The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
Calcineurin (PP2B) LxVP 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_PxIxI_1) and LxVP (DOC_PP2B_LxvP_1) motifs.
ELMs with same tags:
ELM Description:
This PP2B-docking motif is defined by four amino acids. There is an absolute requirement for leucine in the first position of the motif and for proline in the last position. However, the viral protein A238L (O36972), which has been shown to prevent recognition of LxvP-containing substrates by calcineurin, contains a lysine residue instead of a proline in the last position (4F0Z) (Grigoriu,2013). The second position can accommodate a variety of amino acids. The third position generally contains a valine, but other hydrophobic residues have also been observed. Some motif instances are immediately preceded by an aromatic residue, which can further strengthen the interaction. Upon binding of Ca2+, the CNA subunit undergoes a conformational change, exposing the hydrophobic motif-binding pocket that is located at the interface of the CNA and CNB subunits. Therefore, LxvP sites can only interact with activated calcineurin. The hydrophobic pocket includes two CNA residues (W352, F356) and three CNB residues (L115, M118, V119), which mediate binding to substrates containing an LxvP motif. Immunosuppressants have been shown to bind to the hydrophobic pocket in a similar way. Upon binding to the hydrophobic pocket, the motif adopts a conformation in which it is almost parallel to the α-helix of CNA binding to CNB. The proline in the motif is predicted to interact with the aromatic residues in CNA.
Pattern: L.VP
Pattern Probability: 0.0003129
Present in taxon: Eukaryota
Interaction Domains:
o See 50 Instances for DOC_PP2B_LxvP_1
o Abstract
Calcineurin (also known as PP2B or PPP3) is a calcium-dependent phosphatase. The calcineurin holoenzyme binds to both substrates and regulators two motifs known as the PxIxIT (DOC_PP2B_PxIxI_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 200 amino acids N-terminal of the LxVP, and the phosphorylation sites regulated by calcineurin are in the extensive disordered 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 facilitate 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 suggest 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 African Swine Fever Virus inhibits the 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 at least 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 the model organism yeast as compared to human are quite distinct due to significant network rewiring (Goldman,2014).
o 8 selected references:

o 10 GO-Terms:

