PROTEIN PHOSPHORYLATION IN SPERM FLAGELLAR MOTILITY

精子鞭毛运动中的蛋白质磷酸化

• 批准号: 3277135
• 负责人: JOSEPH S TASH
• 金额: $ 19.47万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277135
• 关键词: affinity chromatography; autoradiography; binding proteins; calcium; calcium binding protein; calmodulin; cilium /flagellum motility; cyclic AMP; dogs; dynein ATPase; enzyme mechanism; enzyme substrate; gel electrophoresis; image processing; immunoelectron microscopy; laboratory mouse; laboratory rabbit; membrane proteins; phosphoprotein phosphatase; phosphoproteins; phosphorylation; protein kinase; protein structure; radionuclides; sea urchins; second messengers; sperm motility; spermatogenesis; swine

The role of protein phosphorylation in the regulation of sperm flagellar motility by cAMP and calcium-calmodulin (Ca2+ -CaM) will be studied. Dynein ( the alpha-heavy chain and 3 smaller proteins) was identified as a substrate for phosphorylation by cAMP-dependent protein kinase (cA-K) and for dephosphorylation by CaM-dependent protein phosphatase (CaM-PrPase). Phosphorylation of dynein stimulated ATPase activity as well as the ability of dynein to propel taxol-stabilized microtubules on a glass substrate. CaM-PrPase was identified in human, dog, pig and sea urchin sperm as well as Chlamydomonas flagella. All sperm flagellar phosphatase was differentially salt-extracted from flagella and dynein and could be reversibly co-sedimented into sucrose gradients with 21S dynein. The phosphatase regulates Ca2+ -dependent swimming parameters in sperm models. These observations represent the first evidence for a direct connection between second messenger phosphorylation and dephosphorylation pathways and the regulation of a functional flagellar component such as dynein. Based on these observations, the hypothesis proposed is that dynein may be a major site of action of second messenger regulation of flagellar function and that components of second messenger pathways may be closely associated with dynein within the framework of the sperm flagellar axoneme. The Specific Aims for this project period are: 1. Isolate and characterize the components of dynein that are substrates for second-messenger-modulated phosphorylation and dephosphorylation and: i) identify and characterize those phosphoproteins that affect dynein ATPase activity and dynein function, and ii) determine whether these phosphoproteins are altered in vivo and in vitro in relation to changes in sperm flagellar movement. 2. Identify and isolate the flagellar components and CaM-PrPase. 3. Isolate and characterize the flagellar CaM-PrPase. 4. Identify non-outer arm dynein flagellar phosphoprotein substrates for CaM-PrPase and cAMP- dependent protein kinase. 5. Characterize these substrates with respect to their cAMP- and Ca2+ -dependent phosphorylation, structural localization and potential interaction with dynein. To achieve these Specific Aims, the major experimental approaches will utilize detergent-permeabilized sperm reactivated with ATP, as well as in vitro microtubule gliding assays for dynein. Each type of assay will be challenged with probes for cAMP and Ca2= -CaM pathways. Digital image analysis will be used to quantitate flagellar and microtubule movement and [Y-32P]Atp will be used to identify phosphoproteins, the flagellar form of CaM-PrPase and non-dynein flagellar phosphoproteins that are substrates for CaM-PrPase and cA-K. This bank will then be used to probe reconstituted flagellar models and isolated dynein to dissect the mechanism of action of these axonemal regulatory elements. The results obtained should yield a greater understanding of the possible aberrations underlying disease states typified by altered and/or abnormal axonemal motility.

蛋白磷酸化在精子鞭毛调控中的作用 将通过cAMP和钙-钙调蛋白（Ca 2 + -CaM）研究运动性。 动力蛋白（α-重链和3种较小的蛋白质）被鉴定为一种 cAMP依赖性蛋白激酶（cA-K）磷酸化底物， 用于通过CaM依赖性蛋白磷酸酶（CaM-PrP）去磷酸化。 磷酸化的动力蛋白刺激ATP酶的活性，以及能力 动力蛋白来推动玻璃基底上的紫杉醇稳定微管。 在人、狗、猪和海胆精子中也发现了钙调素 衣原体鞭毛 所有精子鞭毛磷酸酶 差异盐提取鞭毛和动力蛋白，可以 与21 S动力蛋白可逆共沉淀到蔗糖梯度中。 的 磷酸酶调节精子模型中钙依赖性游泳参数。 这些观测结果首次证明了 第二信使磷酸化和去磷酸化途径之间的联系， 对功能性鞭毛成分如动力蛋白的调节。 基于 根据这些观察结果，提出的假设是，动力蛋白可能是一种 第二信使调节鞭毛功能的主要作用部位 第二信使通路的组成部分可能与 精子鞭毛轴丝框架内含有动力蛋白。 的 本项目期间的具体目标是：1。 分离和表征 动力蛋白的成分是第二信使调节的底物， 磷酸化和去磷酸化，以及：i）鉴定和表征 影响动力蛋白ATP酶活性和动力蛋白 功能，以及ii）确定这些磷蛋白是否在 体内和体外与精子鞭毛运动变化的关系。 2. 鉴定并分离鞭毛组分和CaM-Pre。 3. 隔离 并表征鞭毛CaM-Pre。 4. 识别非外臂 动力蛋白鞭毛磷蛋白底物CaM-Pre和cAMP- 依赖性蛋白激酶 5. 表征这些基质， cAMP和Ca 2+依赖性磷酸化，结构定位 以及与动力蛋白的潜在相互作用。 为了实现这些具体目标， 主要的实验方法将利用洗涤剂渗透的精子 用ATP重新激活，以及体外微管滑动试验， 动力蛋白 每种类型的试验将用cAMP探针进行挑战， Ca 2 = -CaM途径。 数字图像分析将用于定量 鞭毛和微管运动和[Y-32 P]Atp将用于鉴定 磷蛋白，鞭毛形式的CaM-Pre蛋白和非动力蛋白鞭毛 作为CaM-Pre和cA-K底物的磷蛋白。 这家银行 然后将用于探测重组鞭毛模型，并分离 动力蛋白来剖析这些轴丝调节的作用机制 元素 所取得的成果应有助于更好地了解 潜在疾病状态的可能畸变，典型表现为改变和/或 异常的轴丝运动