Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
Biochem J. 2011 Mar 15;434(3):399-413. doi: 10.1042/BJ20101244.
An improved understanding of the roles of protein kinases in intracellular signalling and disease progression has driven significant advances in protein kinase inhibitor discovery. Peptide inhibitors that target the kinase protein substrate-binding site have continued to attract attention. In the present paper, we describe a novel JNK (c-Jun N-terminal kinase) inhibitory peptide PYC71N, which inhibits JNK activity in vitro towards a range of recombinant protein substrates including the transcription factors c-Jun, ATF2 (activating trancription factor 2) and Elk1, and the microtubule regulatory protein DCX (doublecortin). Analysis of cell culture studies confirmed the actions of a cell-permeable version of PYC71 to inhibit c-Jun phosphorylation during acute hyperosmotic stress. The analysis of the in vitro data for the kinetics of this inhibition indicated a substrate-inhibitor complex-mediated inhibition of JNK by PYC71N. Alanine-scanning replacement studies revealed the importance of two residues (PYC71N Phe9 or Phe11 within an FXF motif) for JNK inhibition. The importance of these residues was confirmed through interaction studies showing that each change decreased interaction of the peptide with c-Jun. Furthermore, PYC71N interacted with both non-phosphorylated (inactive) JNK1 and the substrate c-Jun, but did not recognize active JNK1. In contrast, a previously characterized JNK-inhibitory peptide TIJIP [truncated inhibitory region of JIP (JNK-interacting protein)], showed stronger interaction with active JNK1. Competition binding analysis confirmed that PYC71N inhibited the interaction of c-Jun with JNK1. Taken together, the results of the present study define novel properties of the PYC71N peptide as well as differences from the characterized TIJIP, and highlight the value of these peptides to probe the biochemistry of JNK-mediated substrate interactions and phosphorylation.
对蛋白激酶在细胞内信号转导和疾病进展中作用的深入了解，推动了蛋白激酶抑制剂发现的重大进展。针对激酶蛋白底物结合位点的肽抑制剂一直备受关注。在本文中，我们描述了一种新型 JNK（c-Jun N 端激酶）抑制肽 PYC71N，它在体外抑制 JNK 对一系列重组蛋白底物的活性，包括转录因子 c-Jun、ATF2（激活转录因子 2）和 Elk1 以及微管调节蛋白 DCX（双皮质蛋白）。细胞培养研究的分析证实了一种可渗透细胞的 PYC71 版本可在急性高渗应激时抑制 c-Jun 磷酸化。对这种抑制的体外数据的动力学分析表明，PYC71N 通过底物-抑制剂复合物抑制 JNK。丙氨酸扫描替换研究揭示了两个残基（FXF 基序内的 PYC71N 苯丙氨酸 9 或苯丙氨酸 11）对 JNK 抑制的重要性。这些残基的重要性通过相互作用研究得到证实，表明每个变化都降低了肽与 c-Jun 的相互作用。此外，PYC71N 与非磷酸化（无活性）JNK1 和底物 c-Jun 相互作用，但不识别活性 JNK1。相比之下，先前表征的 JNK 抑制肽 TIJIP [JIP（JNK 相互作用蛋白）的截断抑制区] 与活性 JNK1 表现出更强的相互作用。竞争结合分析证实 PYC71N 抑制了 c-Jun 与 JNK1 的相互作用。总之，本研究的结果定义了 PYC71N 肽的新特性以及与表征的 TIJIP 的差异，并强调了这些肽在探测 JNK 介导的底物相互作用和磷酸化的生物化学方面的价值。
