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Gossypol Disrupts Calcineurin Activation at Multiple Sites
Author(s) -
Carruthers Nicholas John,
Dowd Michael K.,
Stemmer Paul M.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1123-d
Subject(s) - gossypol , chemistry , calcineurin , binding site , ic50 , phosphatase , serine , calmodulin , stereochemistry , biochemistry , enzyme , in vitro , transplantation , medicine
Calcineurin (CN), the Ca 2+ /calmodulin (CaM) dependant serine/threonine phosphatase is the target for the immunosuppressant drugs FK506 and cyclosporine A (CsA). These established CN inhibitors each require an immunophilin protein cofactor. Gossypol, a polyphenol produced by the cotton plant, exists in two enantiomeric forms which are reported to have different potency for cell toxicity. Racemic gossypol inhibits CN (IC 50 =15 μM), in a noncompetitive, reversible manner, and is independent of any cofactor. We hypothesize that gossypol has a specific binding site on CN that overlaps the FK506/CsA binding site and that gossypol enantiomers will have different potency in CN inhibition. (+)− and (−) −gossypol inhibited CN phosphatase activity with IC 50 s of 23 and 14 μM respectively, indicating that the gossypol binding site was not specific. A CaM‐independent form of CN was less sensitive to inhibition by gossypol than native CN (IC 50 =41 and 18 μM, respectively) indicating that gossypol may interfere with CaM binding. A fluorescence polarization based assay demonstrated that 100 μM gossypol reduced the affinity of Fl‐CaM for CN (from Kd=2.4 to 250 nM). Inhibition of CN phosphatase activity by gossypol could not be overcome by excess CaM or by using CaM‐independent CN indicating that gossypol acts at multiple sites. Gossypol decreased the affinity of CN for FK506 only in the presence of CaM, indicating that the binding sites for FK506 and gossypol on CN do not overlap. The inhibition of CN by gossypol via multiple binding sites without stereo‐specificity indicates that gossypol is not a specific CN inhibitor.