CYCLIC-AMP - DEPENDENT PROTEIN KINASE REGULATION OF SPERM MOTILITY
HOROWITZ, JILL ANN
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cAMP-dependent phosphorylation of sperm proteins has been implicated in the initiation and/or regulation of sperm motility. Since cAMP-dependent protein kinase (R(,2)C(,2)) is believed to be the sole cellular mediator of all cAMP dependent processes, this enzyme is likely to play a central role in this process and in microtubule-based, dynein ATPase-mediated motility in general. To understand the role of the cAMP-dependent protein kinases in sperm motility the following studies were carried out: (i) characterization and localization of cAMP-dependent kinase regulatory subunits (R) in rat epididymal sperm; (ii) analysis of the nature of the interaction of the sperm flagellum and the R subunit of type II cAMP-dependent kinase (RII); (iii) the flagellar proteins capable of binding to R; and (iv) potential substrates for C-dependent phosphorylation in sperm flagellum.;The majority of cAMP binding activity in rat caudal epididymal sperm was associated with the demembranated tailpiece (flagellum). This cAMP binding activity was identified as the regulatory subunit of cAMP-dependent protein kinase by reconstitution of cAMP-dependent protein kinase activity after the addition of exogenous purified catalytic subunit from bovine heart. The flagellar enzyme was tightly bound to sperm tails via the R subunit. The catalytic subunit was released into solution upon the addition of cAMP. The majority of cAMP-dependent kinase in sperm was shown to be of the type II class. Selective solubilization of flagella using chaotropic reagents showed that RII is probably associated with the 9+2 microtubule structure (axoneme). RII may be released from the flagella by dithiothreitol without concomitant release or dynein or tubulin, and the structure of the flagellum remains intact. RII appears to have a limited site of interaction with the axoneme. The major proteolytic cleavage fragment which contains the cAMP binding sites and the C-terminal 70% of the polypeptide can be released from the axoneme by mild proteolysis. Possible RII binding proteins were detected by RII overlay/immunoblot techniques. cAMP-dependent kinase catalytic subunit was specifically observed to phosphorylate several flagellar polypeptides of molecular weight = 150, 55, 44 and approximately 15 kDaltons. The flagellar RII was observed to react with monoclonal antibodies which recognize both neural and non-neural subclasses of RII but not with those which recognize only the neural subclass. (Abstract shortened with permission of author.).
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