The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
Calcineurin (PP2B) PxIxIT docking motif
Functional site description:
Calcineurin, also referred to as PPP3 or PP2B, is a metalloenzyme that dephosphorylates phosphoserine and phosphothreonine residues. Calcineurin controls Ca2+-dependent processes in all human tissues, most notably activating the adaptive immune response, though it also plays a key role in the regulation of inflammation, axonal guidance, the Ca2+-dependent migration of neutrophils, synaptic plasticity, and apoptosis. Calcineurin acts on a select group of protein substrates containing one or more SLiMs from two motif classes, the PxIxIT (DOC_PP2B_PxIxIT_1) and LxVP (DOC_PP2B_LxvP_1) motifs.
ELMs with same tags:
ELM Description:
PxIxIT sequences are found in most, but not all substrates and regulators of calcineurin. The PxIxIT mnemonic only approximates the motifs which are highly degenerate in most positions of the sequence. The core sequences typically share a position 1 proline, followed by two hydrophobic residues in positions 3 and 5, a hydrophilic residue in position 6, and a variety of amino acids in positions 2 and 4 (the “x” positions”). Only two PxIxIT sequences lacking a P in position 1 are known, in human AKAP79 and yeast RCN1. Structures of different PxIxIT-calcineurin complexes (3LL8; 4F0Z; 6UUQ; 6NUU) show nearly identical interactions binding by β-augmentation. Positions 2, 4 and 6 cannot have Pro due to the β-strand H-bonding requirements. PxIxITs vary widely in their affinity for calcineurin, with motifs from five different yeast substrates having Kds ranging from ~15-250 μM.
Recent systematic analyses of amino acid substitutions in four PxIxIT peptides determined that the “x” positions and flanking residues (i.e. positions before and after the PxIxIT core) influence affinity for calcineurin: basic residues at position 2 (K or R) increase affinity; hydrophobic or acidic residues at position -1 increase or decrease affinity, respectively; acidic or phosphorylated residues at position 9 increase affinity (Nguyen,2019). The experimentally validated PxIxIT peptides are consistent with these findings.
PxIxIT peptides form contacts along the edge of two β sheets in the calcineurin A subunit (Li,2004). This hydrophobic docking groove is remote from the catalytic cleft, which explains the ability of PxIxIT to associate with calcineurin A in the absence of the B subunit, and to interact equally with the active and inactive forms of the enzyme. Thus, PxIxIT is critical for tethering calcineurin to substrates or regulators, such as the AKAP79 scaffold protein, but PxIxIT based-inhibitors do not interact with the catalytic centre, or interfere with dephosphorylation of substrates that lack a PxIxIT site.
Pattern Probability: 0.0011290
Present in taxon: Eukaryota
Interaction Domains:
  • Metallophos (PF00149) Calcineurin-like phosphoesterase (Stochiometry: 1 : 1)
  • Calcineurin-like phosphoesterase domain, ApaH type (IPR004843) This domain is found in a diverse range of phosphoesterases, including bis(5'-nucleosyl)-tetraphosphatase (apaH), nucleotidases, sphingomyelin phosphodiesterases and 2'-3' cAMP phosphodiesterases, as well as nucleases such as bacterial SbcD or archaeal/yeast Mre11 (Stochiometry: 1 : 1)
o See 27 Instances for DOC_PP2B_PxIxIT_1
o Abstract
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).

o 17 selected references:

o 14 GO-Terms:

o 27 Instances for DOC_PP2B_PxIxIT_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
Q6VV64 Kcnk18
210 215 KPVEEAIPQIVIDAGVDELL TP 3 Mus musculus (House mouse)
P53968 CRZ1
331 336 THAAPVTPIISIQEFNEGHF TP 2 Saccharomyces cerevisiae (Baker"s yeast)
Q99332 FRT1
72 77 SKSSSRLPVIAVNDNPVVPR TP 2 Saccharomyces cerevisiae (Baker"s yeast)
P40485 SLM1
673 678 SELQDQVPNIYIQTPINDFK TP 3 Saccharomyces cerevisiae (Baker"s yeast)
O95644 NFATC1
118 123 GAPALESPRIEITSCLGLYH TP 1 Homo sapiens (Human)
Q60591 Nfatc2
111 116 AGASGPSPRIEITPSHELMQ TP 1 Mus musculus (House mouse)
Q05193-3 DNM1
844 849 VPSRPNRAPPGVPRITISDP TP 4 Homo sapiens (Human)
O36972 Mal-047
206 211 HRRFKKKPKIIITGCEDNVY TP 4 African swine fever virus Malawi LIL 20/1
Q65142 5EL
205 210 HRWFKKKPKIIITGCKNNVY TP 5 African swine fever virus
O77638 NFATC1
110 115 GAPALESPRIEITSYLGLHH TP 3 Sus scrofa (Pig)
P19634 SLC9A1
715 720 YEPKEDLPVITIDPASPQSP TP 5 Homo sapiens (Human)
P53805 RCAN1
209 214 HAATDTTPSVVVHVCESDQE TP 5 Homo sapiens (Human)
O95180 CACNA1H
2264 2269 RAEHLTVPSFAFEPLDLGVP TP 2 Homo sapiens (Human)
P12270 TPR
2083 2088 RPPHPLPPRLTIHAPPQELG TP 5 Homo sapiens (Human)
419 424 PGTFWTNPQFKISLPEGDDP TP 2 Homo sapiens (Human)
408 413 LQYETSNPTVQLKIPQLRVS TP 2 Homo sapiens (Human)
P06731 CEACAM5
287 292 STQELFIPNITVNNSGSYTC TP 2 Homo sapiens (Human)
P21333 FLNA
2167 2172 CDLSLKIPEISIQDMTAQVT TP 2 Homo sapiens (Human)
Q14872 MTF1
548 553 NSVLTNNPTITITPTPNTAI TP 2 Homo sapiens (Human)
Q9H252 KCNH6
182 187 YRTISQIPQFTLNFVEFNLE TP 2 Homo sapiens (Human)
Q16799 RTN1
316 321 KPSPDTVPTVTVSEPEDDSP TP 2 Homo sapiens (Human)
1060 1065 DPASRTNPDLILTSPDFALS TP 2 Homo sapiens (Human)
64 69 AAVSSSAPEIVVSKPEHNNS TP 2 Homo sapiens (Human)
1903 1908 SRVGMKVPTISVTSFHSQSS TP 2 Homo sapiens (Human)
O14511 NRG2
616 621 YSLATQVPTFEITSPNSAHA TP 2 Homo sapiens (Human)
P49790 NUP153
485 490 PITSSSLPTFNFSSPEITTS TP 5 Homo sapiens (Human)
528 533 VPRNRPPPLVEITPRDQLLN TN 2 Homo sapiens (Human)
Please cite: The Eukaryotic Linear Motif resource: 2022 release. (PMID:34718738)

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