Calcineurin-phospholipid interactions. Identification of the phospholipid-binding subunit and analyses of a two-stage binding process.
Photoaffinity labeling of calcineurin by 1,2-distearoyl-sn-glycero-3-phospho-N-(4-azido-3-[125I]iodo-2- hydroxybenzoyl)ethanolamine resulted in preferential labeling of its regulatory B subunit. Photolabeling of B was greatly enhanced by Ca2+ which further supports the hypothesis that the phospholipid-binding site of calcineurin is located on this Ca2(+)-binding subunit. Extending the time of incubation of calcineurin with the photoprobe prior to photolysis also elevated labeling of the B subunit, probably as a result of time-dependent changes in protein conformation. Support for these conformational changes was obtained when time-dependent preincubation of calcineurin with acidic phospholipids enhanced subsequent tryptic degradation of its B subunit. Activity measurements and analyses of the reversibility of phospholipid-binding provided evidence for a two-stage mechanism of calcineurin-phospholipid interactions. Initial binding of calcineurin to phospholipids is rapid, Ca2(+)-sensitive, reversible, and leads to stimulation of the phosphatase toward a number of its substrates. A subsequent slow phase strengthens the association and appears to correlate with the phospholipid-promoted conformational change of the B subunit; the corresponding time-dependent effects on enzymatic activity are, again, substrate-dependent.
Politino
M
M
Department of Chemistry, Ohio State University, Columbus 43210.
King
M M
MM
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D009532:Nickel / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000138:chemical synthesis / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity
D000345:Affinity Labels; D001386:Azides; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D046911:Macromolecular Substances; D010714:Phosphatidylethanolamines; D010743:Phospholipids; C063903:1,2-distearoyl-sn-glycero-3-phospho-N-(4-azido-3-iodo-2-hydroxybenzoyl)ethanolamine; D009532:Nickel; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium