Identification of Phospho-proteins Regulating Sperm Function

调节精子功能的磷酸蛋白的鉴定

• 批准号: 9333123
• 负责人: SRINIVASAN VIJAYARAGHAVAN
• 金额: $ 18.73万
• 依托单位: KENT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-17 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333123
• 关键词: Adenylate Cyclase; Alleles; Antibodies; Bicarbonates; Binding; Biochemical; Catalytic Domain; Chemicals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotides; Detection; Enzymes; Epididymis; Event; Fertility; Fertilization; Genes; Genetic Engineering; Germ Cells; Goals; Hand; Impairment; Intracellular Second Messenger; Knock-in; Knock-in Mouse; Knockout Mice; Knowledge; Lead; Male Infertility; Mass Spectrum Analysis; Modification; Mus; Mutant Strains Mice; Phosphoproteins; Phosphorylation; Phosphotransferases; Process; Protein Subunits; Proteins; Role; Second Messenger Systems; Sperm Capacitation; Sperm Maturation; Sperm Motility; Testis; Validation; adenylate kinase; analog; cell motility; chemical genetics; genetic approach; inorganic phosphate; male; mutant; novel; novel strategies; reproductive tract; sperm cell; sperm function; sperm protein; zygote

Abstract It has been long known that protein kinase A (PKA) activated by cAMP is essential for sperm motility and fertility. Targeted disruption of the sperm specific catalytic subunit of PKA (Cα2) or the soluble adenylyl cyclase (sAC) results in male infertility. Sperm from both these knock out mice have impaired motility and lack the ability to undergo capacitation and hyperactivation required for fertilization. While the role of PKA in sperm function is well established little is known about the protein substrates of PKA. Determination of the identities of these proteins will enable us to understand mechanisms underlying sperm motility and fertility. We will use a novel chemical-genetic approach to identify sperm PKA substrates. This approach uses a genetically engineered modification of the ATP binding domain of the catalytic subunit of PKA such that the modified enzyme recognizes specific ATP analogues that are not accepted by other kinases. Phosphorylation of substrates in the presence of the ATP analog enables their identification with relative ease. We have, in hand, the knock-in mice harboring the modified gene for the catalytic subunit of PKA and mice with a targeted disruption of the sperm sAC. We are generating double mutant mice expressing the analog sensitive PKA on a sAC null background. We propose two specific aims. In the first aim sperm proteins from the analog sensitive knock-in mice and the double-mutant mice will be phosphorylated with an ATP analog. Phosphorylated proteins will be detected by analog-specific antibodies followed by their identification using tandem MS. In the second aim we will confirm that the proteins are valid PKA substrates and determine how changes in their phosphorylation occur during sperm maturation in the epididymis and during sperm activation events required for fertilization. Successful execution of these aims should lead to a mechanistic understanding of how cAMP and PKA regulate sperm function.

抽象的 众所周知，CAMP激活的蛋白激酶A（PKA）是ES科学的精子运动 和生育能力。 环酶（SAC）导致男性不育症。 经历施肥的能力和过度激活。 关于PKA的蛋白质底物知之甚少。 这些蛋白质将终结精子运动和生育能力的终结机制。 我们将使用一种新型的化学遗传学方法来识别精子PKA底物 PKA催化亚基的ATP结合结构域的遗传发动机修饰，使您 改良的酶识别其他激酶不接受的特定ATP类似物。 在ATP模拟的存在下的底物可以相对容易地识别 手，带有针对PKA和小鼠催化亚基的修饰基因的敲入小鼠 精子囊的破坏。 囊无背景。 我们在模拟敏感的小鼠中提出了两个特定目标。 双突变小鼠将用ATP类似物磷酸化。 通过模拟特异性抗体检测到它们在第二个目标中使用Tandem MS检测。 将确认蛋白质是有效的PKA底物，并确定其磷酸化的变化如何变化 发生在精子成熟期间和精子激活事件期间所需的延期。 这些目标的成功执行应导致对CAMP和PKA的机械理解 调节精子功能。

项目成果

Cyclic AMP and glycogen synthase kinase 3 form a regulatory loop in spermatozoa.

• DOI: 10.1002/jcp.26557
• 发表时间: 2018-09
• 期刊: Journal of cellular physiology
• 影响因子: 5.6
• 作者: Dey S;Goswami S;Eisa A;Bhattacharjee R;Brothag C;Kline D;Vijayaraghavan S
• 通讯作者: Vijayaraghavan S

Regulators of the protein phosphatase PP1γ2, PPP1R2, PPP1R7, and PPP1R11 are involved in epididymal sperm maturation.

• DOI: 10.1002/jcp.27130
• 发表时间: 2019-03
• 期刊: Journal of cellular physiology
• 影响因子: 5.6
• 作者: Goswami S;Korrodi-Gregório L;Sinha N;Bhutada S;Bhattacharjee R;Kline D;Vijayaraghavan S
• 通讯作者: Vijayaraghavan S

The protein YWHAE (14-3-3 epsilon) in spermatozoa is essential for male fertility.

• DOI: 10.1111/andr.12865
• 发表时间: 2021-01
• 期刊: Andrology
• 影响因子: 4.5
• 作者: Eisa A;Dey S;Ignatious A;Nofal W;Hess RA;Kurokawa M;Kline D;Vijayaraghavan S
• 通讯作者: Vijayaraghavan S

Roles of glycogen synthase kinase 3 alpha and calcineurin in regulating the ability of sperm to fertilize eggs.

• DOI: 10.1096/fj.201902163r
• 发表时间: 2020-01
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Dey S;Eisa A;Kline D;Wagner FF;Abeysirigunawardena S;Vijayaraghavan S
• 通讯作者: Vijayaraghavan S