Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
SAXS/WAXS, Australian Synchrotron, Clayton, Victoria, Australia.
FEBS J. 2019 Dec;286(24):4910-4925. doi: 10.1111/febs.15002. Epub 2019 Jul 30.
Scribble is a crucial adaptor protein that plays a pivotal role during establishment and control of cell polarity, impacting many physiological processes ranging from cell migration to immunity and organization of tissue architecture. Scribble harbours a leucine-rich repeat domain and four PDZ domains that mediate most of Scribble's interactions with other proteins. It has become increasingly clear that post-translational modifications substantially impact Scribble-ligand interactions, with phosphorylation being a major modulator of binding to Scribble. To better understand how Scribble PDZ domains direct cell polarity signalling and how phosphorylation impacts this process, we investigated human Scribble interactions with MCC (Mutated in Colorectal Cancer). We systematically evaluated the ability of all four individual Scribble PDZ domains to bind the PDZ-binding motif (PBM) of MCC as well as MCC phosphorylated at the -1 Ser position. We show that Scribble PDZ1 and PDZ3 are the major interactors with MCC, and that modifications to Ser at the -1 position in the MCC PBM only has a minor effect on binding to Scribble PDZ domains. We then examined the structural basis for these observations by determining the crystal structures of Scribble PDZ1 domain bound to both the unphosphorylated MCC PBM as well as phosphorylated MCC. Our structures indicated that phospho-Ser at the -1 position in MCC is not involved in major contacts with Scribble PDZ1, and in conjunction with our affinity measurements suggest that the impact of phosphorylation at the -1 position of MCC must extend beyond a simple modulation of the affinity for Scribble PDZ domains.
Scribble 是一种关键的衔接蛋白，在细胞极性的建立和控制中发挥着关键作用，影响着从细胞迁移到免疫和组织架构组织等多种生理过程。Scribble 含有一个富含亮氨酸的重复结构域和四个 PDZ 结构域，这些结构域介导了 Scribble 与其他大多数蛋白质相互作用。越来越明显的是，翻译后修饰对 Scribble-配体相互作用有很大影响，磷酸化是与 Scribble 结合的主要调节因子。为了更好地理解 Scribble PDZ 结构域如何指导细胞极性信号转导以及磷酸化如何影响这一过程，我们研究了人 Scribble 与 MCC（结直肠癌突变）的相互作用。我们系统地评估了所有四个单独的 Scribble PDZ 结构域结合 MCC 的 PDZ 结合基序（PBM）以及在 -1 位丝氨酸磷酸化的 MCC 的能力。结果表明，Scribble PDZ1 和 PDZ3 是与 MCC 主要相互作用的结构域，而 MCC PBM 中 -1 位丝氨酸的修饰对与 Scribble PDZ 结构域的结合仅有微小影响。然后，我们通过确定 Scribble PDZ1 结构域与未磷酸化的 MCC PBM 以及磷酸化的 MCC 的晶体结构来研究这些观察结果的结构基础。我们的结构表明，MCC 中 -1 位的磷酸丝氨酸不参与与 Scribble PDZ1 的主要接触，结合我们的亲和力测量结果表明，MCC -1 位的磷酸化的影响必须超出对 Scribble PDZ 结构域亲和力的简单调节。
