CAMP SIGNALING AND GENE TRANSCRIPTION IN HAPLOID CELLS

环AMP依赖性蛋白激酶（PKA）磷酸化丝氨酸， 底物蛋白的苏氨酸残基以修饰细胞功能， 基因表达和细胞周期进程。RIIalpha调节 PKA亚基在分化的单倍体中以升高的水平表达， 细胞，并在cAMP信号传导中发挥核心作用。 哺乳动物精子发生 在精子中，RII α与 鞭毛和调节cAMP依赖的精子细胞激活 能动性在生殖细胞发育的早期阶段， RII α与细胞核相关，并将调节PKA依赖性 在一些实施方案中，所述方法包括使单倍体特异性转录因子如CREMtau磷酸化。 细胞核PKA的激活可能是导致广泛的 基因在单倍体细胞中的表达。两种生殖细胞特异性mRNA亚型 RII α的表达在不同阶段受到不同程度的调节， 同步睾丸我们假设RII α mRNA亚型 代表鞭毛和细胞质/核蛋白亚型所需的 适当的亚细胞定位，并建议（i）表征 两种RII α同种型之间的结构（序列）差异， 鉴定相应生殖细胞特异性RII α A-激酶锚定 蛋白（AKAP）和（ii）确认精确的阶段特异性调节 RII α亚型在睾丸中的分布。使用该基因的5'启动子区， RIIalpha基因和瞬时转染实验，我们已经确定了一个DNA 负责雄激素激活的调节区， 通过PKA的转录反馈抑制。这个新的DNA区域也 与生殖细胞核蛋白相互作用。我们建议研究信号 cAMP和雄激素信号通路之间的转导串扰 通过（iii）瞬时转染转录报告基因， PKA表达载体，并将转录因子克隆到允许的 细胞系，并表征生殖细胞核蛋白， 启动子序列。我们还建议（iv）分析RII α启动子 使用转基因小鼠进行生殖细胞发育调控。长期 本研究的目的是为了解男性 与运动或异常生殖细胞有关的不育问题 发展，并提供雄激素和cAMP信号的基本信息 正常和肿瘤组织使用的转导串扰机制。

Cyclic-AMP-dependent protein kinase (PKA) phosphorylates serine and threonine residues of substrate proteins to modify cellular functions such as gene expression and cell cycle progression. The RIIalpha regulatory subunit of PKA is expressed at elevated levels in differentiating haploid germinal cells and occupies a central role in cAMP signaling during mammalian spermatogenesis. In spermatozoa, RIIalpha is associated with the flagella and regulates cAMP-dependent activation of sperm cell motility. In earlier steps of germ cell development, non-flagellar RIIalpha is associated with the nucleus and will regulate PKAdependent phosphorylation of haploid specific transcription factors such as CREMtau. Activation of nuclear PKA could be the genetic switch which leads to broad expression of genes in haploid cells. Two germ cell specific mRNA isoforms for RIIalpha are observed to be differentially regulated in stage- synchronized testis. We hypothesize that the RIIalpha mRNA isoforms represent flagellar and cytoplasmic/nuclear protein isoforms required for appropriate sub cellular localization and propose to (i) characterize the structural (sequence) differences between the two RIIalpha isoforms and to identify corresponding germ cell specific RIIalpha A-kinase anchoring proteins (AKAPs) and (ii) to confirm the precise stage specific regulation of RIIalpha isoforms in testis. Using the 5' promoter region of the RIIalpha gene and transient transfection assays, we have identified a DNA regulatory region which is responsible for androgen activation and transcriptional feedback inhibition by PKA. This novel region of DNA also interacts with germ cell nuclear proteins. We propose to study signal transduction crosstalk between the cAMP and androgen signaling pathways through (iii) transient transfection of transcriptional reporter genes, PKA expression vectors, and cloned transcription factors into permissive cell lines, and to characterize the germ cell nuclear proteins which bind to promoter sequences. We also propose to (iv) analyze RIIalpha promoter regulation in developing germ cells using transgenic mice. The long term goal of this research is to provide a foundation for understanding male infertility problems related to motility or abnormal germ cell development, and to provide basic information on androgen and cAMP signal transduction crosstalk mechanisms used by normal and neoplastic tissues.