Department of Physiology, Anatomy and Genetics, Oxford University, Oxford OX1 3PT, United Kingdom.
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709.
Proc Natl Acad Sci U S A. 2021 May 11;118(19). doi: 10.1073/pnas.2012908118.
To avoid conflicting and deleterious outcomes, eukaryotic cells often confine second messengers to spatially restricted subcompartments. The smallest signaling unit is the Ca nanodomain, which forms when Ca channels open. Ca nanodomains arising from store-operated Orai1 Ca channels stimulate the protein phosphatase calcineurin to activate the transcription factor nuclear factor of activated T cells (NFAT). Here, we show that NFAT1 tethered directly to the scaffolding protein AKAP79 (A-kinase anchoring protein 79) is activated by local Ca entry, providing a mechanism to selectively recruit a transcription factor. We identify the region on the N terminus of Orai1 that interacts with AKAP79 and demonstrate that this site is essential for physiological excitation-transcription coupling. NMR structural analysis of the AKAP binding domain reveals a compact shape with several proline-driven turns. Orai2 and Orai3, isoforms of Orai1, lack this region and therefore are less able to engage AKAP79 and activate NFAT. A shorter, naturally occurring Orai1 protein that arises from alternative translation initiation also lacks the AKAP79-interaction site and fails to activate NFAT1. Interfering with Orai1-AKAP79 interaction suppresses cytokine production, leaving other Ca channel functions intact. Our results reveal the mechanistic basis for how a subtype of a widely expressed Ca channel is able to activate a vital transcription pathway and identify an approach for generation of immunosuppressant drugs.
为了避免冲突和有害的结果，真核细胞通常将第二信使局限在空间受限的亚区室中。最小的信号单元是 Ca 纳米区，它在 Ca 通道打开时形成。来自储存操纵性 Orai1 Ca 通道的 Ca 纳米区刺激蛋白磷酸酶钙调神经磷酸酶激活转录因子活化 T 细胞核因子（NFAT）。在这里，我们表明，直接与支架蛋白 AKAP79（A 激酶锚定蛋白 79）偶联的 NFAT1 被局部 Ca 内流激活，提供了一种选择性募集转录因子的机制。我们确定了 Orai1 与 AKAP79 相互作用的 N 端区域，并证明该位点对于生理兴奋-转录偶联是必不可少的。对 AKAP 结合结构域的 NMR 结构分析揭示了一种具有多个脯氨酸驱动的转弯的紧凑形状。Orai2 和 Orai3 是 Orai1 的同工型，缺乏该区域，因此与 AKAP79 的结合能力较弱，无法激活 NFAT。一种较短的、自然发生的 Orai1 蛋白，来源于翻译起始的替代，也缺乏 AKAP79 相互作用位点，无法激活 NFAT1。干扰 Orai1-AKAP79 相互作用可抑制细胞因子的产生，而不影响其他 Ca 通道的功能。我们的结果揭示了广泛表达的 Ca 通道亚型如何能够激活重要的转录途径的机制基础，并确定了一种生成免疫抑制剂药物的方法。
