Calcium- and calmodulin-sensitive interactions of calcineurin with phospholipids.

Physical association of calcineurin with phosphatidylserine (PS) or phosphatidylglycerol (PG) was observed by molecular exclusion chromatography; the enzyme did not associate with phosphatidylethanolamine or phosphatidylcholine. The interactions with PS and PG were enhanced by Ca2+ which implicates a regulatory role for the Ca2+-binding subunit in this process. Addition of PG or PS to standard calcineurin assays elicited profound changes in enzymatic activity; phosphatidylcholine and phosphatidylethanolamine were without effect. Up to 23-fold stimulation of the calmodulin-independent activity was observed with phosphorylated histone H1 or synapsin I as the substrates. In contrast, the activity toward p-nitrophenyl phosphate and tyrosine phosphate was found to be inhibited. A characterization and comparison of the two opposite responses showed that: the phospholipids had insignificant effects on the Km for substrates, the phospholipid specificity for activation and inhibition was nearly indistinguishable, half-maximal activation and inhibition were obtained at similar concentrations of PG (K0.5 = 0.21 and 0.14 mg/ml, respectively), and calmodulin enhanced the responses to PG (K0.5 = 0.064 and 0.033 mg/ml for activation and inhibition, respectively) to similar extents. Together, these observations demonstrate that the two substrate-dependent responses of calcineurin are due to the association of the phosphatase with phospholipids and not a result of substrate-phospholipid interactions. This suggests that Ca2+- and calmodulin-stimulated interactions of calcineurin with acidic phospholipids may play a role in regulating the substrate specificity of this multifunctional phosphatase.