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.
