PROTEIN PHOSPHORYLATION IN SPERM FLAGELLAR MOTILITY

蛋白质磷酸化在精子鞭毛调控中的作用 将研究cAMP和钙-钙调蛋白(Ca~(2+)-CaM)的运动性。 动力蛋白(α-重链和3个较小的蛋白质)被鉴定为 CAMP依赖蛋白激酶(CA-K)磷酸化的底物和 用于CaM依赖的蛋白磷酸酶(CaM-PrPase)的去磷酸化。 动力蛋白的磷酸化刺激ATPase活性和能力 动力蛋白在玻璃衬底上推动紫杉醇稳定的微管。 在人、狗、猪和海胆的精子中也发现了CaM-PrPase 为鞭毛衣藻。所有精子鞭毛磷酸酶均为 分别从鞭毛和动力蛋白中提取盐分，可以 与21S动力蛋白可逆地共沉淀成蔗糖梯度。这个 磷酸酶调节精子模型中钙离子依赖的游泳参数。 这些观察结果是直接联系的第一个证据。 在第二信使磷酸化和去磷酸化途径之间以及 对鞭毛功能成分的调节，如动力蛋白。基座 根据这些观察，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 并且第二信使通路的组件可以紧密地关联 在精子鞭毛轴丝的框架内含有动力蛋白。这个 本项目期的具体目标是：1.分离和鉴定 作为第二信使调节底物的动力蛋白的成分 磷酸化和去磷酸化以及：i)鉴定和表征 那些影响动力蛋白ATPase活性和动力蛋白的磷酸蛋白 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. 鉴定和分离鞭毛成分和CaM-PrPase。3.隔离 并对鞭毛CaM-PrPase进行了表征。4.识别非外臂 CaM-PrPase和cAMP-的动力蛋白鞭毛磷蛋白底物 依赖蛋白激酶。5.尊重地描述这些底物 对其依赖的cAMP和钙离子依赖的磷酸化、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性的精子 用三磷酸腺苷重新激活，以及体外微管滑动试验 动力蛋白。每种类型的化验都将受到cAMP和cAMP探针的挑战 CA2=-Cam通路。将使用数字图像分析来量化 鞭毛和微管运动和[Y-32P]ATP将被用来识别 CaM-PrPase和非动力蛋白鞭毛的磷蛋白 作为CaM-PrPase和CA-K底物的磷酸蛋白。这家银行 将被用于探测重组鞭毛模型，并分离出 Dynein来剖析这些轴线调节的作用机制 元素。所获得的结果应该会使我们更好地理解 以改变和/或改变为代表的疾病状态潜在的可能的异常 轴丝运动异常。

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.