C ALCINEURIN REGULATION BY REDOX PROCESSES

First indications of calcineurin susceptibility to oxidation came from the study showing that some sulfhydryl reagents, including N-ethylmaleimide (NEM) and p-hydroxymercuribenzoic acid, inhibited its metal-dependent and –independent activities [King, 1986; Gupta et al., 1990]. Inactivation of calcineurin could be traced to modification of one or two thiols. Another evidence for redox sensitivity of calcineurin provided the fact that calcineurin was one of the six major proteins in neutrophil homogenate binding to immobilized phenylarsine oxide (PAO), a specific reagent for vicinal thiol groups

[Schmachtel, 1996]. Direct involvement of redox processes in the regulation of calcineurin activity was first shown in the pioneering work of Klee and associates [Wang et al., 1996b].

Investigating the process of calcium- and calmodulin-dependent calcineurin inactivation in rat brain homogenates [Stemmer et al., 1995] Wang et al. found that Cu, Zn-SOD protected calcineurin against this inactivation. In addition, yeast lacking Cu, Zn-SOD had the same phenotype as calcineurin-null strains, and calcineurin activity in lysates of SOD1-mutants was about 1/10 of wild-type strains. Subsequent studies of several groups revealed that exogenous H2O2 could inhibit calcineurin activity in neutrophils [Carballo et al., 1999], NK cells [Furuke et al., 1999] and Jurkat T-lymphocytes [Reiter et al., 1999], and one study on cell lysates and purified calcineurin revealed that in general calcineurin activity is increased by antioxidants and decreased by oxidants [Sommer et al., 2000]. Calcineurin was also inhibited after thiol depletion of the culture medium [Furuke et al., 1999]. H2O2 blocked also downstream calcineurin-NFAT or calcineurin-NFκB signaling in these cases.

The discovery of calcineurin sensitivity to superoxide prompted the efforts to discover the situations, where such an inhibition could take place in vivo. One example provided regulation of gene expression in hippocampus, where the increase of CREB protein phosphorylation after prolonged electrical stimulation of neurons could be traced to the inhibition of calcineurin activity reversed by antioxidants [Bito et al., 1996]. Particular interest focused on the regulation of calcineurin activity during FALS, a disease characterized by mutations of Cu, Zn-SOD. Although one study found no effect of certain FALS mutations on calcineurin activity [Lee et al., 1999], others provided a correlation between the severity of the disease caused by particular mutation and the degree of calcineurin inhibition [Ferri et al., 2000; Volkel et al., 2001; Ferri et al., 2001].

Regarding the mechanism of calcineurin sensitivity towards ROS Klee and associates initially proposed that the iron in the binuclear metal center of calcineurin (postulated to have Fe²⁺-Zn²⁺ composition) is the target as evidenced by the protective effect of ascorbate and the reversal of inactivation by a mix of Fe²⁺ , ascorbate and DTT [Wang et al., 1996]. This hypothesis was however contradicted by the studies of Rusnak and colleagues [Rusnak et al., 1999a; Rusnak and Reiter, 2000]. EPR spectroscopy of the purified bovine calcineurin showed the presence of Fe³⁺ in the binuclear center of calcineurin [Yu et al., 1995; Yu et al., 1997]. This calcineurin was active towards pNPP and RII peptide, although the activity towards RII was lower than reported by Wang et al. Moreover, the reduction of Fe³⁺ by dithionite led to the loss of the EPR signal and the simultaneous loss of calcineurin activity towards pNPP [Yu et al., 1997]. Additionally, a mixed valence Fe³⁺-Fe²⁺ binuclear center of

calcineurin was obtained by metal exchange in the same group [Yu et al., 1995; Yu et al., 1997]. This calcineurin was equally sensitive to H2O2 as well as to dithionite [Yu et al., 1997], and it was proposed that this metal center represents the redox-sensitive calcineurin form in vivo, the hypothesis still contested in favor of the Fe²⁺-Zn²⁺ center [Aramburu et al., 2000].

The involvement of calcineurin thiols in redox regulation of calcineurin remained unattended, although some indications, including reversibility of calcineurin inhibition by DTT [Reiter et al., 1999] and inhibition after cellular thiol depletion [Furuke et al., 1999] pointed to a possible role of calcineurin cysteines in regulation of its activity. It should be noted, however, that site-directed mutagenesis of several calcineurin cysteines (Cys197, Cys228, Cys166 and Cys88) produced protein with the activity equal to that of the wild type enzyme [Reiter et al., 1999]. In summary, although it is recognized that redox reactions modulate calcineurin activity, the mechanism of ROS action remains unclear.

AIMS OF THIS STUDY.

The recognition of the crucial role, which redox modulation of enzyme activity plays in cellular signal transduction, led to a fast development of research concentrated on redox modifications of proteins. Calcineurin represents one of the major protein phosphatases, and is critical for regulation of numerous physiological processes. Therefore elucidation of the mechanisms, by which redox processes modulate calcineurin activity, appears to be of importance for our understanding of how cellular phosphorylation/dephosphorylation cascades respond to environmental changes. Previous research identified two possible redox-sensitive targets on calcineurin, protein thiols and its binuclear metal center, but the exact mechanisms of redox modulation of calcineurin activity remained unclear. In addition, the question, which oxidant is the most relevant redox modulator of calcineurin in vivo, is left open. In this regard, this study pursued the following aims:

• To investigate the mechanisms, by which thiol-modifying agents and oxidants modulate the activity of isolated calcineurin

• To investigate the effects of superoxide and other oxidants on calcineurin activity in cellular extracts and in isolated form

• To elucidate the involvement of the enzyme binuclear metal center in redox regulation of calcineurin

• To compare the effects of oxidants on calcineurin activity in vitro and in intact cells