Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation.
Signal transduction pathways regulate various aspects of mammalian sperm function. When human sperm were incubated in a medium supporting capacitation, proteins became tyrosine-phosphorylated in a time-dependent manner. This phosphorylation was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation was also reduced when sperm were incubated either in the presence of increasing concentrations of extracellular Ca2+ or in a medium containing the Ca2+ ionophore A23187. This Ca2+-induced dephosphorylation was calmodulin-dependent, suggesting that calcineurin was involved. In this regard, the calcineurin inhibitor deltamethrin inhibited the Ca2+ ionophore-induced dephosphorylation. A limited number of Mr 80,000-105,000 polypeptides were the most prominent phosphotyrosine-containing proteins present in human sperm. Unlike mouse sperm, which contains a tyrosine-phosphorylated isoform of hexokinase, a phosphotyrosine-containing hexokinase in human sperm was not detected. Most of the tyrosine-phosphorylated proteins were Triton X-100-insoluble and were localized to the principal piece of the flagellum, the region where the cytoskeletal fibrous sheath is found. Prominent phosphotyrosine-containing proteins of Mr 82,000 and 97,000 were identified as the human homologues of mouse sperm AKAP82, the major fibrous sheath protein, and pro-AKAP82, its precursor polypeptide, respectively. These proteins are A Kinase Anchor Proteins, polypeptides that sequester protein kinase A to subcellular locations. Taken together, these results suggest that protein tyrosine phosphorylation may be part of a signal transduction cascade(s) regulating events pertaining to capacitation and/or motility in mammalian sperm and that an interrelationship between tyrosine kinase and cAMP signaling pathways exists in these cells.
Carrera
A
A
Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia 19104-6080, USA.
Moos
J
J
Ning
X P
XP
Gerton
G L
GL
Tesarik
J
J
Kopf
G S
GS
Moss
S B
SB
D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology; D008297:Male; D051379:Mice; D010617:Phenanthridines / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011506:Proteins / Q000378:metabolism*; D029589:Seminal Plasma Proteins; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology
D000470:Alkaloids; D053119:Benzophenanthridines; D002147:Calmodulin; D004791:Enzyme Inhibitors; D007529:Isoflavones; D010617:Phenanthridines; D047428:Protein Kinase Inhibitors; D011506:Proteins; D029589:Seminal Plasma Proteins; D019000:Phosphotyrosine; D000001:Calcimycin; D019833:Genistein; C016299:chelerythrine; D017868:Cyclic AMP-Dependent Protein Kinases; D019311:Staurosporine; D002118:Calcium