o 50 Instances for DOC_PP2B_LxvP_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P19634 SLC9A1
684 687 LEQKINNYLTVPAHKLDSPT TP 5 Homo sapiens (Human)
58 61 REPPGGQLLAVPAVSVDRKG TP 2 Homo sapiens (Human)
O95180 CACNA1H
2261 2264 QASCRAEHLTVPSFAFEPLD TP 2 Homo sapiens (Human)
Q96GX8 C16orf74
33 36 APVLNDKHLDVPDIIITPPT TP 2 Homo sapiens (Human)
Q15842 KCNJ8
405 408 SIRRNNSSLMVPKVQFMTPE TP 3 Homo sapiens (Human)
O43318 MAP3K7
422 425 KTASFGNILDVPEIVISGNG TP 2 Homo sapiens (Human)
O88942 Nfatc1
388 391 KGAFCEQYLSVPQASYQWAK TP 2 Mus musculus (House mouse)
Q13469 NFATC2
369 372 RNSAPESILLVPPTWPKPLV TP 8 Homo sapiens (Human)
589 592 VPTYPQLTLEVPQAPEVLRS TP 2 Homo sapiens (Human)
P26368 U2AF2
358 361 TINQTPVTLQVPGLMSSQVQ TP 2 Homo sapiens (Human)
938 941 ARESKSTTLTVPEQQRTTHH TP 2 Homo sapiens (Human)
558 561 DQGRSRSMLEVPRSISVPPS TP 2 Homo sapiens (Human)
327 330 SRNLSMDSLVVPLPNTSESF TP 2 Homo sapiens (Human)
713 716 EIGKAVGFLSVPKSLSSDST TP 2 Homo sapiens (Human)
P31629 HIVEP2
1007 1010 EFGKHSEFLTVPAGSYSLSV TP 2 Homo sapiens (Human)
292 295 QQTERNQSLLVPANPYHTAE TP 2 Homo sapiens (Human)
651 654 PEGLRPLTLQVPQGWAVLTG TP 2 Homo sapiens (Human)
81 84 QVEWNPQLLEVPPQTQFDYT TP 2 Homo sapiens (Human)
964 967 PKQEGPLHLQVPALTTFSDQ TP 2 Homo sapiens (Human)
168 171 GTKTTKQYLNVPPSPKVEDR TP 2 Homo sapiens (Human)
283 286 STCQNEITLQVPNPSELRAK TP 2 Homo sapiens (Human)
433 436 VVPDLPVFLPVPPNPIATFN TP 2 Homo sapiens (Human)
207 210 LTEKREELLSVPKEFLLLGP TP 2 Homo sapiens (Human)
393 396 EANFSANTLSVPRWSPQIPR TP 7 Homo sapiens (Human)
311 314 PQLPKVDLLTVPAVDTQMET TP 2 Homo sapiens (Human)
614 617 SEAPKMHTLQVPENHSVALN TP 2 Homo sapiens (Human)
Q01850 CDR2
296 299 QSLLEEMFLTVPESHRKPLK TP 2 Homo sapiens (Human)
O94875 SORBS2
342 345 YCSTYRQHLDVPRDSPRAIS TP 2 Homo sapiens (Human)
O14490 DLGAP1
318 321 QQERSCQYLQVPQDEWTGYT TP 2 Homo sapiens (Human)
O00763 ACACB
395 398 ASTVVAQTLQVPTLPWSGSG TP 2 Homo sapiens (Human)
Q96QT6 PHF12
377 380 LQSVKRRSLKVPDAIKSQYQ TN 2 Homo sapiens (Human)
2 5 MLNVPSQSFPAPRSQQRVAS TN 2 Homo sapiens (Human)
P78549 NTHL1
82 85 EKGEGAEPLKVPVWEPQDWQ TN 2 Homo sapiens (Human)
Q6ZSN1 Putative uncharacterized protein FLJ45355
120 123 SQGVKRGWLSVPTSRGVPPP TN 2 Homo sapiens (Human)
O95180 CACNA1H
2326 2329 PEKRRGLYLTVPQCPLEKPG TN 2 Homo sapiens (Human)
1085 1088 PRGLPPTSLQVPAAYPGILS TN 2 Homo sapiens (Human)
253 256 LDQDGDKQLSVPEFISLPVG TN 2 Homo sapiens (Human)
Q99504 EYA3
81 84 SAKPYAHILSVPVSETAYPG TN 2 Homo sapiens (Human)
2 5 MLAVPEMGLQGLYIGSSPER TN 2 Homo sapiens (Human)
Q8TF68 ZNF384
53 56 APPHYPTLLTVPASVSLPSG TN 2 Homo sapiens (Human)
108 111 QSPAEVFTLSVPNISLPAPS TN 2 Homo sapiens (Human)
O60346 PHLPP1
1584 1587 EKEKQQHLLQVPAEASDEGI TN 2 Homo sapiens (Human)
242 245 GPSQVTTFLTVPLTFATPSP TN 2 Homo sapiens (Human)
Q05315 CLC
4 7 MSLLPVPYTEAASLSTGSTV TN 2 Homo sapiens (Human)
O35303 Dnm1l
645 648 QKGHAVNLLDVPVPVARKLS TP 5 Rattus norvegicus (Norway rat)
P00515 PRKAR2A
83 86 SESEDEEDLDVPIPGRFDRR TP 1 Bos taurus (Cattle)
P36054 RCN1
101 104 QRNLTKQYLKVPESEKMFLI TP 4 Saccharomyces cerevisiae S288c
Q14934 NFATC4
378 381 KEVAGMDYLAVPSPLAWSKA TP 4 Homo sapiens (Human)
Q12968 NFATC3
393 396 KDSCGDQFLSVPSPFTWSKP TP 8 Homo sapiens (Human)
O95644 NFATC1
387 390 KGGFCDQYLAVPQHPYQWAK TP 14 Homo sapiens (Human)
Please cite: The Eukaryotic Linear Motif resource: 2022 release. (PMID:34718738)

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