MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, UK.
Cell Signal. 2012 Nov;24(11):2085-94. doi: 10.1016/j.cellsig.2012.06.018. Epub 2012 Jul 11.
PCTAIRE-1 (cyclin-dependent kinase [CDK] 16) is a highly conserved serine/threonine kinase that belongs to the CDK family of protein kinases. Little is known regarding PCTAIRE-1 regulation and function and no robust assay exists to assess PCTAIRE-1 activity mainly due to a lack of information regarding its preferred consensus motif and the lack of bona fide substrates. We used positional scanning peptide library technology and identified the substrate-specificity requirements of PCTAIRE-1 and subsequently elaborated a peptide substrate termed PCTAIRE-tide. Recombinant PCTAIRE-1 displayed vastly improved enzyme kinetics on PCTAIRE-tide compared to a widely used generic CDK substrate peptide. PCTAIRE-tide also greatly improved detection of endogenous PCTAIRE-1 activity. Similar to other CDKs, PCTAIRE-1 requires a proline residue immediately C-terminal to the phosphoacceptor site (+1) for optimal activity. PCTAIRE-1 has a unique preference for a basic residue at +4, but not at +3 position (a key characteristic for CDKs). We also demonstrate that PCTAIRE-1 binds to a novel cyclin family member, cyclin Y, which increased PCTAIRE-1 activity towards PCTAIRE-tide >100-fold. We hypothesised that cyclin Y binds and activates PCTAIRE-1 in a way similar to which cyclin A2 binds and activates CDK2. Point mutants of cyclin Y predicted to disrupt PCTAIRE-1-cyclin Y binding severely prevented complex formation and activation of PCTAIRE-1. We have identified PCTAIRE-tide as a powerful tool to study the regulation of PCTAIRE-1. Our understanding of the molecular interaction between PCTAIRE-1 and cyclin Y further facilitates future investigation of the functions of PCTAIRE-1 kinase.
PCTAIRE-1（细胞周期蛋白依赖性激酶 [CDK] 16）是一种高度保守的丝氨酸/苏氨酸激酶，属于蛋白激酶 CDK 家族。关于 PCTAIRE-1 的调节和功能知之甚少，并且由于缺乏有关其首选共识基序的信息以及缺乏真正的底物，因此没有可靠的测定法来评估 PCTAIRE-1 的活性。我们使用定位扫描肽文库技术鉴定了 PCTAIRE-1 的底物特异性要求，随后设计了一种肽底物，称为 PCTAIRE-tide。与广泛使用的通用 CDK 底物肽相比，重组 PCTAIRE-1 对 PCTAIRE-tide 的酶动力学显示出极大的改善。PCTAIRE-tide 还大大提高了内源性 PCTAIRE-1 活性的检测。与其他 CDKs 相似，PCTAIRE-1 要求在磷酸受体位点（+1）的 C 末端紧邻脯氨酸残基以获得最佳活性。PCTAIRE-1 对+4 位置的碱性残基具有独特的偏好，但不在+3 位置（CDKs 的一个关键特征）。我们还证明，PCTAIRE-1 与一种新型细胞周期蛋白家族成员 cyclin Y 结合，这使 PCTAIRE-1 对 PCTAIRE-tide 的活性增加了> 100 倍。我们假设 cyclin Y 以类似于 cyclin A2 结合和激活 CDK2 的方式结合并激活 PCTAIRE-1。预测会破坏 PCTAIRE-1-cyclin Y 结合的 cyclin Y 点突变体严重阻止了 PCTAIRE-1 的复合物形成和激活。我们已经将 PCTAIRE-tide 鉴定为研究 PCTAIRE-1 调节的有力工具。我们对 PCTAIRE-1 和 cyclin Y 之间分子相互作用的理解进一步促进了对 PCTAIRE-1 激酶功能的未来研究。
