Bencivenga Debora, Tramontano Annunziata, Borgia Alessia, Negri Aide, Caldarelli Ilaria, Oliva Adriana, Perrotta Silverio, Della Ragione Fulvio, Borriello Adriana
a Department of Biochemistry; Biophysics and General Pathology ; Second University of Naples ; Naples , Italy.
Cell Cycle. 2014;13(23):3768-82. doi: 10.4161/15384101.2014.965999.
p27Kip1 is a critical modulator of cell proliferation by controlling assembly, localization and activity of cyclin-dependent kinase (CDK). p27Kip1 also plays important roles in malignant transformation, modulating cell movement and interaction with the extracellular matrix. A critical p27Kip1 feature is the lack of a stable tertiary structure that enhances its "adaptability" to different interactors and explains the heterogeneity of its function. The absence of a well-defined folding underlines the importance of p27Kip1 post-translational modifications that might highly impact the protein functions. Here, we characterize the metabolism and CDK interaction of phosphoserine10-p27Kip1 (pS10- p27Kip1), the major phosphoisoform of p27Kip1. By an experimental strategy based on specific immunoprecipitation and bidimensional electrophoresis, we established that pS10-p27Kip1 is mainly bound to cyclin E/CDK2 rather than to cyclin A/CDK2. pS10- p27Kip1 is more stable than non-modified p27Kip1, since it is not (or scarcely) phosphorylated on T187, the post-translational modification required for p27Kip1 removal in the nucleus. pS10-p27Kip1 does not bind CDK1. The lack of this interaction might represent a mechanism for facilitating CDK1 activation and allowing mitosis completion. In conclusion, we suggest that nuclear p27Kip1 follows 2 almost independent pathways operating at different rates. One pathway involves threonine-187 and tyrosine phosphorylations and drives the protein toward its Skp2-dependent removal. The other involves serine-10 phosphorylation and results in the elongation of p27Kip1 half-life and specific CDK interactions. Thus, pS10-p27Kip1, due to its stability, might be thought as a major responsible for the p27Kip1-dependent arrest of cells in G1/G0 phase.
p27Kip1是细胞增殖的关键调节因子，它通过控制细胞周期蛋白依赖性激酶（CDK）的组装、定位和活性来发挥作用。p27Kip1在恶性转化、调节细胞运动以及与细胞外基质的相互作用中也起着重要作用。p27Kip1的一个关键特征是缺乏稳定的三级结构，这增强了其对不同相互作用分子的“适应性”，并解释了其功能的异质性。缺乏明确的折叠结构突出了p27Kip1翻译后修饰的重要性，这些修饰可能对蛋白质功能产生重大影响。在此，我们对磷酸化丝氨酸10-p27Kip1（pS10-p27Kip1）的代谢及与CDK的相互作用进行了表征，pS10-p27Kip1是p27Kip1的主要磷酸化异构体。通过基于特异性免疫沉淀和双向电泳的实验策略，我们确定pS10-p27Kip1主要与细胞周期蛋白E/CDK2结合，而非与细胞周期蛋白A/CDK2结合。pS10-p27Kip1比未修饰的p27Kip1更稳定，因为它在T187位点不被（或几乎不被）磷酸化，而T187位点的磷酸化是p27Kip1从细胞核中去除所必需的翻译后修饰。pS10-p27Kip1不与CDK1结合。这种相互作用的缺失可能是促进CDK1激活并使有丝分裂完成的一种机制。总之，我们认为细胞核中的p27Kip1遵循两条几乎独立且速率不同的途径。一条途径涉及苏氨酸-187和酪氨酸磷酸化，并促使该蛋白被Skp2依赖性去除。另一条途径涉及丝氨酸-10磷酸化，导致p27Kip1半衰期延长及特定的CDK相互作用。因此，由于其稳定性，pS10-p27Kip1可能被认为是细胞在G1/G0期因p27Kip1而停滞的主要原因。
