Factors responsible for the Ca²⁺‐dependent inactivation of calcineurin in brain

Abstract

The Ca2+-dependent protein phosphatase activity of crude rat brain extracts measured in the presence of okadaic acid, exhibits the characteristic properties of the calmodulin-stimnlated protein phosphatase, calcineurin. It is stimulated more than 200-fold by Ca 2+ and inhibited by the calmodnlin-binding peptide, MI3, and by the immunosuppressive drug, FKS06. It is insensitive to rapamycin at concentrations up to 1 pM. Its specific activity, based on calcineurin concentration determined by quantitative analysis of Western blots exposed to anti-bovine brain IgG, is ten to twenty times that of purified rat brain calcineurin assayed under similar conditions. Unlike the purified enzyme it is rapidly and irreversibly inactivated in a time-, temperature-, and Ca2+/ calmodulin-dependent fashion without evidence of extensive proteolytic degradation. The enzyme is converted to a state which does not lose activity by removal of low molecular weight material by gel filtration. Reconstitution of a labile enzyme is achieved by the addition of the low molecular weight-containing fraction elnted from the gel filtration column. These observations indicate that calcineurin in crude brain extracts is under the control of Ca2+/calmodulin-dependent positive and negative regulatory mechanisms which involve unidentified endogenous factor(s).

Regulation of the Calmodulin-stimulated Protein Phosphatase, Calcineurin

“…In the recently published crystal structures of calcineurin (28,29), these two metal ions are modeled on the structure of the [Fe 3ϩ -Zn 2ϩ ] kidney bean purple acid phosphatase. The high specific activity of calcineurin in crude extracts in the absence of added metals suggests that the crude enzyme has retained its natural cofactors (22). Inactivation of crude calcineurin by the superoxide anion and its protection and reactivation by ascorbate strongly suggest that reduced iron is required for activity (23).…”

“…In crude tissue extracts, calcineurin exhibits a high phosphatase activity that is almost completely dependent on calmodulin and does not depend on added metals for activity but is subject to a time-and Ca 2ϩ /calmodulin-dependent inactivation facilitated by small heat-stable inactivators (22). The search for factors responsible for the high phosphatase activity and instability of crude calcineurin led to the finding that, in crude extracts, calcineurin is protected against inactivation by superoxide dismutase (23).…”

“…Because it is a poor substrate for protein phosphatases 1, 2A, and 2C it is well suited to quantitate the Ca 2ϩ /calmodulin-dependent, metal-independent, okadaic acid-insensitive calcineurin activity in crude tissue extracts provided that the incubation time is reduced to 1-2 min to minimize Ca 2ϩ -dependent calcineurin inactivation (21)(22)(23)(24). The conveniently measurable p-nitrophenyl-phosphatase activity has been employed to study its catalytic mechanism and to propose a catalysis involving the protonation of the phosphoester bond by a metal-activated water molecule followed by the cleavage of the bond by a second metal-activated water molecule, without the formation of a covalent intermediate (25).…”

Neural functions of calcineurin in synaptic plasticity and memory: Figure 1.

“…CaN has very high affinity for Ca 2+ and is activated by nanomolar concentrations of Ca 2+ (Cohen and Klee 1988). It can also be reversibly inactivated after prolonged Ca 2+ /CaM exposure (Stemmer et al 1995;Shen et al 2008) by, for instance, oxidation of a critical amino acid in the CaM binding domain of CaNA that blocks Ca 2+ /CaM binding and CaN activation (Carruthers and Stemmer 2008).…”

Modulation of calcineurin activity in mouse brain by chronic oral administration of cyclosporine A

“…It has been shown that CN phosphatase activity is also sensitive to oxidative stress and may be modulated by the intracellular redox potential (27,28). Stemmer reported that a stabilising factor in crude bovine brain extracts protected CN from inactivation.…”