Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm.
Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.
Tash
J S
JS
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Krinks
M
M
Patel
J
J
Means
R L
RL
Klee
C B
CB
Means
A R
AR
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001667:Binding, Competitive; D019703:Calcineurin; D002118:Calcium / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004285:Dogs; D005407:Flagella / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D007118:Immunoassay; D008297:Male; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D012617:Sea Urchins; D013081:Sperm Motility; D013094:Spermatozoa / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure; D013552:Swine
D000906:Antibodies; D002148:Calmodulin-Binding Proteins; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium