State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Nature. 2013 Jan 3;493(7430):111-5. doi: 10.1038/nature11699. Epub 2012 Dec 2.
Ionic protein-lipid interactions are critical for the structure and function of membrane receptors, ion channels, integrins and many other proteins. However, the regulatory mechanism of these interactions is largely unknown. Here we show that Ca(2+) can bind directly to anionic phospholipids and thus modulate membrane protein function. The activation of T-cell antigen receptor-CD3 complex (TCR), a key membrane receptor for adaptive immunity, is regulated by ionic interactions between positively charged CD3ε/ζ cytoplasmic domains (CD3(CD)) and negatively charged phospholipids in the plasma membrane. Crucial tyrosines are buried in the membrane and are largely protected from phosphorylation in resting T cells. It is not clear how CD3(CD) dissociates from the membrane in antigen-stimulated T cells. The antigen engagement of even a single TCR triggers a Ca(2+) influx and TCR-proximal Ca(2+) concentration is higher than the average cytosolic Ca(2+) concentration. Our biochemical, live-cell fluorescence resonance energy transfer and NMR experiments showed that an increase in Ca(2+) concentration induced the dissociation of CD3(CD) from the membrane and the solvent exposure of tyrosine residues. As a consequence, CD3 tyrosine phosphorylation was significantly enhanced by Ca(2+) influx. Moreover, when compared with wild-type cells, Ca(2+) channel-deficient T cells had substantially lower levels of CD3 phosphorylation after stimulation. The effect of Ca(2+) on facilitating CD3 phosphorylation is primarily due to the charge of this ion, as demonstrated by the fact that replacing Ca(2+) with the non-physiological ion Sr(2+) resulted in the same feedback effect. Finally, (31)P NMR spectroscopy showed that Ca(2+) bound to the phosphate group in anionic phospholipids at physiological concentrations, thus neutralizing the negative charge of phospholipids. Rather than initiating CD3 phosphorylation, this regulatory pathway of Ca(2+) has a positive feedback effect on amplifying and sustaining CD3 phosphorylation and should enhance T-cell sensitivity to foreign antigens. Our study thus provides a new regulatory mechanism of Ca(2+) to T-cell activation involving direct lipid manipulation.
离子型蛋白质-脂质相互作用对于膜受体、离子通道、整合素和许多其他蛋白质的结构和功能至关重要。然而，这些相互作用的调节机制在很大程度上是未知的。在这里，我们表明 Ca(2+) 可以直接与阴离子磷脂结合，从而调节膜蛋白的功能。T 细胞抗原受体-CD3 复合物（TCR）是适应性免疫的关键膜受体，其激活受质膜中带正电荷的 CD3ε/ζ 胞质结构域（CD3(CD)）与带负电荷的磷脂之间离子相互作用的调节。关键的酪氨酸残基埋藏在膜中，在静止的 T 细胞中很大程度上免受磷酸化。目前尚不清楚在抗原刺激的 T 细胞中 CD3(CD) 如何从膜上解离。即使单个 TCR 的抗原结合也会引发 Ca(2+) 内流，并且 TCR 近端 Ca(2+) 浓度高于平均细胞溶质 Ca(2+) 浓度。我们的生化、活细胞荧光共振能量转移和 NMR 实验表明，Ca(2+) 浓度的增加诱导 CD3(CD) 从膜上解离，酪氨酸残基暴露于溶剂中。结果，Ca(2+) 内流显著增强了 CD3 酪氨酸磷酸化。此外，与野生型细胞相比，Ca(2+) 通道缺陷型 T 细胞在刺激后 CD3 磷酸化水平明显降低。Ca(2+) 促进 CD3 磷酸化的作用主要归因于该离子的电荷，这一事实可以通过用非生理离子 Sr(2+) 替代 Ca(2+) 产生相同的反馈作用来证明。最后，(31)P NMR 光谱显示 Ca(2+) 在生理浓度下与阴离子磷脂的磷酸基团结合，从而中和磷脂的负电荷。这种 Ca(2+) 调节途径不是启动 CD3 磷酸化，而是对 CD3 磷酸化的放大和维持具有正反馈作用，应该增强 T 细胞对外来抗原的敏感性。因此，我们的研究提供了一种新的 Ca(2+) 调节 T 细胞激活的机制，涉及直接的脂质操作。
