p19ARF prevents G1 cyclin-dependent kinase activation by interacting with MDM2 and activating p53 in mouse fibroblasts.
p19ARF encoded by the INK4a tumor suppressor gene locus functions upstream of p53 to induce cell cycle arrest. p19ARF can interact with MDM2 and p53 in cells ectopically overexpressing these three components, but the biochemical cascades from p19ARF to cell cycle arrest has not been fully elucidated. In this study, we generated stably transfected NIH3T3 cells that express exogenous p19ARF under the control of a heavy metal-inducible metalothionine promoter. Cells arrested in G1 by ectopically expressed p19ARF contained considerably reduced G1 cyclin dependent kinase (cdk2 and cdk4) activities. The expression of cyclin A (a regulatory subunit of cdk2) markedly decreased, while cyclin D1, the major cdk4 partner in fibroblasts, expressed at a slightly higher level and formed complexes with cdk2 and cdk6 in addition to cdk4. Induction of p19ARF activated p53 by increasing its stability, and allowed the expression of p21Cip1, which bound to all of the cyclin D1-cdk complexes (cyclin D1-cdk2, -cdk4, and -cdk6) thereby inhibiting their kinase activities. p19ARF formed complexes with several cellular proteins including mouse MDM2. The majority of MDM2 was found in the complex with p19ARF, while no p53 was detected in association with p19ARF. Thus, we propose that p19ARF neutralizes MDM2 by sequestration from p53, which results in activation of p53, inhibition of G1 cyclin-cdk activities, and G1 arrest.
Kurokawa
K
K
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan.
Tanaka
T
T
Kato
J
J
D016475:3T3 Cells; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology*; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins; D011506:Proteins / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D016203:CDC2 Protein Kinase