sGSK Group, Institute of Molecular & Cell Biology, A*STAR, Singapore, Singapore.
FB Laboratory, Institute of Molecular & Cell Biology, A*STAR, Singapore, Singapore.
Nat Commun. 2021 Sep 7;12(1):5315. doi: 10.1038/s41467-021-25011-w.
Human PAK4 is an ubiquitously expressed p21-activated kinase which acts downstream of Cdc42. Since PAK4 is enriched in cell-cell junctions, we probed the local protein environment around the kinase with a view to understanding its location and substrates. We report that U2OS cells expressing PAK4-BirA-GFP identify a subset of 27 PAK4-proximal proteins that are primarily cell-cell junction components. Afadin/AF6 showed the highest relative biotin labelling and links to the nectin family of homophilic junctional proteins. Reciprocally >50% of the PAK4-proximal proteins were identified by Afadin BioID. Co-precipitation experiments failed to identify junctional proteins, emphasizing the advantage of the BioID method. Mechanistically PAK4 depended on Afadin for its junctional localization, which is similar to the situation in Drosophila. A highly ranked PAK4-proximal protein LZTS2 was immuno-localized with Afadin at cell-cell junctions. Though PAK4 and Cdc42 are junctional, BioID analysis did not yield conventional cadherins, indicating their spatial segregation. To identify cellular PAK4 substrates we then assessed rapid changes (12') in phospho-proteome after treatment with two PAK inhibitors. Among the PAK4-proximal junctional proteins seventeen PAK4 sites were identified. We anticipate mammalian group II PAKs are selective for the Afadin/nectin sub-compartment, with a demonstrably distinct localization from tight and cadherin junctions.
人 PAK4 是一种普遍表达的 p21 激活激酶，其作用于 Cdc42 的下游。由于 PAK4 在细胞-细胞连接处富集，我们用激酶的局部蛋白质环境来探测其位置和底物。我们报告说，表达 PAK4-BirA-GFP 的 U2OS 细胞鉴定出一组 27 个靠近 PAK4 的蛋白质，这些蛋白质主要是细胞-细胞连接处的成分。Afadin/AF6 的相对生物素标记最高，并与同亲性连接蛋白的 nectin 家族相连。反过来，>50%的靠近 PAK4 的蛋白质被 Afadin BioID 识别。共沉淀实验未能鉴定出连接蛋白，强调了 BioID 方法的优势。从机制上讲，PAK4 依赖于 Afadin 进行其连接定位，这与果蝇的情况相似。一个高度接近 PAK4 的蛋白质 LZTS2 与 Afadin 在细胞-细胞连接处免疫定位。虽然 PAK4 和 Cdc42 是连接的，但 BioID 分析没有产生传统的钙粘蛋白，表明它们的空间分离。为了鉴定细胞 PAK4 的底物，我们随后评估了用两种 PAK 抑制剂处理后 12 分钟内磷酸化蛋白质组的快速变化。在靠近 PAK4 的 27 个连接蛋白中，有 17 个被鉴定为 PAK4 位点。我们预计哺乳动物 II 组 PAK 对 Afadin/nectin 亚区室具有选择性，与紧密连接和钙粘蛋白连接具有明显不同的定位。
