Department of Molecular Structural Biology, Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany.
PLoS One. 2012;7(1):e29672. doi: 10.1371/journal.pone.0029672. Epub 2012 Jan 4.
Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation.
A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated.
The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.
自然杀伤 (NK) 细胞通过多种胚系编码的激活受体的参与，有助于抵御感染和转化的细胞。单独刺激 Fc 受体 CD16 就足以激活 NK 细胞，而其他受体，如 2B4（CD244）和 DNAM-1（CD226），则协同作用。受体结合后，蛋白激酶在控制 NK 细胞效应功能的信号转导网络中发挥主要作用。然而，尚未系统地表征人类基因组（激酶组）中所有编码激酶中哪些参与 NK 细胞激活。
激酶选择性磷酸蛋白质组学方法确定了在人 NK 细胞中表达的 188 种激酶。CD16 交联以及 2B4 和 DNAM-1 的交联总共揭示了 109 种不同激酶上的 313 个独特激酶磷酸化位点。CD16 或 2B4 和 DNAM-1 共同结合后，总共 21 种激酶的磷酸化位点受到类似的调节。其中，FYN、KCC2G（CAMK2）、FES 和 AAK1 的磷酸化增加，MARK2 的磷酸化减少，在 CD16 结合后和 2B4 和 DNAM-1 共同结合后均被重复观察到。值得注意的是，只有 PAK4 上的一个磷酸化位点受到差异调节。
本研究鉴定了 NK 细胞激酶组的重要部分，并在原代淋巴细胞中定义了新的磷酸化位点。在 NK 细胞激活的早期阶段观察到的受调控的磷酸化暗示这些激酶参与 NK 细胞信号转导。总之，这项研究表明，不同激活受体下游存在一个很大程度上共享的信号通路，并为进一步阐明 NK 细胞效应反应的调节提供了有价值的资源。
