Calmodulin calcineurin activation

With the identification of calcineurin-mediated NF-ATc translocation in T cells, a model of the cytosolic events involved in T-cell activation has emerged, as oudined in Fig. 4. Stimulation of the TCR results in a series of membrane-associated events culminating in a rise in intracellular Ca2+, which binds to and activates calmodulin/calcineurin. Activated calcineurin then causes the cytoplasmic-to-nuclear translocation of NF-ATc, an event potentially mediated via the dephosphorylation of critical serine and/or threonine residues on the NF-ATc protein (Beals et al., 1997a, 1997b). Once nuclear, NF-ATc binds DNA with its nuclear partner, NF-ATn to drive production of IL-2 and other early genes, leading to lymphocyte activation and proliferation. 

Upon binding calcium ions, the small acidic protein known as calmodulin can activate enzymes by binding to a wide variety of proteins containing cahnodulin-binding domains. Such proteins include cAMP phosphodiesterase, calmodulin-dependent nitric oxide synthase, calmodulin kinases, the plasma membrane calcium pump, calcineurin, and calmodulin-dependent inositol-(l,4,5)-trisphosphate 3-kinase. See also Activation Autoinhibition... 

Calcineurin activity was measured at 30°C in a total volume of 150 fiL containing 50 mM Tris-HCl (pH 7.0), 0.1 mM EGTA, 0.5 mM dithiothreitol, 0.01% Brij, 0.3 mg/mL bovine serum albumin 0.1 yiM calmodulin, 1 mM MnCl2, 1 mM CaCl2, and variable amounts of calcineurin (0.11-0.44 /u.g/150 /u.L). Aliquots (20 /u.L) were taken at intervals and the reaction was stopped with 70 fiL of 0.5% perchloric acid containing 0.1% Triton X-100 and the internal standard, probenecid (10 nmol/mL). After centrifugation, a 40 fiL aliquot of the supemate was directly applied on the HPLC column. The rate of enzyme activity was calculated from the linear appearance of product over the first 6 minutes. 

The oxidation state of the Fe ion of calcineurin is still the subject of some debate. In a recent study, Klee and colleagues proposed that the functional redox state was the reduced Fe -Zn form [48]. This proposal was based on data which showed that a portion of calcineurin activity in crude brain extract was susceptible to inactivation following incubation with Ca + and calmodulin and that inactivation could be prevented by the addition of superoxide dismutase. Inactivation was more pronounced imder aerobic than in anaerobic conditions. Additionally, incubation with and calmodulin in the presence of Snm ascorbate protected calcineurin from inactivation while reactivation could be achieved by addition of 0.5 mM Fe(NH4)2(S04)2 in the presence of 5mM dithiothreitol and 5mM ascorbate. These data were taken to propose that the active redox state was Fe +-Zn " and that inactivation was due to oxidation of the cluster to the Fe +-Zn + state. Direct confirmation of the oxidation states was not provided. 

Carruthers, N.J. Stemmer, P.M. Methionine oxidation in the calmodulin-binding domain of calcineurin disrupts cahnoduUn binding and calcineurin activation. Biochemistry 2008,47, 3085-3095. 

Ca2+ [66], making calcium monitoring a vital tool for cellular biologists. For example, calmodulin-dependent protein phosphatase, calcineurin can be monitored using a probe based on the regulatory domain of the nuclear factor of activated T cells [69], This approach can be taken further to monitor multiple calcium-dependent events in real time [70],... 

Protein kinase Cd, Akt kinase, calcium/calmodulin-dependent protein kinase IV, mitogen-activated protein kinase kinase (MEKK-1), focal adhesion kinase (FAK), protein phosphatase (PP)2A, calcineurin... 

As explained, the immimomodulation signal starts with binding the antigen on T-cell receptor, which ultimately results in Ca2+ release. Ca2+ induces calmodulin activation, which in turn activates calcineurin, a Ca2+-dependent phosphatase. This event leads to translocation of the cytoplasmic component of the transcription factor, which is required for IL-2 gene expression and T-cell activation. 

Protein phosphatase 2B is also known as calcineurin. Calcineurin is made up of a catalytic A subunit and a regulatory B subimit that shows similarity to calmodulin. A binding site for Ca Vcalmoduhn, required for activation of calcineurin, is found in the catalytic subunit. 

Deactivation of calmodulin-dependent enzymes may involve calmodulin-binding proteins such as calcineurin,229 although calcineurin has been shown to possess phosphatase activity.230... 

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