Hsueh Kai-Wei, Fu Shu-Ling, Chang Chirn-Bin, Chang Yu-Ling, Lin Chao-Hsiung
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan.
Biochim Biophys Acta. 2013 Feb;1834(2):508-15. doi: 10.1016/j.bbapap.2012.11.005. Epub 2012 Nov 28.
Crosstalk between Aurora-A kinase and p53 has been proposed. While the genetic amplification of Aurora-A has been observed in many human cancers, how p53 is regulated by Aurora-A remains ambiguous. In this study, Aurora-A-mediated phosphorylation of p53 was analyzed by mass spectrometry in order to identify a new phosphorylation site. Subsequently, the functional consequences of such phosphorylation were examined.
In vitro phosphorylation of p53 by Aurora-A was performed and the phosphorylated protein was then digested with trypsin and enriched for phosphopeptides by immobilized metal affinity chromatography. Subsequently, a combination of β-elimination and Michael addition was applied to the phosphopeptides in order to facilitate the identification of phosphorylation sites by MS. The functional consequences of the novel phosphorylation of p53 on the protein-protein interactions, protein stability and transactivation activity were then examined using co-immunoprecipitation, Western blotting and reporter assays.
Ser-106 of p53 was identified as a novel site phosphorylated by Aurora-A. A serine-to-alanine mutation at this site was found to attenuate Aurora-A-mediated phosphorylation in vitro. In addition, phosphate-sensitive Phos-tag SDS-PAGE was used to confirm that the Ser-106 of p53 is in vivo phosphorylated by Aurora-A. Finally, co-immunoprecipitation studies suggested that Ser-106 phosphorylation of p53 decreases its interaction with MDM2 and prolongs the half-life of p53.
The inhibition of the interaction between p53 and MDM2 by a novel Aurora-A-mediated p53 phosphorylation was identified in this study and this provides important information for further investigations into the interaction between p53 and Aurora-A in terms of cancer biology.
已有研究提出极光激酶A（Aurora-A kinase）与p53之间存在相互作用。虽然在许多人类癌症中都观察到了极光激酶A的基因扩增，但极光激酶A如何调节p53仍不明确。在本研究中，通过质谱分析极光激酶A介导的p53磷酸化，以鉴定一个新的磷酸化位点。随后，研究了这种磷酸化的功能后果。
进行极光激酶A对p53的体外磷酸化，然后用胰蛋白酶消化磷酸化蛋白，并通过固定金属亲和色谱法富集磷酸肽。随后，对磷酸肽应用β-消除和迈克尔加成相结合的方法，以促进通过质谱鉴定磷酸化位点。然后使用免疫共沉淀、蛋白质免疫印迹和报告基因检测来研究p53新磷酸化对蛋白质-蛋白质相互作用、蛋白质稳定性和反式激活活性的功能后果。
p53的丝氨酸106（Ser-106）被鉴定为极光激酶A磷酸化的新位点。发现该位点的丝氨酸到丙氨酸突变在体外减弱了极光激酶A介导的磷酸化。此外，使用对磷酸盐敏感的Phos-tag SDS-PAGE证实p53的Ser-106在体内被极光激酶A磷酸化。最后，免疫共沉淀研究表明，p53的Ser-106磷酸化降低了其与MDM2的相互作用，并延长了p53的半衰期。
本研究发现了一种新的极光激酶A介导的p53磷酸化对p53与MDM2之间相互作用的抑制作用，这为进一步研究p53与极光激酶A在癌症生物学方面的相互作用提供了重要信息。
