The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Accession:
Functional site class:
14-3-3 binding phosphopeptide motif
Functional site description:
The 14-3-3 proteins are a family of conserved regulatory molecules that are involved in diverse cellular processes through the interaction with hundreds of different proteins. In mammals, seven isoforms are present. 14-3-3 proteins form either homo- or heterodimers that target certain phosphoserine/threonine-containing motifs with a low micromolar affinity. Binding to a small set of unmodified proteins has also been reported. Phosphorylation-dependent and independent binding occurs via the same deep ligand-binding groove. There are canonical arginine-containing motifs and a non-canonical motif group that are difficult to classify but utilize additional hydrophobic interactions. The canonical Arg-containing 14-3-3 binding peptides are phosphorylated by members of basophilic kinases. Sites phosphorylated by Proline-directed kinases cannot be bound by 14-3-3 proteins, hence there is no overlap with the basophilic kinase signalling pathways.
ELMs with same func. site: LIG_14-3-3_CanoR_1  LIG_14-3-3_CterR_2 
ELM Description:
The canonical motif has at least one Arg residue located at the -4,-3 or -2 position relative to the phospho-residue which aids in the placement of the peptide in the binding groove. Arg stabilizes the phospho-peptide conformation by forming hydrogen bonds as well as an intramolecular salt bridge with the phosphate group. Asp/Glu are not tolerated for at least 3 positions preceding the phosphorylated residue. Pro and Gly at position -1 are also not tolerated since they weaken the interaction between the phospho-residue and the 14-3-3 binding pocket. Proline is also disallowed at position +1 (Panni,2011). At this position, the -NH group of the peptide backbone forms a hydrogen bond with the 14-3-3 protein as part of local geometry crucial to orientate the adjacent phosphorylated sidechain (Molzan,2012). Therefore, there is no physical space for a Pro.
The canonical binding motif requires at least one favourable hydrophobic interaction following the P-site residue as this part of the 14-3-3 groove is markedly hydrophobic. This residue can be located at three different distances following the phosphosite. These alternatives arrangements are non-exclusive. In the first option, the +1 residue is a bulky hydrophobic residue with an extensive packing face (5D3E, 4DAU). If there is no hydrophobic residue at +1, other residues are tolerated with the exception of the disallowed Arg, Ile, Gly, Lys and Asn (Panni,2011). In the second option, there is a Pro at +2. Pro is strongly favoured in the +2 position as it introduces an exit kink into the peptide chain and contacts hydrophobic walls of the groove (3O8I, 3MHR, 2C74, 2BR9, 2BTP, 3UAL, 4IEA, 4IHL). If option 1 or 3 are not present, option 3 is a requirement for a bulky hydrophobic residue in the +4 to +6 range which loops back down into the hydrophobic wall of the groove (5D2D, 4WRQ, 2C1J). Variant 2 and 3 have been reported more often than variant 1.
Pattern: R[^DE]{0,2}[^DEPG]([ST])(([FWYLMV].)|([^PRIKGN]P)|([^PRIKGN].{2,4}[VILMFWYP]))
Pattern Probability: 0.0044767
Present in taxon: Eukaryota
Interaction Domain:
14-3-3 (PF00244) 14-3-3 protein (Stochiometry: 1 : 1)
PDB Structure: 1QJB
o See 62 Instances for LIG_14-3-3_CanoR_1
o Abstract
The 14-3-3 proteins (PF00244) are a highly conserved group of proteins, present in most or all eukaryotic organisms. In the mammalian proteome seven isoforms can be found, whereas only two isoforms are present in yeasts, insects and nematodes (Obsil,2011). 14-3-3 proteins can form both homo-and heterodimers (1A4O, 5IQP). Each monomer consists of nine antiparallel alpha-helices with a deep central binding cavity that can accommodate peptides from a multitude of diverse binding partners. Consequently, 14-3-3 proteins are shown to be involved in important cellular processes such as signal transduction, cell-cycle control (Peng,1997), apoptosis (Zha,1996), stress response and malignant transformation (16125003). The 14-3-3 proteins posses no catalytic activity and thus they perform their functional tasks only by binding and modulating the activity of their partner proteins. Mechanisms of 14-3-3 action include the induction of conformational changes, the occlusion of sequence-specific or structural features, scaffolding complexes (16091624) and change of cellular localization (Obsilova,2005).
