title: Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.
abstract: Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways.
authors.affiliation: Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas 77843-4466, USA.
authors.forename: Lauro
authors.initials: L
authors.lastname: González-Fernández
authors.forename: Beatriz
authors.initials: B
authors.lastname: Macías-García
authors.forename: Shavahn C
authors.initials: SC
authors.lastname: Loux
authors.forename: Dickson D
authors.initials: DD
authors.lastname: Varner
authors.forename: Katrin
authors.initials: K
authors.lastname: Hinrichs
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006736:Horses; D008297:Male; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013075:Sperm Capacitation / Q000187:drug effects / Q000502:physiology; D013081:Sperm Motility / Q000187:drug effects / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D013449:Sulfonamides; C017967:W 7; D051416:Focal Adhesion Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium
keywords: calcium; capacitation; equine; protein tyrosine phosphorylation; sperm