Identifying New Substrates and Functions for an Old Enzyme: Calcineurin

Abstract

Biological processes are dynamically regulated by signaling networks composed of protein kinases and phosphatases. Calcineurin, or PP3, is a conserved phosphoserine/phosphothreonine-specific protein phosphatase and member of the PPP family of phosphatases. Calcineurin is unique, however, in its activation by Ca²⁺ and calmodulin. This ubiquitously expressed phosphatase controls Ca²⁺-dependent processes in all human tissues, but is best known for driving the adaptive immune response by dephosphorylating the nuclear factor of the activated T-cells (NFAT) family of transcription factors. Therefore, calcineurin inhibitors, FK506 (tacrolimus), and cyclosporin A serve as immunosuppressants. We describe some of the adverse effects associated with calcineurin inhibitors that result from inhibition of calcineurin in nonimmune tissues, illustrating the many functions of this enzyme that have yet to be elucidated. In fact, calcineurin has essential roles beyond the immune system, from yeast to humans, but since its discovery more than 30 years ago, only a small number of direct calcineurin substrates have been shown (∼75 proteins). This is because of limitations in current methods for identification of phosphatase substrates. Here we discuss recent insights into mechanisms of calcineurin activation and substrate recognition that have been critical in the development of novel approaches for identifying its targets systematically. Rather than comprehensively reviewing known functions of calcineurin, we highlight new approaches to substrate identification for this critical regulator that may reveal molecular mechanisms underlying toxicities caused by calcineurin inhibitor-based immunosuppression.

Figure 1.

Overview of calcineurin structure. (A) Schematic of canonical (α, β2, and γ) and β1 CN isoforms. BBH, CN-B binding helix; CBD, calmodulin-binding domain; AIS, autoinhibitory sequence; AID, autoinhibitory domain; MLS, membrane localization sequence; EF, calcium-binding helix-loop-helix domain (EF1, 2 are lower affinity calcium-binding domains and EF3, 4 are higher affinity calcium-binding domains). (B) Ribbon diagram of CN heterodimer from PDB entry 4ORC (Li et al. 2016). CN-A subunit is in gray and CN-B subunit is in wheat. Calcium ions are colored in green, AIS in magenta, and AID in red. The catalytic site is represented by zinc (magenta) and iron (lime) metal ions.

Figure 2.

Schematic of proposed activation mechanism for canonical and calcineurin-β1 isozymes. Ca²⁺ ions binding CN-B and CaM (calmodulin) are in white. The magenta box represents the autoinhibitory sequence (AIS) sequence. For canonical calcineurin isozymes, Ca²⁺ binding to CN-B and calmodulin binding to CN-A relieves autoinhibition by the AIS and autoinhibitory domain (AID). For the calcineurin β1 isozyme, activation by calcium and calmodulin entail removal of the AIS and LAVP sequence from the substrate-binding groove. Activation may also involve localization of calcineurin-β1 to membranes via lipid modifications at the carboxy-terminal end. For all calcineurin isozymes, cleavage of the carboxy-terminal domains by calpain or caspases results in constitutively active enzyme.

Figure 3.

Calcineurin interacts with substrates via SLiMs, PxIxIT, and LxVP. (A) Space-filled diagram of calcineurin heterodimer complexed with the calcineurin-inhibiting domain from the viral protein A238L (PBD entry 4FOZ; Grigoriu et al. 2013). CN-A subunit is in gray and CN-B subunit is in wheat. The A238L peptide is in green with its PxIxIT-SLiM colored in orange and the LxVP-SLiM colored in purple. The calcineurin catalytic site is marked in red. (B) Space-filled diagram of calcineurin heterodimer complexed with FK506-FKBP12 (PDB entry ITCO; Griffith et al. 1995). CN-A and -B subunits and the calcineurin catalytic site are colored and in A. The FK506-FKBP12 complex is in magenta and occupies the same groove as the LxVP-SLiM in A.

Figure 4.

Schematic for proposed mechanism of substrate engagement by calcineurin. The substrate is shown as a dark brown line with PxIxIT (yellow) and LxVP (purple) SLiMs and the phosphate group to be removed (green circle). The PxIxIT SLiM can interact with calcineurin under nonactivating conditions, whereas the LxVP SLiM engages with calcineurin only after enzyme activation by Ca²⁺ and calmodulin. The immunosuppressants (IS) complexes, either CsA-cyclophilin or FK506-FKBP (both denoted as IS, red) with immunophilin (IMPH, orange) occlude the LxVP pocket to inhibit substrate binding.