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Table 1.

Effect of various kinase inhibitors on the motility of intact sea urchin spermatozoa

Kinase or pathway targetedSubstanceIC50 (μmol l–1)Type of action or targetConcentration range testedInhibition of motility
AKT AKT inhibitor Phosphatidyl-inositol ester analog 1–10 μmol l–1 None 
ERK pathway CGP85793 ND Grb2-SH2 domain competitor 1–100 μmol l–1 None 
 CGP78850 ND Grb2-SH2 domain competitor 1–100 μmol l–1 Partial at 100 μmol l–1 
PKA H-89 0.048 ATP-site competitor 1–100 μmol l–1 Total at ⩾30 μmol l–1 
 KT 5720 0.056 ATP-site competitor 0.1–10 μmol l–1 None 
 dbcAMP NA Activator 1–10 mmol l–1 None 
 IBMX NA Activator 0.1–1 mmol l–1 None 
 Rp-cAMPS 11 cAMP competitor 0.2–2 mmol l–1 None 
PKC Chelerythrine 0.66 Inhibits PKC translocation 0.5–50 μmol l–1 Total at ⩾2 μmol l–1 
 Calphostin C 0.05 Diacylglycerol competitor 0.15–15 μmol l–1 Total at ⩾10 μmol l–1 
 Gö6976 0.002–0.006 Ca2+ dependent PKC α1 and β1 0.05–50 μmol l–1 Partial at ⩾0.5 μmol l–1 
PI3K Wortmannin 0.005 Catalytic subunit 1–100 nmol l–1 None 
PTK Tyrphostin A47 2.4 Receptor type PTK 1–50 μmol l–1 Partial at ⩾10 μmol l–1 
 Tyrphostin A1 >100 Inactive analog 0.1–100 μmol l–1 None 
 PP2 0.005 Non-receptor type PTK 0.2–2 μmol l–1 None 
 Herbymicin A 8–12 Non-receptor type PTK 10 μmol l–1 None 
 Genistein 2–6 ATP-site competitor 100 μmol l–1 None 
 poly-Glu-Tyr NA Competitor for Tyr phosphorylation 10 μg ml–1 None 
Broad spectrum Staurosporine 0.001–0.03 ATP-site competitor 0.2–2 μmol l–1 Total at ⩾1 μmol l–1 
 H-7 3–6 ATP-site competitor 10–100 μmol l–1 None 
Kinase or pathway targetedSubstanceIC50 (μmol l–1)Type of action or targetConcentration range testedInhibition of motility
AKT AKT inhibitor Phosphatidyl-inositol ester analog 1–10 μmol l–1 None 
ERK pathway CGP85793 ND Grb2-SH2 domain competitor 1–100 μmol l–1 None 
 CGP78850 ND Grb2-SH2 domain competitor 1–100 μmol l–1 Partial at 100 μmol l–1 
PKA H-89 0.048 ATP-site competitor 1–100 μmol l–1 Total at ⩾30 μmol l–1 
 KT 5720 0.056 ATP-site competitor 0.1–10 μmol l–1 None 
 dbcAMP NA Activator 1–10 mmol l–1 None 
 IBMX NA Activator 0.1–1 mmol l–1 None 
 Rp-cAMPS 11 cAMP competitor 0.2–2 mmol l–1 None 
PKC Chelerythrine 0.66 Inhibits PKC translocation 0.5–50 μmol l–1 Total at ⩾2 μmol l–1 
 Calphostin C 0.05 Diacylglycerol competitor 0.15–15 μmol l–1 Total at ⩾10 μmol l–1 
 Gö6976 0.002–0.006 Ca2+ dependent PKC α1 and β1 0.05–50 μmol l–1 Partial at ⩾0.5 μmol l–1 
PI3K Wortmannin 0.005 Catalytic subunit 1–100 nmol l–1 None 
PTK Tyrphostin A47 2.4 Receptor type PTK 1–50 μmol l–1 Partial at ⩾10 μmol l–1 
 Tyrphostin A1 >100 Inactive analog 0.1–100 μmol l–1 None 
 PP2 0.005 Non-receptor type PTK 0.2–2 μmol l–1 None 
 Herbymicin A 8–12 Non-receptor type PTK 10 μmol l–1 None 
 Genistein 2–6 ATP-site competitor 100 μmol l–1 None 
 poly-Glu-Tyr NA Competitor for Tyr phosphorylation 10 μg ml–1 None 
Broad spectrum Staurosporine 0.001–0.03 ATP-site competitor 0.2–2 μmol l–1 Total at ⩾1 μmol l–1 
 H-7 3–6 ATP-site competitor 10–100 μmol l–1 None 

Immotile concentrated spermatozoa were first diluted in IM in the absence or presence of kinase inhibitor and then in ASW with or without the inhibitor at the same concentration. Motility was assessed by light microscopy with a dark-field illumination as detailed in the Materials and methods, and evaluations of motility inhibition reported here are for the end of the 15-min observation period. ND, not determined; NA, not applicable

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