DOC_PP2B_PxIxIT_1

Calcineurin (also known as PP2B or PPP3) is a calcium-dependent phosphatase. The calcineurin holoenzyme binds to both substrates and regulators through two motifs known as the PxIxIT (DOC_PP2B_PxIxIT_1) and LxVP (DOC_PP2B_LxvP_1) docking motifs. PxIxIT and LxVP sequences were first defined in the nuclear factor of activated T-cells (NFAT) family of transcription factors. Subsequently, PxIxIT and LxVP motifs were identified in multiple substrates of human and budding yeast calcineurin (Wigington,2020; Goldman,2014; Brauer,2019). The holoenzyme is active only in the presence of (i) Ca2+ which binds to the B subunit and (ii) Ca2+-bound calmodulin which binds to an amphipathic helix in the A subunit (Rusnak,2000).
The name calcineurin generally refers to the complete heterodimeric holoenzyme consisting of a calcineurin A (CNA; Q08209), the catalytic subunit, and calcineurin B (CNB; P63098), the Ca2+-binding regulatory subunit. In addition to the phosphatase domain, the CNA subunit contains three regulatory domains including a CNB-binding domain, a calmodulin-binding domain, and an auto-inhibitory domain. The auto-inhibitory domain can bind to the substrate-binding pocket of the CNA catalytic subunit, resulting in basal auto-inhibition. The CNB subunit contains four Ca2+-binding EF-hand motifs.
The PxIxIT motifs bind the CNA subunit and the LxVP motifs bind a composite interface consisting of both the A and B subunits. The PxIxIT motif can associate with calcineurin A in the absence of the B subunit and interacts equally with the active and inactive forms of the enzyme. Thus, the PxIxIT can tether inactive calcineurin to substrates awaiting calcineurin activation. Conversely, under basal conditions, the LxVP binding pocket is occluded by the auto-inhibitory sequences, and becomes available for substrate binding only after enzyme activation with Ca2+ and Ca2+/calmodulin. This mechanism maintains calcineurin in the inactive state when Ca2+ levels are low. Structural analysis of the β isoform of human calcineurin A shows that a C-terminal FSVL peptide binds the LxVP-binding pocket.
PxIxIT and LxVP motifs play distinct roles during dephosphorylation. By binding under both basal and signalling conditions, PxIxIT motifs target calcineurin to substrates/regulators or to protein complexes that contain substrates. For example, the human scaffold protein, AKAP79, co-binds calcineurin and two substrates (the L-type Ca2+ channel and PKA RII regulatory subunit) that lack PxIxITs but contain LxVP motifs. In contrast, the LxVP motif, which can only bind active calcineurin, is hypothesised to help orient the phosphosite towards the catalytic centre of calcineurin for dephosphorylation. Several CN binders contain both PxIxIT and LxVP motifs and the relative positioning, intervening regions and affinity of the motifs can encode distinct outcomes. In NFAT, for example, the PxIxIT is located at 200 amino acids N-terminal of the LxVP, and the phosphorylation sites regulated by calcineurin are in the extensive disordered polypeptide region between the two motifs. Whereas, Regulator of calcineurin 1 (RCAN1) also contains a PxIxIT and LxVP motif that form an extended interface, however, in this case, the co-operative binding of both motifs facilitates a high-affinity interaction blocking substrate recruitment and allowing RCAN to act as a calcineurin inhibitor (PDH:6UUQ). The diversity of binding modes to calcineurin suggests that other mechanisms of substrate engagement by calcineurin likely await discovery.
PxIxIT interactions are essential for dephosphorylation of most substrates and mutating the PxIxIT site in a substrate disrupts its dephosphorylation. Furthermore, peptides or small molecules that compete with native PxIxIT sequences for binding to calcineurin inhibit dephosphorylation of many substrates in vitro and in vivo (Nguyen,2019; Matsoukas,2015; Aramburu,1999). Cyclosporin A and FK506, in complex with their respective small binding protein (immunophilin), block the LxVP-binding pocket of calcineurin. Because Cyclosporin A and FK506 inhibit calcineurin from multiple species and abrogate all known calcineurin-mediated dephosphorylation events, engagement of the LxVP-binding pocket by substrates is also thought to be essential for dephosphorylation. Both docking motifs can cooperate to drive the phosphatase activity of calcineurin, for example, both motifs in NFAT proteins are required for efficient dephosphorylation (Rodriguez,2009). A238L, a protein produced by the African Swine Fever Virus, inhibits calcineurin using the same mechanism (Grigoriu,2013). The A238L peptide binds tightly to calcineurin (Kd=4nM) via a PxIxIT and a non-canonical LxVP motif.
The best-studied cellular function of calcineurin involves the regulation of the T cell activation in the immune response via dephosphorylation of NFAT family transcription factors, enabling NFAT nuclear translocation (reported in complex with calcineurin) and activation of interleukin IL-2. The widely prescribed immunosuppressant drugs, FK506 (tacrolimus) and cyclosporin A inhibit calcineurin by blocking its ability to bind a short linear peptide motif in NFAT (LxVP), which is a required step in recognition and dephosphorylation by the phosphatase. These immunosuppressant drugs find use after organ transplantations and, in the case of tacrolimus, for ectopic treatment in atopic dermatitis.
Calcineurin is evolutionarily conserved across Eukaryotes and seems to be ubiquitously expressed (Rusnak,2000). The PxIxIT and LxVP binding surfaces on calcineurin are highly conserved, suggesting that this characteristic mode of substrate recognition is retained throughout the fungal and animal kingdoms (Roy,2009). The PxIxIT peptides form contacts along the edge of two β sheets in the calcineurin A subunit. This interaction is similar to that of the RVxF SLiM with the catalytic subunit of PP1, demonstrating that these critical binding sites are evolutionarily related (Li,2004). Interestingly, calcineurin substrates in yeast and humans are distinct due to significant network rewiring (Goldman,2014).