Bicarbonate-dependent serine/threonine protein dephosphorylation in capacitating boar spermatozoa.
This study investigates the dynamics of serine/threonine (S/T) protein phosphorylation in sperm incubated under capacitating (C) conditions using the boar as a model system. For the first time, this approach has identified multiple dephosphorylation events that occur in a bicarbonate-dependent fashion. Different phospho-(S/T) kinase substrate antibodies were used, and dephosphorylation of 5 S/T phosphoproteins was observed in C sperm compared with noncapacitated (N) cells. Specifically, dephosphorylation of 96-, 90-, 64-, and 55-kd proteins was detected by immunoblotting using 2 phospho-Akt substrate antibodies and a phosphoprotein kinase A substrate antibody. In addition, dephosphorylation of a 105-kd protein was detected using a phospho-ATM/ATR substrate antibody. In contrast, no dephosphorylation was observed using a phosphoprotein kinase C substrate antibody, and increased tyrosine phosphorylation of 32- and 20-kd proteins was detected in C compared with N sperm. Immunolocalization experiments revealed subtle changes in the pattern expression as well as a reduction of phosphorylation in C sperm. Whereas sperm incubated in N medium containing dibutyryl cAMP (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) did not show protein dephosphorylation, incubation in C medium with dbcAMP/IBMX showed dephosphorylation as well as increased phosphorylation of other proteins (p68, p51, and p29). Finally, calyculin A, a phosphatase inhibitor, prevented dephosphorylation of p96, p90, p64, and p55 but not p105. Based on these data, we propose 2 pathways of protein dephosphorylation that are active during capacitation and independent of cAMP. Together, this provides direct evidence for more complex S/T phosphorylation dynamics than has been previously described for sperm undergoing capacitation.
Alnagar
Fahima A
FA
Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff, UK.
Brennan
Paul
P
Brewis
Ian A
IA
D015056:1-Methyl-3-isobutylxanthine; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D003994:Bucladesine; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D008297:Male; D008387:Marine Toxins; D010080:Oxazoles; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000201:enzymology*; D013552:Swine
D001639:Bicarbonates; D008387:Marine Toxins; D010080:Oxazoles; D003994:Bucladesine; C059041:calyculin A; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D015056:1-Methyl-3-isobutylxanthine