p19^ARF prevents G1 cyclin-dependent kinase activation by interacting with MDM2 and activating p53 in mouse fibroblasts

Abstract

p19^ARF encoded by the INK4a tumor suppressor gene locus functions upstream of p53 to induce cell cycle arrest. p19^ARF can interact with MDM2 and p53 in cells ectopically overexpressing these three components, but the biochemical cascades from p19^ARF to cell cycle arrest has not been fully elucidated. In this study, we generated stably transfected NIH3T3 cells that express exogenous p19^ARF under the control of a heavy metal-inducible metalothionine promoter. Cells arrested in G1 by ectopically expressed p19^ARF 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 p19^ARF activated p53 by increasing its stability, and allowed the expression of p21^Cip1, which bound to all of the cyclin D1-cdk complexes (cyclin D1-cdk2, -cdk4, and -cdk6) thereby inhibiting their kinase activities. p19^ARF formed complexes with several cellular proteins including mouse MDM2. The majority of MDM2 was found in the complex with p19^ARF, while no p53 was detected in association with p19^ARF. Thus, we propose that p19^ARF neutralizes MDM2 by sequestration from p53, which results in activation of p53, inhibition of G1 cyclin-cdk activities, and G1 arrest.