SASH1 is a novel binding partner to disassemble Caskin1 tandem SAM homopolymer through heterogeneous SAM ‐ SAM interaction

作者：Yanhui Wang, Qiangou Chen, Cang Wu, Yuzhen Ding, Yuan Lin, Ziyi Wang, Yu Chen, Jianchao Li, Zhongmin Liu, Kang Xiao, Wei Liu · 发表于：FEBS Journal · 年份：2024 · DOI：10.1111/febs.17354 · 被引用次数：2 · 研究领域：RNA Research and Splicing、Signaling Pathways in Disease、RNA and protein synthesis mechanisms

Calcium/calmodulin-dependent serine protein kinase (CASK) interaction protein 1/2 (Caskin1/2) is essential neuronal synaptic scaffold protein in nervous system development. Knockouts of Caskin1/2 display severe deficits in novelty recognition and spatial memory. The tandem sterile alpha motif (SAM) domains of Caskin1/2, also conserved in their Drosophila homolog Ckn, are known to form homopolymers, yet their dynamic regulation mechanism remains unclear. In this study, SAM and SH3 domain-containing protein 1 (SASH1) was first identified as a novel binding partner of Caskin1/2 through yeast two-hybrid (Y2H) screening. The SAM-SAM interaction between SASH1 and Caskin1 was biochemically characterized by size-exclusion chromatography (SEC), isothermal titration calorimetry (ITC), and glutathione-S-transferase (GST) pull-down and co-immunoprecipitation (co-IP) assays. Structural insights from AlphaFold2-predicted models of the Caskin1-SAMs/SASH1-SAM1 complex, along with mutagenesis validations, revealed key residues at the end-helix (EH)/mid-loop (ML) interface for this interaction. More interestingly, the Caskin1-SAMs homopolymer can be disrupted by the SAM-SAM interaction, which was consistently verified by using sedimentation, transmission electron microscopy (TEM), and immunofluorescence (IF) staining in heterologous cell lines. In summary, our findings provide a solid biochemical basis for the Caskin1/SASH1 interaction and propose a potential mechanism for regulating Caskin1/2...