The serine-/threonine-specific protein phosphatase calcineurin is conserved from yeast to man and was first detected in bovine brain extracts by Wang and Desai and independently by Watterson and Vanaman in 1976 (Wang and Desai 1976; Watterson and Vanaman 1976). Klee and Krinks were the first to purify calcineurin in 1978 (Klee and Krinks 1978), and Klee also coined the name “calcineurin,” since the protein was extracted from brain tissue and it could bind Ca2+ (Klee et al. 1979). However, the function of calcineurin was not known at this time. It was assumed that calcineurin might be a regulatory subunit of the phosphodiesterase, as it could inhibit phosphodiesterase activity. In the early 1980s, the real function as a phosphatase was demonstrated. Stewart et al. found that the protein phosphatase 2B isolated from rabbit skeletal muscle was identical to the previously identified calcineurin (Stewart et al. 1982). Since then, many studies elucidated the expression and the enzymatic activity of calcineurin in various organisms (reviewed in Rusnak and Mertz 2000). In the early 1990s, another milestone was reached in calcineurin research: it was discovered that the immunosuppressive drugs cyclosporine A (CsA) and tacrolimus (FK506), both used particularly in transplant medicine since the late 1970s and mid-1980s, respectively, inhibit calcineurin activity (Liu et al. 1991). At this time it was also discovered that CsA and FK506 could block the dephosphorylation of the transcription factor NFAT (nuclear factor of activated T-cells) (Flanagan et al. 1991). Adding one and one together, it was quite easy to conclude that the dephosphorylation of NFAT is mediated by calcineurin. Subsequently, the role of calcineurin and NFAT during immune response and during other cellular processes was intensively studied (Rusnak and Mertz 2000). Additionally, research on calcineurin and NFAT and its orthologues, respectively, was expanded to nonmammalian organisms (Thewes 2014a). Especially the identification of the yeast calcineurin substrate Crz1, which is a functional orthologue of NFAT, also opened a new research area in lower eukaryotes (Stathopoulos and Cyert 1997).
Structure and Activation of Calcineurin
The activation of calcineurin occurs stepwise, mediated by an increase in the cytosolic Ca2+ concentration (Fig. 1b) (Li et al. 2011). At low cytosolic Ca2+ concentrations, calcineurin is inactive. An increase of the intracellular Ca2+ concentration ([Ca2+]i) leads to binding of Ca2+ ions to the EF hands of CNB, resulting in a partially active form. Further increase of [Ca2+]i leads to activation of calmodulin, which, in turn, can bind to the CaM-binding domain. This binding leads to a displacement of the autoinhibitory domain and to full activation of the phosphatase (Fig. 1b). Activated calcineurin specifically dephosphorylates its target proteins at serine or threonine residues. After binding, the phosphoryl group of the target protein is probably directly transferred to a water molecule (Rusnak and Mertz 2000).
Besides regulation via the intracellular calcium concentration, several other mechanisms affecting calcineurin activity (inhibition or activation) have been identified (Rusnak and Mertz 2000). It has been shown that phospholipids – depending on the lipid – can activate or inhibit calcineurin. Further, arachidonic acid and unsaturated, long-chain fatty acids can activate calcineurin. As the active site of the catalytic calcineurin subunit contains a dinuclear Fe2+-Zn2+ center, calcineurin activity can be also influenced by the redox state of the cell (Namgaladze et al. 2002).
