Yeast Two-Hybrid Screen Identifies PKA-Riα Interacting Proteins during Mouse Spermiogenesis

Abstract

cAMP-dependent protein kinase (PKA) signaling plays various roles during mammalian spermatogenesis, ranging from the regulation of gene expression to the modulation of sperm motility. However, the molecular mechanisms that govern the multifaceted functions of PKA during spermatogenesis remain largely unclear. We previously found that PKA regulatory subunit I α (RIα) and catalytic subunit α (Cα) co-sediment with polyribosomal fractions of mouse testis lysate on sucrose gradient and the stimulation of PKA activity facilitates protein synthesis in post-meiotic elongating spermatids, indicating that type I PKA is intricately associated with protein translation machinery and regulates protein synthesis during mouse spermiogenesis. Since PKA activity is often regulated by interacting proteins that form complexes with its regulatory subunits, the identification of PKA-RIα interacting proteins in post-meiotic spermatogenic cells will facilitate our understanding of its regulatory roles in protein synthesis and spermiogenesis. In the present study, we applied a yeast two-hybrid screen to identify PKA-Riα-binding proteins using a cDNA library generated from mouse round and elongating spermatids. Numerous proteins were found to potentially interact with PKA-RIα, including proteostasis modulators, metabolic enzymes, cytoskeletal regulators, and mitochondrial proteins, many of which are specifically expressed in testes. Consistently, the examination of MENA (mouse ENA/VASP homolog) in developing mouse testes suggested that post-meiotic spermatogenic cells express a short isoform of MENA that interacts with PKA-RIα in yeast two-hybrid assay. The identification of PKA-RIα interacting proteins provides us solid basis to further explore how PKA signaling regulates protein synthesis and cellular morphogenesis during mouse spermatogenesis.