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

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 homopolymerization via SAM‐SAM interactions. More importantly, the principle governing SAM homopolymer depolymerization is generalized via suggesting two distinct types of heterogeneous SAM‐SAM interactions, offering fresh insights into SAM domain‐mediated homopolymerization and depolymerization.