Different interacting and target proteins have been identified in different organisms (Thewes 2014a; Adler et al. 2011; Goldman et al. 2014). Calcineurin is highly selective for its targets, primarily by recognizing the degenerate docking motifs PxIxIT and LxVP. However, although the PxIxIT motif binds substrates to calcineurin and increases the local substrate concentration, the site is not essential for dephosphorylation. The LxVP site is thought to be necessary for substrate orientation. Both motifs are difficult to identify in silico due to their variable orientation and degeneracy in their sequence (Goldman et al. 2014). Additionally, in contrast to other phosphatases, in silico predictions of putative target proteins usually fail for calcineurin, as the phosphatase seems to prefer unstructured regions instead of specific motifs for dephosphorylation (Li et al. 2013). Therefore, systematic approaches – using, e.g., phosphoproteomics – were used to identify interacting and target proteins of calcineurin (Goldman et al. 2014). As can be seen in Fig. 2, in S. cerevisiae calcineurin is the central player in a complex signaling network. Many different interacting proteins as well as real substrates of calcineurin have been identified, belonging to many different cellular processes. A similar diversity of interacting proteins and substrates has been detected in humans and other organisms (Thewes 2014a; Li et al. 2011). Although the interacting proteins and substrates of calcineurin are not always identical in higher and lower eukaryotes, the cellular processes in which calcineurin is involved are conserved among different species (Thewes 2014b). One example of conserved calcineurin function is polarized growth of cells in different fungi and in neurons of mammals (see also below). The best-investigated calcineurin targets are probably the family of the nuclear factor of activated T-cells (NFAT1–NFAT4) in mammals (Müller and Rao 2010) and the zinc-finger transcription factor Crz1 in yeast and its orthologues from other organisms (Thewes 2014a).
Calcineurin Function in Different Organisms
The overall significance of calcineurin varies in different organisms. Whereas the knockout of both CNA and CNB is lethal in some organisms (e.g., mice and D. discoideum), the knockout of the subunits in other organisms is feasible, resulting only in mild phenotypes (defects) under normal growth conditions (e.g., S. cerevisiae) (Rusnak and Mertz 2000; Thewes 2014b).
Furthermore, as the diversity of interacting and substrate proteins of calcineurin is quite high, the function of the phosphatase is also quite diverse. In lower eukaryotes calcineurin is involved in the regulation of stress response, cell wall integrity, drug resistance, glucose metabolism, pathogenesis, ion homeostasis, and development (Fig. 2 and (Thewes 2014b) and references therein). In higher eukaryotes, calcineurin is especially involved in immune response and cancer, mainly mediated by NFAT (Müller and Rao 2010). However, in higher eukaryotes calcineurin is also involved in several developmental processes. Among those are developmental processes of the brain (neural induction, dendrite outgrowth and branching, synapse density, neurodegeneration); development of the vasculature, the heart (coronary arteries, valves), the kidney (nephrogenic zone, glomeruli), the bones (formation and growth), and the muscles; and the regulation of fin outgrowth in zebra fish or tadpole tail generation in frogs (Thewes 2014b). Some of these processes are also mediated via NFAT, whereas other processes are NFAT independent (Thewes 2014b). Of particular interest is that – in higher and lower eukaryotes – calcineurin is involved in the development of “filamentous structures” such as axons, blood vessels, or hyphae (Thewes 2014b). Another parallel between higher and lower eukaryotes is that the regulation of filamentous structures by calcineurin is partially mediated by members of the NFAT family in mammals and its functional orthologues of the Crz1 family in fungi and other lower eukaryotes (Thewes 2014a).
Since its discovery in the late 1970s, calcineurin has become one of the best-studied proteins in different organisms, resulting in over 700 publications per year (in the last 10 years) dealing with calcineurin. This “popularity” of the phosphatase is mainly caused by its involvement in the immune response, particularly after organ transplantation. Current research focuses on the safety and efficacy of traditional (CsA, FK506) as well as newly identified calcineurin inhibitors. Further, more and more cellular processes with calcineurin-involvement are elucidated. One highlight of current research is the idea to target the testis-specific mammalian CNA-γ-isoform for the development of a male contraceptive (Miyata et al. 2015). In filamentous fungi a specific phosphorylated serine-/proline-rich region of calcineurin has been discovered, which is absent in humans, suggesting the possibility of harnessing this novel site for innovative antifungal drug design (Juvvadi et al. 2013). Taken together, in addition to calcineurin’s involvement in immunological processes, the phosphatase has become very attractive in many other research areas.
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