Abstract

The Ca2+-dependent phosphatase Calcineurin, also known as PP2B and CaN, is involved in a number of quite diverse signalling pathways in the cells of different tissues. However, dephosphorylation by calcineurin is not used as broadly in cell regulation as is the PP1 phosphatase. Calcineurin is evolutionarily conserved (all Eukaryotes) and seems to be ubiquitously expressed (Rusnak and Mertz, 2000). It participates in signal transduction pathways governing the development and function of the immune, nervous, cardiovascular and musculoskeletal systems (Aramburu et al., 2004). The most studied cellular function of calcineurin involves the regulation of T cell gene expression by dephosphorylation of NFAT family transcription factors, enabling NFAT nuclear translocation (reportedly in complex with calcineurin) and activation of interleukin IL-2. In yeast, the localization of PP2B substrates are dispersed, with examples of PP2B substrates present in the nucleus (Stathopoulos-Gerontides et al., 1999), on the cytosolic side of the plasma membrane (Bultynck et al., 2006) and cytosolic tail-anchored proteins of the endoplasmic reticulum (Heath et al., 2004). The function of PP2B substrates in yeast is associated with various environmental stimuli that are stressful to the cell (Boustany and Cyert, 2002). (Unstressed PP2B-minus cells are viable in the laboratory). Survival response includes correct polarization of the actin cytoskeleton.

Calcineurin is a heterodimeric protein consisting of calcineurin A (CNA), the catalytic subunit, and calcineurin B (CNB), the Ca2+-binding subunit. In addition to the enzymatic site, the calcineurin A subunit contains three regulatory domains that include the calcineurin B-binding domain, the calmodulin-binding domain, and the autoinhibitory domain. The calcineurin B subunit contains four Ca2+-binding EF-hand motifs. The autoinhibitory domain binds to the substrate-binding pocket of the catalytic domain, resulting in basal autoinhibition. Binding of Ca2+/calmodulin to the enzyme results in a conformational change that relieves autoinhibition and leads to activation of the phosphatase (Rusnak and Mertz,2000). Activated calcineurin then dephosphorylates target S/T phosphorylation sites. Calcineurin signalling is effectively blocked by cyclosporin A and tacrolimus (FK506), which act to stabilise a complex of calcineurin with the proline isomerases cyclophilin or FKBP12. The stable drug-immunophilin complex blocks nuclear translocation of NFATs, thereby suppressing T cell activation (Snyder et al., 1998). These immunosuppressant drugs find use after organ transplant and, in the case of tacrolimus, ectopically for atopic dermatitis.



Calcineurin does not necessarily dephosphorylate all sites on the substrate. Most of the residues dephosphorylated by calcineurin are in SP or TP sites, possibly explaining the associated proline isomerase. A docking site has been described in NFATs with conserved sequence PVIVIT (Li et al., 2007). The motif binds to calcineurin by beta-augmentation (at a site that is equivalent to the RVxF-binding site of PP1). A related but less tightly conserved variant P.(I).(IV) occurs in other known calcineurin substrates and the motif pattern seems to be more relaxed in yeast than in vertebrates.