human cancer, perhaps second only to the loss of p53. Using insect cells infected with baculovirus vectors and NIH 3T3 fibroblasts infected with ARF retrovirus, we determined that mouse p19ARF can interact directly with p53, as well as with the p53 regulator mdm2. ARF can bind p53-DNA complexes, and it depends upon functional p53 to transcriptionally induce mdm2 and the cyclin-dependent kinase inhibitor p21Cip1, and to arrest cell proliferation. Binding of p19ARF to p53 requires the ARF N-terminal domain (amino acids 1–62) that is necessary and sufficient to induce cell cycle arrest. Overexpression of p19ARF in wild type or ARF-null mouse embryo fibroblasts increases the half-life of p53 from 15 to ≈75 min, correlating with an increased p53-dependent transcriptional response and growth arrest. Surprisingly, when overexpressed at supra-physiologic levels after introduction into ARF-null NIH 3T3 cells or mouse embryo fibroblasts, the p53 protein is handicapped in inducing this checkpoint response. In this setting, reintroduction of p19ARF restores p53’s ability to induce p21Cip1 and mdm2, implying that, in addition to stabilizing p53, ARF modulates p53-dependent function through an additional mechanism.