The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Accession:
Functional site class:
EPIYA ligand motif for CSK-SH2
Functional site description:
The Csk SH2 domain binds an unusual motif EPIYA that is notable for being the subject of pathogen hijack. The motif was first found in certain bacterial effector proteins that are secreted into the host cytosol. The bacterial proteins usually have repeats of EPIYA motifs. The best described vertebrate cellular motif is found in Pragmin, a regulator of tyrosine kinase activity.
ELM Description:
The EPIYA motif is defined by the sequence of 13 instances with experimental evidence that include eukaryotic (Human and Rat) and bacterial (three species) proteins. The regular expression of this motif agrees with the motif sequences of cellular Pragmin and p140Cap proteins in mammals and repeating motifs in CagA, BepE, BepF and LspA1 effector proteins from H. pylori, B. henselae and H. ducreyi. EPIYA is considered to bind the CSK SH2 domain but, due to the unusal pattern for an SH2-interacting motif, other interactions should not be ruled out.

Positions -3, -2 and 0 (pTyr) are fixed, since no variation was found in the analyzed sequences. Position -1 was variable between the hydrophobic residues leucine and isoleucine, being the isoleucine conserved among CagA proteins (all repeats), Pragmin homologs and LspA1 (both repeats) proteins. Leucine at position -1 is conserved in p140Cap homologs and both BepD and BepE proteins. Position +1 has an alanine corresponding to the residue found in Pragmin and p140Cap homologues as well as repeats A, C and D from CagA protein as defined elsewhere (Higashi,2002). Repeat B is variable containing an alanine or threonine residue at position +1: both options (EPIYA or EPIYT) have been experimentally validated (Tsutsumi,2003; Selbach,2009). In addition to that, a glycine residue is present in the position +1 in BepD and BepE proteins. This defines the regular expression of the motif for Csk binding as EP[IL]Y[TAG]. Note that there is no solved structure for the complexed motif and therefore the motif pattern is likely to be revised in future when the interaction(s) are better understood.
Pattern: EP[IL]Y[TAG]
Pattern Probability: 0.0000025
Present in taxon: Vertebrata
Interaction Domain:
SH2 (PF00017) SH2 domain (Stochiometry: 1 : 1)
o See 13 Instances for LIG_CSK_EPIYA_1
o Abstract
Several pathogenic bacteria use SH2 domains (Pfam:PF00017) in host proteins to mediate binding of tyrosine-phosphorylated effector proteins (Backert,2005). For example, Helicobacter pylori CagA (Q9RF15) binds to the SH2 domain of C-terminal Src kinase (Csk) (with a KD<1120 nM; Tsutsumi,2003) as well as to both SH2 domains in SHP-2 (Higashi,2002); Tarp protein from Chlamydia trachomatis serovar D (O84462) binds to the SH2 domain of SHC1 (with a KD<200 nM; Mehlitz,2010) while the homologous Tarp protein from C. trachomatis serovar L2 binds to the SH2 domain of Nck (Mehlitz,2010); as a last example, AnkA protein from Anaplasma phagocytophilum (A0S0L6) binds to the SH2 domain of SHP-1 (IJdo,2007).

In all the mentioned cases the interaction is dependent on a tyrosine phosphorylation that occurs inside the host cell after the translocation of the bacterial protein. As in the case of CagA, the phosphotyrosines have been described as promiscuous binders of up to five partner proteins, as shown by a SILAC experiment using peptides of 15 amino acids long with tyrosine in the central position of different bacterial effectors like Bartonella henselae BepD (Q5QT02), BepE (Q5QT01) and BepF (Q5QT00), or Enteropathogenic Escherichia coli Tir protein (Selbach,2009). Nevertheless, the adjacent sequence to the tyrosine can give the specificity to certain proteins, which can explain the presence of different tyrosines able to be phosphorylated along the sequence of these proteins.

The specificity for the binding of the mentioned Csk protein, a non-receptor tyrosine kinase involved in cell growth, differentiation, migration and immune response (Roskoski R,2015), is defined by the positions -3 to +1 of the tyrosine. This linear motif – consensus EPIYA - is found in predicted intrinsically disordered polypeptide (IDP) and recognizes the SH2 domain in an unknown way (Nesic,2010), though it is dependent on the presence of a serine at the position 109 in rat Csk (P32577; Tsutsumi,2003; Safari,2011). At time of creating this entry, there is no structure of a solved EPIYA-SH2 complex.

