Philipsen Lars, Reddycherla Amarendra V, Hartig Roland, Gumz Janine, Kästle Matthias, Kritikos Andreas, Poltorak Mateusz P, Prokazov Yury, Turbin Evgeny, Weber André, Zuschratter Werner, Schraven Burkhart, Simeoni Luca, Müller Andreas J
Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany.
Leibniz Institute for Neurobiology, Brenneckestrasse 6, 39118 Magdeburg, Germany.
Sci Signal. 2017 Jan 17;10(462):eaaf4736. doi: 10.1126/scisignal.aaf4736.
The enzymatic activity of the Src family tyrosine kinase p56 (Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr and Tyr Phosphorylation of Tyr and the conformational opening of Lck are believed to activate the kinase, whereas Tyr phosphorylation is thought to generate a closed, inactive conformation of Lck. We investigated whether the conformation of Lck and its phosphorylation state act in concert to regulate the initiation of T cell receptor (TCR) signaling. With a sensitive biosensor, we used fluorescence lifetime imaging microscopy (FLIM) to investigate the conformations of wild-type Lck and its phosphorylation-deficient mutants Y394F and Y505F and the double mutant Y394F/Y505F in unstimulated T cells and after TCR stimulation. With this approach, we separated the conformational changes of Lck from the phosphorylation state of its regulatory tyrosines. We showed that the conformational opening of Lck alone was insufficient to initiate signaling events in T cells. Rather, Lck additionally required phosphorylation of Tyr to induce T cell activation. Consistent with the FLIM measurements, an optimized immunofluorescence microscopy protocol revealed that the TCR-stimulated phosphorylation of Lck at Tyr occurred preferentially at the plasma membrane of Jurkat cells and primary human T cells. Our study supports the hypothesis that T cell activation through the TCR complex is accompanied by the de novo activation of Lck and that phosphorylation of Tyr plays a role in Lck function that goes beyond inducing an open conformation of the kinase.
Src家族酪氨酸激酶p56（Lck）的酶活性受到两个酪氨酸残基（Tyr和Tyr）差异磷酸化的严格控制。Tyr的磷酸化和Lck的构象开放被认为可激活该激酶，而Tyr的磷酸化则被认为会产生Lck的封闭、无活性构象。我们研究了Lck的构象及其磷酸化状态是否协同作用以调节T细胞受体（TCR）信号传导的起始。我们使用灵敏的生物传感器，通过荧光寿命成像显微镜（FLIM）研究了野生型Lck及其磷酸化缺陷突变体Y394F和Y505F以及双突变体Y394F/Y505F在未刺激的T细胞和TCR刺激后的构象。通过这种方法，我们将Lck的构象变化与其调节性酪氨酸的磷酸化状态区分开来。我们发现，仅Lck的构象开放不足以启动T细胞中的信号事件。相反，Lck还需要Tyr的磷酸化来诱导T细胞活化。与FLIM测量结果一致，优化的免疫荧光显微镜检测方案显示，TCR刺激后Lck在Tyr处的磷酸化优先发生在Jurkat细胞和原代人T细胞的质膜上。我们的研究支持以下假设：通过TCR复合物激活T细胞伴随着Lck的从头激活，并且Tyr的磷酸化在Lck功能中发挥的作用超出了诱导激酶开放构象的范围。
