Elia Andrew E H, Cantley Lewis C, Yaffe Michael B
Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Science. 2003 Feb 21;299(5610):1228-31. doi: 10.1126/science.1079079.
We have developed a proteomic approach for identifying phosphopeptide binding domains that modulate kinase-dependent signaling pathways. An immobilized library of partially degenerate phosphopeptides biased toward a particular protein kinase phosphorylation motif is used to isolate phospho-binding domains that bind to proteins phosphorylated by that kinase. Applying this approach to cyclin-dependent kinases (Cdks), we identified the polo-box domain (PBD) of the mitotic kinase polo-like kinase 1 (Plk1) as a specific phosphoserine (pSer) or phosphothreonine (pThr) binding domain and determined its optimal binding motif. This motif is present in known Plk1 substrates such as Cdc25, and an optimal phosphopeptide containing the motif disrupted PBD-substrate binding and localization of the PBD to centrosomes. This finding reveals how Plk1 can localize to specific sites within cells in response to Cdk phosphorylation at those sites and provides a structural mechanism for targeting the Plk1 kinase domain to its substrates.
我们开发了一种蛋白质组学方法，用于鉴定调节激酶依赖性信号通路的磷酸肽结合结构域。偏向特定蛋白激酶磷酸化基序的部分简并磷酸肽固定文库用于分离与该激酶磷酸化的蛋白质结合的磷酸结合结构域。将该方法应用于细胞周期蛋白依赖性激酶（Cdks），我们鉴定出有丝分裂激酶波罗样激酶1（Plk1）的波罗盒结构域（PBD）是一种特异性磷酸丝氨酸（pSer）或磷酸苏氨酸（pThr）结合结构域，并确定了其最佳结合基序。该基序存在于已知的Plk1底物如Cdc25中，包含该基序的最佳磷酸肽破坏了PBD与底物的结合以及PBD定位于中心体。这一发现揭示了Plk1如何响应细胞内这些位点的Cdk磷酸化而定位到特定位点，并提供了一种将Plk1激酶结构域靶向其底物的结构机制。
