title: Changes of PKA and PDK1 in the principal piece of boar spermatozoa treated with a cell-permeable cAMP analog to induce flagellar hyperactivation.
abstract: A cAMP-induced protein tyrosine phosphorylation and flagellar hyperactivation are controlled via complicated signaling cascades in mammalian spermatozoa. For instance, these events seem to be regulated positively by the PKA-mediated signaling and negatively by the PI3K/PDK1-mediated signaling. In this article, we have shown molecular changes of PKA and PDK1 in cAMP analog (cBiMPS)-treated boar spermatozoa in order to disclose possible roles of these kinases in protein tyrosine phosphorylation and hyperactivation. Ejaculated spermatozoa were incubated with cBiMPS, and then they were used for biochemical analyses of sperm kinases by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The first 30-min incubation with cBiMPS highly activated PKA of the principal piece to the accompaniment of autophosphorylation on Thr-197 of catalytic subunits. However, protein tyrosine phosphorylation and hyperactivation were fully induced in the sperm samples after the 180-min incubation. A potentially active form of PDK1 (54/55-kDa phospho-PDK1) was detected in the principal piece of the spermatozoa during the 90-min incubation. Another potentially active form (59-kDa phospho-PDK1) gradually increased during the same incubation period. However, the PDK1 suddenly became inactive by the dephosphorylation after the 180-min incubation, namely coincidently with full induction of protein tyrosine phosphorylation and hyperactivation. Additionally, existence of PI3K-dependently suppressing mechanisms for protein tyrosine phosphorylation was confirmed in the principal piece by pharmacological experiments with LY294002 and biochemical analyses with anti-PI3K p85 antibodies. These findings suggest that dephosphorylation of PDK1 may be a molecular switch for enhancement of protein tyrosine phosphorylation and flagellar hyperactivation in boar spermatozoa.
authors.affiliation: Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, Kobe, Japan. : harayama@kobe-u.ac.jp
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Harayama
authors.forename: Kazumi
authors.initials: K
authors.lastname: Nakamura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002463:Cell Membrane Permeability / Q000187:drug effects; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000031:analogs & derivatives; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004004:Dichlororibofuranosylbenzimidazole / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005407:Flagella / Q000187:drug effects* / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D012689:Sequence Homology, Nucleic Acid; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism; D034421:Sus scrofa; D013873:Thionucleotides / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D013873:Thionucleotides; C071644:5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate; D004004:Dichlororibofuranosylbenzimidazole; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017027:Protein Tyrosine Phosphatases