Functional and Physical Interactions of the ARF Tumor Suppressor with p53 and Mdm2

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 95; no. 14; pp. 8292 - 8297
• Main Authors: Kamijo, Takehiko, Weber, Jason D., Zambetti, Gerard, Zindy, Frederique, Roussel, Martine F., Sherr, Charles J.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 07.07.1998; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: 3T3 Cells; Animals; Antibodies; Biological Sciences; Cancer; Cell growth; Deoxyribonucleic acid; DNA; Gene Expression Regulation, Neoplastic; Genes; Genes, Tumor Suppressor; Humans; Infections; Mice; Mutation; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; NIH 3T3 cells; Nuclear Proteins; Plasmids; Proteins; Proteins - genetics; Proteins - metabolism; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-mdm2; Retroviridae; Transfection; Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism; Tumors; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.14.8292

The INK4a-ARF locus encodes two proteins, p16INK4aand 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 p19ARFcan 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 p19ARFto p53 requires the ARF N-terminal domain (amino acids 1-62) that is necessary and sufficient to induce cell cycle arrest. Overexpression of p19ARFin 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 p19ARFrestores p53's ability to induce p21Cip1and mdm2, implying that, in addition to stabilizing p53, ARF modulates p53-dependent function through an additional mechanism.