De novo phosphorylation and conformational opening of the tyrosine kinase Lck act in concert to initiate T cell receptor signaling
De novo phosphorylation and conformational opening of the tyrosine kinase Lck act in concert to initiate T cell receptor signaling
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DOI：
10.1126/scisignal.aaf4736
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发表时间：
2017-01-17
影响因子：
7.3
通讯作者：
Mueller, Andreas J.
中科院分区：
文献类型：
--
作者：
Philipsen, Lars;Reddycherla, Amarendra V.;Mueller, Andreas J.
Src家族酪氨酸激酶p56（Lck）（Lck）的酶活性由两个酪氨酸残基Tyr（394）和Tyr（505）的差异磷酸化严格控制。Tyr（394）的磷酸化和Lck的构象开放被认为激活激酶，而Tyr（505）磷酸化被认为产生Lck的封闭的无活性构象。我们研究了Lck的构象及其磷酸化状态是否共同调节T细胞受体（TCR）信号的起始。我们利用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（YF）-F-394和（YF）-F-505以及双突变体（YF）-F-394/（YF）-F-505在未刺激的T细胞中和在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酪氨酸的磷酸化状态分开。我们发现Lck单独的构象开放不足以启动T细胞中的信号传导事件。相反，Lck另外需要Tyr（394）的磷酸化以诱导T细胞活化。与FLIM测量一致，优化的免疫荧光显微术方案揭示了TCR刺激的Lck在Tyr（394）处的磷酸化优先发生在Jurkat细胞和原代人T细胞的质膜处。我们的研究支持了这样的假设，即通过TCR复合物的T细胞活化伴随着Lck的从头活化，并且Tyr（394）的磷酸化在Lck功能中起作用，其超出了诱导激酶的开放构象。
The enzymatic activity of the Src family tyrosine kinase p56(Lck) (Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr(394) and Tyr(505). Phosphorylation of Tyr(394) and the conformational opening of Lck are believed to activate the kinase, whereas Tyr(505) 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 (YF)-F-394 and (YF)-F-505 and the double mutant (YF)-F-394/(YF)-F-505 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(394) 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(394) 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(394) plays a role in Lck function that goes beyond inducing an open conformation of the kinase.