Yeast Two-Hybrid Screen Identifies PKA-Riα Interacting Proteins during Mouse Spermiogenesis.
Yeast Two-Hybrid Screen Identifies PKA-Riα Interacting Proteins during Mouse Spermiogenesis.
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作者：
Shi K;Yang L;Zhuang X;Zhang L;Qi H
cAMP依赖性蛋白激酶（PKA）信号在哺乳动物精子发生过程中起着多种作用，从基因表达的调节到精子运动的调节。然而，PKA在精子发生过程中多方面功能的分子机制仍不清楚。我们以前发现PKA调节亚基I α（RIα）和催化亚基α（Cα）与小鼠睾丸裂解物的多聚核糖体组分在蔗糖梯度上共沉淀，PKA活性的刺激促进减数分裂后伸长精子细胞的蛋白质合成，表明I型PKA与蛋白质翻译机制密切相关，并在小鼠精子发生过程中调节蛋白质合成。由于PKA的活性通常受与其调节亚基形成复合物的相互作用蛋白的调节，因此在减数分裂后的生精细胞中鉴定PKA-RIα相互作用蛋白将有助于我们理解其在蛋白质合成和精子发生中的调节作用。在本研究中，我们应用酵母双杂交筛选PKA-Riα结合蛋白，使用小鼠圆形和伸长精子细胞产生的cDNA文库。许多蛋白质被发现可能与PKA-RIα相互作用，包括蛋白质稳态调节剂、代谢酶、细胞骨架调节剂和线粒体蛋白，其中许多蛋白质在睾丸中特异性表达。因此，在酵母双杂交试验中，对小鼠睾丸发育过程中MENA（mouse ENA/VASP homolog）的检测提示，减数分裂后的生精细胞表达一种短的MENA亚型，该亚型可与PKA-RIα相互作用。PKA-RIα相互作用蛋白的鉴定为进一步研究PKA信号通路对小鼠精子发生过程中蛋白质合成和细胞形态发生的调控提供了坚实的基础。
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.
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