Human Testis Phosphoproteome Reveals Kinases as Potential Targets in Spermatogenesis and Testicular Cancer
Human Testis Phosphoproteome Reveals Kinases as Potential Targets in Spermatogenesis and Testicular Cancer
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DOI：
10.1074/mcp.ra118.001278
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发表时间：
2019-03-15
影响因子：
7
通讯作者：
Jansen, Bastiaan J. H.
中科院分区：
文献类型：
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作者：
Castillo, Judit;Knol, Jaco C.;Jansen, Bastiaan J. H.
精子发生是一个复杂的细胞分化过程，包括显著的遗传、细胞、功能和结构变化。它需要严格的调控，因为任何精子发生过程的干扰都会导致生育能力不足以及后代的疾病。为了增加我们对精子发育过程中信号转导的了解，我们对人类男性性腺中发生的磷酸化事件进行了大规模的鉴定。采用TiO 2金属氧化物亲和层析结合LC-MS/MS技术对人精子发生成熟期睾丸磷酸化蛋白质组进行分析。共鉴定了2661种蛋白质的8187种磷酸肽，是迄今为止人类睾丸磷酸蛋白的最完整的报道。磷酸化事件富集在功能上与精子发生相关的蛋白质中，以及与雄性性腺中的高度活跃的过程，如转录和翻译调节、细胞骨架组织、DNA包装、细胞周期和凋亡。此外，还鉴定了174种磷酸化激酶。通过鉴定的磷酸肽谱的数量和激酶激活环的磷酸化状态来预测睾丸中最活跃的人类蛋白激酶。细胞周期蛋白依赖性激酶12（CDK 12）和p21激活的激酶4（PAK 4）的潜在功能已通过计算机蛋白质-蛋白质相互作用分析，睾丸组织中的免疫检测和人胚胎癌细胞系中的功能测定进行了探索。CDK 12与高尔基体标记物的共定位表明这种蛋白激酶在精子形成过程中的潜在关键作用。PAK 4在人精原细胞中表达，并在胚胎癌细胞对凋亡的反应中发挥作用。总之，我们的蛋白质发现分析证实，蛋白激酶的磷酸化调节在精子分化中非常活跃，并为开发调节男性生育力和肿瘤行为的药物的潜在靶点的详细表征和验证打开了一扇窗户。
Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular, functional and structural changes. It requires tight regulation, because disturbances in any of the spermatogenic processes would lead to fertility deficiencies as well as disorders in offspring. To increase our knowledge of signal transduction during sperm development, we carried out a largescale identification of the phosphorylation events that occur in the human male gonad. Metal oxide affinity chromatography using TiO2 combined with LC-MS/MS was conducted to profile the phosphoproteome of adult human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active human protein kinases in the testis were predicted both by the number of phosphopeptide spectra identified and the phosphorylation status of the kinase activation loop. The potential function of cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4 (PAK4) has been explored by in silico protein-protein interaction analysis, immunodetection in testicular tissue, and a functional assay in a human embryonal carcinoma cell line. The colocalization of CDK12 with Golgi markers suggests a potential crucial role of this protein kinase during sperm formation. PAK4 has been found expressed in human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our protein discovery analysis confirms that phosphoregulation by protein kinases is highly active in sperm differentiation and opens a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility and tumor behavior.