ASSAYING PROTEIN PHOSPHATASES IN SPERM AND FLAGELLA

Sperm and flagellar motility mechanism depends on protein phosphorylation. Dynein has been indicated as a potential site of convergence for regulation through phosphorylation/dephosphorylation by protein kinases and phosphatases. Phosphorylation of dynein has been correlated with increased ATPase activity and an increase in the velocity of dynein-mediated gliding of microtubules in vitro. An important role for protein phosphatases in motility regulation has been suggested by adding protein phosphatases or their inhibitors to flagellar models. A significant decrease in the gliding velocity of microtubules in vitro by dynein has been observed by preincubation of dynein with serinekhreonine protein phosphatase, SPP, recently purified from sea urchin sperm. The enzyme specifically dephosphorylates a single dynein-associated subunit phosphorylated by cyclic adenosine monophosphate (cAMP)-dependent protein kinase. This chapter describes the assay for serine/threonine protein phosphatases using the dephosphorylation of type II regulatory subunit (RII) peptide phosphorylated by cAMP-dependent protein kinase as substrate and the considerations when designing experiments to examine the effect of protein phosphatases and kinases on the gliding velocity of microtubules in vitro by dynein. The methods presented are purification of the catalytic subunit of cAMP-dependent protein kinase, phosphorylation and purification of RII substrate, and protein phosphatase assay. The ability to assay dynein function by examining the propulsion of taxol-stabilized microtubules on glass surfaces offers a tool for examining the role of protein phosphorylation. All experiments examining effects on gliding of manipulation in solution are conducted in parallel with incubations using dynein that has been treated with [32P]ATP and analyzed by sodium dodecyl phosphate–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, to confirm changes in protein phosphorylation that are associated with changes in gliding velocity. A peptide substrate radiolabeled to a high specific activity is essential for the high sensitivity and reproducibility of the protein phosphatase activity. © 1995, Academic Press Inc.