Small ubiquitin-like modifier (SUMO) typically conjugates to target proteins through isopeptide linkage to the ε-amino group of lysine residues. This posttranslational modification (PTM) plays pivotal roles in modulating protein function. Cofilins are key regulators of actin cytoskeleton dynamics and are well-known to undergo several different PTMs. Here, we show that cofilin-1 is conjugated by SUMO1 both in vitro and in vivo. Using mass spectrometry and biochemical and genetic approaches, we identify the N-terminal α-amino group as the SUMO-conjugation site of cofilin-1. Common to conventional SUMOylation is that the N-α-SUMOylation of cofilin-1 is also mediated by SUMO activating (E1), conjugating (E2), and ligating (E3) enzymes and reversed by the SUMO deconjugating enzyme, SENP1. Specific to the N-α-SUMOylation is the physical association of the E1 enzyme to the substrate, cofilin-1. Using F-actin co-sedimentation and actin depolymerization assays in vitro and fluorescence staining of actin filaments in cells, we show that the N-α-SUMOylation promotes cofilin-1 binding to F-actin and cofilin-induced actin depolymerization. This covalent conjugation by SUMO at the N-α amino group of cofilin-1, rather than at an internal lysine(s), serves as an essential PTM to tune cofilin-1 function during regulation of actin dynamics.

小泛素样修饰剂 (SUMO) 通常通过与赖氨酸残基的 ε-氨基的异肽连接来与靶蛋白结合。这种翻译后修饰 (PTM) 在调节蛋白质功能中发挥着关键作用。Cofilins 是肌动蛋白细胞骨架动力学的关键调节因子，众所周知会经历几种不同的 PTM。在这里，我们证明 cofilin-1 在体外和体内均与 SUMO1 缀合。使用质谱、生化和遗传学方法，我们确定 N 末端 α-氨基为 cofilin-1 的 SUMO 缀合位点。传统 SUMO 化的共同之处在于，cofilin-1 的 N-α-SUMO 化也由 SUMO 激活 (E1)、缀合 (E2) 和连接 (E3) 酶介导，并被 SUMO 解缀酶 SENP1 逆转。N-α-SUMO 化的具体特征是 E1 酶与底物 cofilin-1 的物理结合。通过体外 F-肌动蛋白共沉淀和肌动蛋白解聚测定以及细胞中肌动蛋白丝的荧光染色，我们发现 N-α-SUMO 化促进 cofilin-1 与 F-肌动蛋白结合以及 cofilin 诱导的肌动蛋白解聚。SUMO 在 cofilin-1 的 N-α 氨基上（而不是在内部赖氨酸上）进行的这种共价缀合是在肌动蛋白动力学调节过程中调节 cofilin-1 功能的重要 PTM。