Participation of the human sperm proteasome in the capacitation process and its regulation by protein kinase A and tyrosine kinase.
The proteasome is a multicatalytic cellular complex present in human sperm that plays a significant role during several steps of mammalian fertilization. Here, we present evidence that the proteasome is involved in human sperm capacitation. Aliquots of highly motile sperm were incubated with proteasome inhibitors MG132 or epoxomicin. The percentage of capacitated sperm, the chymotrypsin-like activity of the proteasome, cAMP content, and the pattern of protein phosphorylation were assayed by using the chlortetracycline hydrochloride assay, a fluorogenic substrate, the cAMP enzyme immunoassay kit, and Western blot analysis, respectively. Our results indicate that treatment of sperm with proteasome inhibitors blocks the capacitation process, does not alter cAMP concentration, and changes the pattern of protein phosphorylation. To elucidate how proteasome activity is regulated during capacitation, sperm were incubated with: 1) tyrosine kinase (TK) inhibitors (genistein or herbimycin A); 2) protein kinase (PK) A inhibitors or activators (H89 and Rp-cAMPS, and 8-Br-cAMP, respectively); or 3) PKC inhibitors (tamoxifen or staurosporin) at different capacitation times. The chymotrypsin-like activity and degree of phosphorylation of the proteasome were then assayed. The results indicate that sperm treatment with TK and PKA inhibitors significantly decreases the chymotrypsin-like activity of the proteasome during capacitation. Immunoprecipitation and Western blot results suggest that the proteasome is phosphorylated during capacitation in a TK- and PKA-dependent pathway. In conclusion, we suggest that the sperm proteasome participates in the capacitation process, and that its activity is modulated by PKs.
Kong
Milene
M
Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile.
Diaz
Emilce S
ES
Morales
Patricio
P
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D016227:Benzoquinones / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017828:Rifabutin / Q000031:analogs & derivatives; D013075:Sperm Capacitation / Q000187:drug effects / Q000502:physiology*; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology*; D013449:Sulfonamides / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D047029:Lactams, Macrocyclic; D007976:Leupeptins; D009842:Oligopeptides; D061988:Proteasome Inhibitors; D013449:Sulfonamides; D013873:Thionucleotides; D017828:Rifabutin; D015124:8-Bromo Cyclic Adenosine Monophosphate; C016957:adenosine-3',5'-cyclic phosphorothioate; C020754:herbimycin; D019833:Genistein; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D046988:Proteasome Endopeptidase Complex; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C078846:epoxomicin