Quantitative Phosphoproteomic Analysis of Testes from Iqcn-Deficient Mice Highlights the Significance of Calmodulin Signaling in Spermiogenesis

Calmodulin (CaM) plays a crucial role in sperm function. Studies have reported that proteins containing the IQ motif interact with CaM, subsequently engaging with downstream target proteins known as calmodulin-binding proteins (CaMBPs). However, no relevant reports have been published detailing which CaMBPs exist and the mechanisms by which they are regulated. In this study, we conducted quantitative proteomic and phosphoproteomic analysis for mouse testes from wild-type (WT) and Iqcn knockout (Iqcn^–/–) mice. The results indicated that Iqcn deficiency substantially rewires the downstream phosphorylation signaling pathway while not causing equivalent changes at protein levels. Among the 577 differentially regulated phosphorylated sites, most of them (494/577) belong to CaMBPs. Gene ontology analysis of these differentially phosphorylated CaMBPs showed enrichment in male gamete generation, actin cytoskeleton organization, and microtubule cytoskeleton organization process, demonstrating that IQCN regulates sperm function by interacting with CaM, which in turn affects the phosphorylation level of CaMBPs. Further kinase-substrate network analysis and the inhibition assay showed that FGFR4 and SYK tyrosine kinases are important for sperm motility and progressive motility. In summary, this study reveals that the interaction between IQCN and CaM regulates the phosphorylation of downstream CaMBPs and is involved in the related processes of spermiogenesis and sperm function.