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 IQ motif interact with CaM, subsequently engaging with downstream target proteins known as calmodulin-binding proteins (CaMBPs). It is reported that the loss of interaction between IQCN and CaM is mainly manifested as decreased motility, leading to fertilization failure and male infertility. However, no relevant reports have been published detailing which CaMBPs exist and the mechanisms by which they are regulated. In this study, we conducted a comprehensive quantitative proteomic and phosphoproteomic analysis of 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 in protein levels. Among the total 577 differentially regulated phosphorylated sites in our results, most of them (494/577) belong to calmodulin-binding proteins (CaMBPs). Gene ontology analysis of these differentially phosphorylated CaMBPs showed enrichment in male gamete generation, actin cytoskeleton organization and microtubule cytoskeleton organization process, demonstrating IQCN regulates sperm function by interacting with CaM, which in turn affects the phosphorylation level of CaMBPs. Further kinase substrate network analysis revealed that most kinases with substrates phosphorylation sites up-regulated were tyrosine kinases, and the inhibition assay showed that FGFR4 and SYK tyrosine kinases are important for sperm motility and progressive motility. In summary, this study reveals the interaction between IQ motif-containing protein IQCN and CaM, which regulates the phosphorylation of downstream CaMBPs and is involved in the related processes of spermiogenesis and sperm function.