Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):E3162-70. doi: 10.1073/pnas.1312531110. Epub 2013 Aug 5.
Brefeldin A-inhibited guanine nucleotide-exchange factors BIG1 and BIG2 activate, through their Sec7 domains, ADP ribosylation factors (Arfs) by accelerating the replacement of Arf-bound GDP with GTP for initiation of vesicular transport or activation of specific enzymes that modify important phospholipids. They are also implicated in regulation of cell polarization and actin dynamics for directed migration. Reciprocal coimmunoprecipitation of endogenous HeLa cell BIG1 and BIG2 with myosin IIA was demonstrably independent of Arf guanine nucleotide-exchange factor activity, because effects of BIG1 and BIG2 depletion were reversed by overexpression of the cognate BIG molecule C-terminal sequence that follows the Arf activation site. Selective depletion of BIG1 or BIG2 enhanced specific phosphorylation of myosin regulatory light chain (T18/S19) and F-actin content, which impaired cell migration in Transwell assays. Our data are clear evidence of these newly recognized functions for BIG1 and BIG2 in transduction or integration of mechanical signals from integrin adhesions and myosin IIA-dependent actin dynamics. Thus, by anchoring or scaffolding the assembly, organization, and efficient operation of multimolecular myosin phosphatase complexes that include myosin IIA, protein phosphatase 1δ, and myosin phosphatase-targeting subunit 1, BIG1 and BIG2 serve to integrate diverse biophysical and biochemical events in cells.
布雷菲德菌素 A 抑制的鸟嘌呤核苷酸交换因子 BIG1 和 BIG2 通过其 Sec7 结构域激活 ADP 核糖基化因子 (Arfs)，加速 Arf 结合的 GDP 被 GTP 取代，从而启动囊泡运输或激活特定的酶，这些酶修饰重要的磷脂。它们还参与细胞极化和肌动蛋白动力学的调节，以实现定向迁移。内源性 HeLa 细胞 BIG1 和 BIG2 与肌球蛋白 IIA 的相互免疫沉淀显然与 Arf 鸟嘌呤核苷酸交换因子活性无关，因为 BIG1 和 BIG2 耗竭的影响可以通过表达 Arf 激活位点后面的同源 BIG 分子 C 末端序列来逆转。BIG1 或 BIG2 的选择性耗竭增强了肌球蛋白调节轻链 (T18/S19) 的特异性磷酸化和 F-肌动蛋白含量，这在 Transwell 测定中损害了细胞迁移。我们的数据清楚地证明了 BIG1 和 BIG2 在整合素黏附物和肌球蛋白 IIA 依赖性肌动蛋白动力学从机械信号的转导或整合中的这些新识别功能。因此，通过锚定或支架组装、组织和包括肌球蛋白 IIA、蛋白磷酸酶 1δ 和肌球蛋白磷酸酶靶向亚基 1 在内的多分子肌球蛋白磷酸酶复合物的有效运作，BIG1 和 BIG2 有助于整合细胞中不同的生物物理和生化事件。
