PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin αII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.

中文翻译：

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Phactr1/PP1 磷酸酶全酶底物特异性的分子基础

PPP 家族磷酸酶如 PP1 几乎没有内在特异性。辅因子可以将 PP1 靶向底物或亚细胞位置，但尚不清楚它们如何赋予 PP1 序列特异性。细胞骨架调节器 Phactr1 是一种神经元富集的 PP1 辅因子，由 G-肌动蛋白控制。结构分析表明，Phactr1 结合重塑了 PP1 的疏水凹槽，在催化位点附近形成了一个新的复合表面。使用磷酸蛋白质组学，我们确定了小鼠成纤维细胞和神经元 Phactr1/PP1 底物，其中包括细胞骨架成分和调节剂。我们确定了 Phactr1/PP1 与其底物 IRSp53 和血影蛋白 αII 的去磷酸化形式结合的高分辨率结构。这些全酶-产物复合物中磷酸盐的反转支持所提出的 PPP 家族催化机制。去磷酸化位点 C 端的底物序列与复合 Phactr1/PP1 表面密切接触，这是有效去磷酸化所必需的。序列特异性解释了为什么与 apo-PP1 或其他 PP1 全酶相比，Phactr1/PP1 对其底物表现出数量级增强的反应性。