Association of the regulatory subunit of a type II cAMP-dependent protein kinase and its binding proteins with the fibrous sheath of rat sperm flagellum.
Demembranated rat sperm flagellar polypeptides capable of binding the regulatory subunit (RII) of a type II cAMP-dependent protein kinase, having apparent subunit molecular masses of 120, 80 and 57 kDa were identified by an RII overlay procedure [Horowitz, J. A., Wasco, W., Leiser, M. & Orr, G. A. (1988) J. Biol. Chem. 263, 2098-2104]. In this study it is shown that all three polypeptides capable of binding RII on a solid-phase blot are tightly associated with the fibrous sheath. Purified fibrous sheath preparations were capable of binding (a) [3H]cAMP and (b) purified catalytic subunits of cAMP-dependent protein kinase forming a functional holoenzyme. The 57-kDa protein was identified as RII by photoaffinity labeling with 8-azido[32P]cAMP. This peptide was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. RII alpha was also shown to form tight, specific complexes with the fibrous sheath demonstrating the presence of functional RII alpha-binding sites. Truncated RII beta fusion proteins were used to identify the N-terminal amino acids at positions 1-50 as a primary determinant for RII-binding protein interaction. Differential extraction of adult testis with buffers containing Triton X-100, urea and sodium dodecyl sulfate revealed the presence of 80-kDa (major) and 120-kDa (minor) RII-binding proteins in particulate extracts. The 80-kDa polypeptide is only expressed at late stages of spermatogenesis, i.e. during spermiogenesis, suggesting a developmental role for RII anchoring to the sperm flagellar fibrous sheath.
Macleod
J
J
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx.
Mei
X
X
Erlichman
J
J
Orr
G A
GA
D000375:Aging / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D008854:Microscopy, Electron; D008970:Molecular Weight; D010766:Phosphorylation; D051381:Rats; D013082:Sperm Tail / Q000378:metabolism* / Q000648:ultrastructure; D013737:Testis / Q000254:growth & development / Q000378:metabolism*
D002352:Carrier Proteins; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D046911:Macromolecular Substances; C517822:Prkar2a protein, rat; C517839:Prkar2b protein, rat; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases