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.

摘要 蛋白激酶A（PKA）是由cAMP激活的，它对精子运动是必需的 和生育能力。靶向破坏精子特异性PKA催化亚基（Cα2）或可溶性腺苷酸 环化酶（sAC）导致男性不育。这两种基因敲除小鼠的精子都有运动能力受损， 接受受精所需的获能和超活化的能力。PKA在精子中的作用 PKA的功能已被充分确立，但对PKA的蛋白质底物知之甚少。确定身份 这些蛋白质的研究将使我们能够了解精子运动和生育能力的机制。 我们将使用一种新的化学遗传学方法来确定精子PKA底物。这种方法使用 PKA的催化亚基的ATP结合结构域的基因工程修饰，使得 修饰的酶识别不被其他激酶接受的特定ATP类似物。磷酸化 在ATP类似物的存在下，底物的分离使得它们的鉴定相对容易。我们亦 另一方面，携带PKA催化亚基修饰基因的敲入小鼠和携带靶向PKA基因的小鼠， 破坏精子sAC。我们正在产生表达类似物敏感PKA的双突变小鼠， sAC空背景。 我们提出两个具体目标。在第一个目标中，来自类似物敏感基因敲入小鼠的精子蛋白 双突变小鼠将被ATP类似物磷酸化。磷酸化蛋白质将被 通过类似物特异性抗体检测，然后使用串联MS进行鉴定。 将证实蛋白质是有效的PKA底物，并确定其磷酸化的变化 发生在精子在附睾中成熟期间和受精所需的精子激活事件期间。 这些目标的成功实现应该导致对cAMP和PKA如何在细胞内形成的机制的理解。 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

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

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

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