DUAL CALCIUM-ION REGULATION OF CALCINEURIN BY CALMODULIN AND CALCINEURIN-B

The dependence of calcineurin on Ca2+ for activity is the result of th e concerted action of calmodulin, which increases the turnover rate of the enzyme and modulates its response to Ca2+ transients, and of calc ineurin B, which decreases the K-m of the enzyme for its substrate. Th e calmodulin-stimulated protein phosphatase calcineurin is under the c ontrol of two functionally distinct, but structurally similar, Ca2+-re gulated proteins, calmodulin and calcineurin B. The Ca2+-dependent act ivation of calcineurin by calmodulin is highly cooperative (Hill coeff icient of 2.8-3), and the concentration of Ca2+ needed for half-maximu m activation decreases from 1.3 to 0.6 mu M when the concentration of calmodulin is increased from 0.03 to 20 mu M. Conversely, the affinity of calmodulin for Ca2+ is increased by more than 2 orders of magnitud e in the presence of a peptide corresponding to the calmodulin-binding domain of calcineurin A. Calmodulin increases the V-max without chang ing the K-m value of the enzyme. Unlike calmodulin, calcineurin B inte racts with calcineurin A in the presence of EGTA, and Ca2+ binding to calcineurin B stimulates native calcineurin up to only 10% of the maxi mum activity achieved with calmodulin. The Ca2+-dependent activation o f a proteolyzed derivative of calcineurin, calcineurin-45, which lacks the regulatory domain, was used to study the role of calcineurin B. R emoval of the regulatory domain increases the V-max of calcineurin, as does binding of calmodulin, but it also increases the affinity of cal cineurin for Ca2+. Ca2+ binding to calcineurin B decreases the K-m val ue of calcineurin without changing its V-max. Like native calcineurin, calcineurin-45 contains two high-affinity Ca2+ sites (K-d < 0.07 mu M ) and one or two low-affinity sites (K-d > 0.07 mu M).