This five residue-long motif also mediates the binding to CSK of the mammalian protein Pragmin (D3ZMK9; Safari,2011; also named as Sgk223 in human) which is a negative regulator of Src family kinases (SFK). This highly conserved motif is in a region of predicted IDP. (EPIYA-like motifs are also reported in the human SRC kinase signaling inhibitor 1 (SNIP or p140Cap; Q9C0H9; Repetto,2013) but it is unclear if the motifs are in an accessible structural context.). Moreover, the motif is conserved in homologous proteins of Pragmin and p140Cap along mammals.

The tyrosine in the EPIYA motif has been shown to be phosphorylated by Csk in the case of Pragmin, so a feed-forward regulatory loop is created (Senda,2016); Csk was also found to be able to phosphorylate EPIYA of LspA1 protein from Haemophilus ducreyi (Q7VLE8) in vitro (Dodd,2014). Finally, CagA is reported to be phosphorylated by members of the Src Family Kinases (SFK) such as c-Src (Selbach,2002) and Lyn (Selbach,2002; Stein,2002).

The interaction of Csk with the phosphorylated LIG_SH2_Csk motif can lead to different and opposing scenarios. The binding of the cytoplasm-localized phosphorylated Pragmin sequesters Csk far from the membrane, preventing the inactivation of c-Src. On the other hand, the protein CagA is initially located next to the intracellular membrane and brings together Csk with c-Src (Safari,2011), a state that allows Csk to phosphorylate c-Src in a conserved tyrosine at position 530 in the human homolog. This leads c-Src to adopt a closed conformation where its kinase activity is inhibited (Roskoski R,2004). In a celullar system where SFKs are inactivated, substrates of this kinase like the Fcγ receptor in the immunoreceptor tyrosine-based activation motif (ITAM) domain are not phosphorylated and phagocytosis is deregulated, a cellular state abused by H. ducreyi using its EPIYA-containing effector protein LspA1 (Dodd,2014). There is surely much more to discover about the mechanisms whereby pathogenic EPIYA hijack perturbs these tyrosine kinase signalling systems.
o 5 selected references:

o 13 GO-Terms:

o 13 Instances for LIG_CSK_EPIYA_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P55980 cagA
CAGA_HELPY
915 919 GQVASPEEPIYTQVAKKVNA TP 2 Helicobacter pylori 26695
1 
P55980 cagA
CAGA_HELPY
896 900 NGLKNSTEPIYAKVNKKKTG TP 1 Helicobacter pylori 26695
1 
Q5QT01 bepE
Q5QT01_BARHN
34 38 RAPSPQPEPLYATVNKRPPR TP 3 Bartonella henselae
1 
Q5QT02 bepD
Q5QT02_BARHN
208 212 RAPSPQAEPLYAQVNKPPRE TP 1 Bartonella henselae
1 
Q7VLE8 lspA1
Q7VLE8_HAEDU
2542 2546 VRKTDEPEPIYGTINKSPEA TP 5 Haemophilus ducreyi 35000HP
1 
Q7VLE8 lspA1
Q7VLE8_HAEDU
2466 2470 SRKLDEPEPIYGTINKSPEA TP 5 Haemophilus ducreyi 35000HP
1 
Q9C0H9-5 SRCIN1
SRCN1_HUMAN
261 265 IIKIYRKEPLYAAFPGSHLT TP 4 Homo sapiens (Human)
1 
D3ZMK9 Pragmin
D3ZMK9_RAT
388 392 PREAVQPEPIYAESAKRKKA TP 5 Rattus norvegicus (Norway rat)
1 
Q9RF15 cagA
Q9RF15_HELPX
1030 1034 VGRSVSPEPIYATIDDLGGP TP 2 Helicobacter pylori
1 
Q9RF15 cagA
Q9RF15_HELPX
996 1000 VGRSVSPEPIYATIDDLGGP TP 2 Helicobacter pylori
1 
Q9RF15 cagA
Q9RF15_HELPX
962 966 VGRSVSPEPIYATIDDLGGP TP 2 Helicobacter pylori
1 
Q9RF15 cagA
Q9RF15_HELPX
909 913 GQVASPEEPIYAQVAKKVNA TP 2 Helicobacter pylori
1 
Q9RF15 cagA
Q9RF15_HELPX
890 894 NNNGLENEPIYAKVNKKKTG TP 2 Helicobacter pylori
1 
Please cite: ELM 2016-data update and new functionality of the eukaryotic linear motif resource. (PMID:26615199)

ELM data can be downloaded & distributed for non-commercial use according to the ELM Software License Agreement