Department of Structural and Functional Biology, University of Naples Federico II, Naples, Italy.
PLoS One. 2013;8(1):e53631. doi: 10.1371/journal.pone.0053631. Epub 2013 Jan 7.
The INK4a/ARF locus on the short arm of chromosome 9 is one of the most frequently altered loci in human cancer. It is generally accepted that ARF is involved in oncogenic checkpoint pathways by sensitizing incipient cancer cells to undergo growth arrest or apoptosis through both p53-dependent and independent pathways. While intensive studies have been focused on ARF activation at the transcriptional level, only recently mechanisms governing ARF turnover have been identified. Here, we show for the first time that p14ARF is a PKC target. Prediction analysis showed many potential phosphorylation sites in PKC consensus sequences within ARF protein, and, among them, the threonine at position 8 was the most conserved. Substitution of this threonine influences both ARF stability and localization. Furthermore, a phosphomimetic ARF mutation reduces the ability to arrest cell growth although the ability to bind MDM2 and stabilize p53 result unaffected. Thus we propose that phosphorylation of ARF in both immortalized and tumor cell lines could be a mechanism to escape ARF surveillance following proliferative and oncogenic stress.
9 号染色体短臂上的 INK4a/ARF 基因座是人类癌症中最常发生改变的基因座之一。普遍认为，ARF 通过 p53 依赖和非依赖途径使初期癌变细胞对生长抑制或凋亡更为敏感，从而参与致癌检查点通路。尽管人们对 ARF 在转录水平上的激活进行了深入研究，但最近才确定了 ARF 周转的调控机制。在这里，我们首次表明 p14ARF 是 PKC 的靶标。预测分析显示 ARF 蛋白中有许多 PKC 保守序列中的潜在磷酸化位点，其中第 8 位的苏氨酸最为保守。该苏氨酸的取代既影响 ARF 的稳定性又影响其定位。此外，尽管 ARF 结合 MDM2 和稳定 p53 的能力不受影响，但磷酸化 ARF 突变会降低抑制细胞生长的能力。因此，我们提出，在永生化和肿瘤细胞系中 ARF 的磷酸化可能是在增殖和致癌应激后逃避 ARF 监控的一种机制。
