Human p14Arf: an exquisite sensor of morphological changes and of short-lived perturbations in cell cycle and in nucleolar function

Abstract

The human Ink4a/Arf tumor suppressor locus encodes two distinct products: p16Ink4a which prevents phosphorylation and inactivation of the retinoblastoma protein and, p14Arf , a nucleolar protein which activates the function of the tumor suppressor p53 protein in the nucleoplasm in response to oncogenic stimulation through an as yet ill-defined mechanism. Here we show that the level of endogenous p14Arf and its balance between the nucleolus and the nucleoplasm in HeLa cells are exquisitely sensitive to changes in cell morphology and to short-lived perturbations in cell cycle and in nucleolar function such as those induced by the cyclin-dependent kinase inhibitor, roscovitine, and the casein kinase II and RNA synthesis inhibitor, DRB. Most remarkably, whereas p14Arf predominantly concentrates in the nucleolus of interphase cells and transiently disappears between metaphase and early G1 under normal growth conditions, it massively and reversibly accumulates in the nucleoplasm of postmitotic and S-phase cells upon shortterm treatment with roscovitine and, at a lesser extent, DRB. In line with the fact that the nuclear level of p53 reaches a peak between mid-G1 and the G1/S border in p53-expressor cells which lack Arf expression, these results provide a clue that, in p53+/Arf+ cells, Arf proteins might serve both to speed and to amplify p53-mediated responses in conditions and cell cycle periods in which the mechanisms involved in p53 stabilization and activation are not fully operational. They further suggest that human endogenous p14Arf might activate p53 pathways in physiologic situations by acting inside the nucleoplasm, especially when normal cell cycle progression and nucleolar function are compromised.

Nucleophosmin (B23) Targets ARF to Nucleoli and Inhibits Its Function

“…Overall, our findings are in complete accordance with an emerging concept that disruption of the nucleolus contributes to p53 signaling and growth suppression (11,56,66). The idea proposed by Rubbi and Milner is that high levels of DNA damage or other cellular stresses perturb the nucleolus, causing release of nucleolar proteins (such as ARF) into the nucleoplasm, which then bind Mdm2 and activate p53.…”

“…However, the fact that ARF resides in nucleoli in cells lacking Mdm2 argued that other mechanisms normally govern ARF localization. We suggest that NPM regulates that process, in agreement with the virtually superimposable subnuclear distributions of ARF and NPM throughout the cell cycle or in response to various drugs (11). Third, and most importantly, ARF's ability to activate p53 and inhibit growth was inversely related to cellular NPM levels and ARF's localization to nucleoli.…”

Nucleolar Sequestration of RelA (p65) Regulates NF-κB-Driven Transcription and Apoptosis

“…2A). Ki-67 is a proliferation marker whose presence in the nucleolus is cell cycle dependent (11). We found that Ki-67 was mainly nucleoplasmic in aspirin-treated cells and showed minimal colocalization with RelA ( Fig.…”

p14ARF Protein Expression Is a Predictor of Both Relapse and Survival in Squamous Cell Carcinoma of the Anterior Tongue

“…In SCC of the cervix (HeLa) cells, levels of endogenous p14 ARF and its distribution between the nucleolus and nucleoplasm are sensitive to changes in cell morphology, cell cycle, and nucleolar function (38). However, the role of p14 ARF localization in its function remains unclear, with studies showing that nucleolar localization is not sufficient for growth inhibition (17) and is dispensable for relocalization and stabilization of Hdm2/ Mdm2 and p53 (16,39).…”