Interaction of the regulatory subunit of a type II cAMP-dependent protein kinase with mammalian sperm flagellum.
We have reported previously (Horowitz, J. A., Toeg, H., and Orr, G. A. (1984) J. Biol. Chem. 259, 832-838) that most of the type II cAMP-dependent protein kinases in rat sperm are associated with the flagellum. We have now identified flagellar polypeptides which are capable of forming tight complexes with the regulatory subunit of type II cAMP-dependent protein kinase (RII). Flagellar RII-binding polypeptides were identified using an RII overlay/immunoblot procedure and had apparent subunit Mr of 120,000, 80,000, and 57,000 in rat and 120,000 and 57,000 in bovine flagella. RII is released from the flagellum by disulfide reducing agents, e.g. 1 mM dithiothreitol (DTT). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie Blue staining of the DTT-released material shows that a limited subpopulation of flagellar polypeptides are solubilized by disulfide-reducing agents. Neither tubulin, the dynein ATPase, or any of the RII-binding proteins are released by 1 mM DTT, and thin section electron microscopy revealed that the morphology of the flagellum is unaltered by reducing conditions. Our data established that RII is not linked to the flagellum via a direct disulfide bridge. We propose that RII is released from the flagellum, a highly disulfide cross-linked structure, due to structural changes in the flagellum which disrupts the interaction between RII and its binding proteins.
Horowitz
J A
JA
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Wasco
W
W
Leiser
M
M
Orr
G A
GA
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D004229:Dithiothreitol / Q000494:pharmacology; D007163:Immunosorbent Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008854:Microscopy, Electron; D008970:Molecular Weight; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D012995:Solubility; D013082:Sperm Tail / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D013094:Spermatozoa / Q000201:enzymology*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C015482:protein kinase modulator; D011494:Protein Kinases; D004229:Dithiothreitol