Phosphorylation of the Arginine-X-X-(Serine/Threonine) motif in human sperm proteins during capacitation: modulation and protein kinase A dependency.
Sperm capacitation is a complex process that involves a protein kinase A (PKA)-dependent tyrosine phosphorylation of proteins. We studied the time-course, the modulation and the cellular localization of the phosphorylation of the Arginine-X-X-(Serine/Threonine) motif, characteristic of PKA substrates, in sperm proteins during capacitation. There was an increased phosphorylation of 80 (p80) and 105 (p105) kDa protein bands in human sperm treated with different capacitation inducers. Phosphorylation of p80 and p105 induced by fetal cord serum ultrafiltrate or the combination of 3-isobutyl-1-methylxanthine and dibutyryl cAMP was prevented by H89 and Rp-adenosine-3',5'-cyclic monophosphorothionate, confirming the involvement of PKA in this effect. Inhibitors of protein kinase C, receptor type tyrosine kinase and mitogen-activated protein kinase kinase did not affect the Arginine-X-X-(Serine/Threonine) motif phosphorylation. Non-receptor type protein tyrosine kinase inhibitors, PP2 and herbimycin A, enzymatic antioxidants and a nitric oxide synthase inhibitor prevented the phosphorylation of p80 and p105 when sperm were incubated with fetal cord serum ultrafiltrate. The phosphorylated Arginine-X-X-Serine/Threonine motif was immunolocalized all along the flagellum and the fluorescent signal was higher in capacitating than in non-capacitating sperm. These results show for the first time the presence of a PKA-dependent phosphorylation of proteins in human sperm capacitation and its upstream modulation by reactive oxygen species and non-receptor type protein tyrosine kinase.
Urology Research Laboratory, Royal Victoria Hospital and Faculty of Medicine, McGill University, Montréal, Québec, Canada H3A 1A1. coflaher@yahoo.com
C
C
O'Flaherty
E
E
de Lamirande
C
C
Gagnon
D020816:Amino Acid Motifs; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D008297:Male; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D001120:Arginine; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C