Nonobstructive azoospermia (NOA) is the most serious form of spermatogenesis abnormalities in male infertility. Genetic factors are important to consider as elements leading to NOA. Although many pathogenic genes have been reported, the causative genes of NOA for many patients are still unknown. In this study, we found ten point mutations in the gene encoding homeodomain-interacting protein kinase 4 (HIPK4) in patients with NOA, and using in vitro studies, we determined a premature termination point mutation (p. Lys490∗, c.1468A>T) that can cause decreased expression of HIPK4. Our phosphoproteomic analysis of Hipk4^−/− testes revealed phosphorylation of multiple proteins regulated by HIPK4 during spermiogenesis. We also confirmed that a substrate of HIPK4 with four downregulated phosphorylation sites matching the xSPx motif is the known manchette-related protein RIMS-binding protein 3, which is required for sperm head morphogenesis. Therefore, we conclude HIPK4 regulates the phosphorylation of manchette protein RIMS-binding protein 3 and plays essential roles in sperm head shaping and male fertility.

非阻塞性无精子症 (NOA) 是男性不育症中最严重的精子发生异常形式。遗传因素是导致 NOA 的重要因素。尽管已经报道了许多致病基因，但许多患者的NOA致病基因仍然未知。在这项研究中，我们在 NOA 患者中发现编码同源域相互作用蛋白激酶 4 (HIPK4) 的基因中有 10 个点突变，并且通过体外研究，我们确定了一个提前终止点突变 (p. Lys490*, c.1468A> T) 可导致 HIPK4 表达降低。我们对Hipk4的磷酸化蛋白质组学分析^-/-睾丸揭示了在精子发生过程中由 HIPK4 调节的多种蛋白质的磷酸化。我们还证实，具有与 xSPx 基序匹配的四个下调磷酸化位点的 HIPK4 底物是已知的曼切特相关蛋白 RIMS 结合蛋白 3，它是精子头部形态发生所必需的。因此，我们得出结论 HIPK4 调节 manchette 蛋白 RIMS 结合蛋白 3 的磷酸化，并在精子头部塑造和男性生育能力中发挥重要作用。