Functional Genomics Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.
Department of Perioperative Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA.
Sci Rep. 2018 Jan 19;8(1):1177. doi: 10.1038/s41598-018-19532-6.
Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and this interaction was blocked by TFP5, an inhibitor that prevents activation of Cdk5. In vitro peptide-based kinase assay revealed that four of six TRPA1 Cdk5 consensus sites acted as substrates for Cdk5, and modeling of the ankyrin repeats disclosed that phosphorylation would occur at characteristic pockets within the (T/S)PLH motifs. Calcium imaging of trigeminal ganglion neurons from genetically engineered mice overexpressing or lacking the Cdk5 activator p35 displayed increased or decreased responsiveness, respectively, to stimulation with the TRPA1 agonist allylisothiocyanate (AITC). AITC-induced chemo-nociceptive behavior was also heightened in vivo in mice overexpressing p35 while being reduced in p35 knockout mice. Our findings demonstrate that TRPA1 is a substrate of Cdk5 and that Cdk5 activity is also able to modulate TRPA1 agonist-induced calcium influx and chemo-nociceptive behavioral responses.
周期蛋白依赖性激酶 5（Cdk5）是一种关键的神经元激酶，在炎症期间上调，随后可以调节对伤害性刺激的敏感性。我们对在伤害感受器中表达丰富的蛋白质中的 Cdk5 磷酸化位点进行了计算机筛选，并确定了化学敏感的离子通道瞬时受体电位阳离子通道亚家族 A 成员 1（TRPA1）可能是 Cdk5 的底物。免疫沉淀全长 TRPA1 被证明可被 Cdk5 磷酸化，而 TFP5（一种阻止 Cdk5 激活的抑制剂）可阻断这种相互作用。体外基于肽的激酶测定显示，TRPA1 的六个 Cdk5 共有序列中有四个可作为 Cdk5 的底物，并且锚蛋白重复序列的建模表明磷酸化将发生在（T/S）PLH 基序内的特征口袋中。过表达或缺乏 Cdk5 激活剂 p35 的基因工程小鼠三叉神经节神经元的钙成像显示，分别对 TRPA1 激动剂异硫氰酸烯丙酯（AITC）的刺激表现出增加或减少的反应性。在过表达 p35 的小鼠中，AITC 诱导的化学伤害性行为也在体内加剧，而在 p35 敲除小鼠中则减少。我们的研究结果表明，TRPA1 是 Cdk5 的底物，并且 Cdk5 活性还能够调节 TRPA1 激动剂诱导的钙内流和化学伤害性行为反应。
