Gossypol inhibits calcineurin phosphatase activity at multiple sites.
Calcineurin, the Ca2+/calmodulin-dependant serine/threonine phosphatase is the target for the immunosuppressant drugs FK506 and cyclosporine-A. These established calcineurin inhibitors each require an immunophilin protein cofactor. Gossypol, a polyphenol produced by the cotton plant, inhibits calcineurin (IC50=15 microM), in a noncompetitive, reversible manner, and is independent of any cofactor. We found that gossypol acts by at least two mechanisms to inhibit calcineurin phosphatase activity. A calmodulin-independent form of calcineurin was less sensitive to inhibition by gossypol than native calcineurin (IC50=41 and 18 microM, respectively) indicating that gossypol may interfere with calmodulin binding. A fluorescence polarization based assay demonstrated that 100 microM gossypol reduced the affinity of calmodulin for calcineurin (from K(d)=2.4 to 250 nM). Inhibition of calcineurin phosphatase activity by gossypol could not be overcome by adding excess calmodulin or by testing the inhibition toward a calmodulin-independent calcineurin indicating that gossypol acts at a site different from the calmodulin-binding site. Gossypol decreased the affinity of calcineurin for immunosuppressant/immunophilin complexes only in the presence of calmodulin, indicating that gossypol blocks the effects of calmodulin binding to calcineurin. In addition, gossypol had a stimulatory effect on native calcineurin in the absence of calmodulin, possibly indicating a calmodulin mimetic effect. Gossypol exists in two enantiomeric forms which are reported to have different potency for cell toxicity. (+) and (-) gossypol had equivalent potency for inhibition of native and calmodulin-independent calcineurin phosphatase activity, and for inhibition of calmodulin binding. The inhibition of calcineurin by gossypol via multiple binding sites without stereo-specificity indicates that gossypol is not a specific calcineurin inhibitor.
Carruthers
Nicholas J
NJ
Wayne State University, Institute of Environmental Health Sciences, Detroit, MI 48201, United States.
Dowd
Michael K
MK
Stemmer
Paul M
PM
D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D065095:Calcineurin Inhibitors; D002147:Calmodulin / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006072:Gossypol / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology; D010455:Peptides / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D011817:Rabbits
D065095:Calcineurin Inhibitors; D002147:Calmodulin; D004791:Enzyme Inhibitors; D010455:Peptides; C065738:MLCK peptide; C120817:calcineurin phosphatase; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; D003546:Cysteine Endopeptidases; C019913:clostripain; D006072:Gossypol