All 14-3-3 isoforms primarily target phosphoserine/-threonine peptide motifs in their interaction partners. The phosphate group orients the peptide and is stabilized by a conserved 14-3-3 Arg-Arg-Tyr triad forming a positively charged pocket within the otherwise amphiphatic 14-3-3 ligand groove (Yang,2006): It is unlikely that phosphomimetic residue substitutions will function due to the number and orientation of the charged functional groups binding the phosphate. Phosphopeptide library screens and crystal structures led to the definition of consensus motifs such as RSxpSxP, RxxxpSxP and pS/pTx1-2-COOH used to classify the number of 14-3-3 mediated interactions (Muslin,1996, Yaffe,1997). The experimental data of Panni et al., 2011 (Panni,2011) are helpful for revising the motif description, including where certain residues are disallowed. In the current ELM entry, we have been able to define a canonical Arg-containing motif where the phosphoresidue is followed by one or more bulky hydrophobic residues with several preferred spacings, and a second shorter C-terminal Arg-containing motif, terminating just after the phosphoresidue. Both motifs are observed in numerous experimentally validated examples, and there are many crystal structures of phosphopeptide/14-3-3 complexes.
Since at least one positively charged arginine precedes the phosphorylated residue, these canonical motifs are compatible with phosphorylation by the very large group of basophilic Ser/Thr kinases including PKA, CaMKII and the Aurora kinases. The blocking effect of Pro at +1 excludes14-3-3 proteins from binding substrates of proline-directed kinases (e.g. MAPK, CDK), even when these are preceded by an Arg residue. Many 14-3-3 binding proteins such as Raf-1 contain several 14-3-3 binding sequences, separated by variable length polypeptide segments (4IHL). Binding studies have shown that a 14-3-3 dimer can cooperatively bind two phosphorylated epitope sites on the same target protein partner. For ADAM22, Raf-1 and CFTR, dual binding resulted in higher affinity, more stable, complexes at which the space between the strong tandem 14-3-3 motifs averaged 14, 18 (4IHL) and 15 amino acids (5D2D, 5D3E), respectively (Yaffe,1997, Stevers,2016).
As well as the classical canonical motifs, it has become clear that there are at least two varieties of non-canonical motifs. In some 14-3-3 binding partners, a second weaker motif (e.g. substituting Lys for Arg) is present in addition to a strong canonical motif. Examples are the proteins KSR1 (Cacace,1999), HDAC4 (Rose,2012, 3UZD), PADI6 (Rose,2012, 4DAT), MDM4 (Pereg,2006) and p53 (20206173, 3LW1). A second non-canonical class of 14-3-3-binding motif lacks the basic residue but compensates with additional hydrophobic interactions using several residues following the phosphorylated residue. A number of such variants have been tested in vitro and bind with affinities equivalent to the canonical motifs, leaving little room for doubt that these interactions are biologically significant. Among 14-3-3 binding partners in which only a non-canonical hydrophobic motif was proved to mediate a good-affinity interaction are the integrins β2 (2V7D) and α4 (23763993) and plant H+ ATPases (17289589, 2O98). As yet, we have not been able to define ELM motifs for the non-canonical hydrophobic 14-3-3 binding variants but it is important to be aware of their existence.
Certain fungal toxins and bacterial virulence factors target 14-3-3 proteins. The fungal phytotoxin fusicoccin is shown to stabilize the protein complex between the plant plasma membrane H+-ATPase (PMA2) and 14-3-3 proteins (Wurtele,2003). Subsequent studies with semi-synthetic fusicoccanes describe the ability of fusicoccanes to enhance human 14-3-3 protein-protein interactions (Anders,2013, 3SMM, Stevers,2016, 5D3F).
Although 14-3-3 interactors are overwhelmingly found to contain a phosphorylated residue, even this rule is not absolute. 14-3-3 proteins can also recognize a few unmodified peptides notably the pathogenic Pseudomonas exoS protein (8460141, Yang,2006, 2C23). The exoS peptide LLDALDL binds in the reverse orientation compared to the phosphorylated peptides and therefore it is possible that there exist cellular equivalents of this binding mode (Ottmann,2007, 2O02).
o 11 selected references:

o 14 GO-Terms:

o 62 Instances for LIG_14-3-3_CanoR_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
Q61097 Ksr1
KSR1_MOUSE
294 302 FPTLTRSKSHESQLGNRIDD TP 1 Mus musculus (House mouse)
2 
Q61097 Ksr1
KSR1_MOUSE
388 394 LPLARLRRTESVPSDINNPV TP 1 Mus musculus (House mouse)
1 
Q8IPH9 Slob
SLOB_DROME
51 60 ANGLKRSNSAIEFDVSALTA TP 1 Drosophila melanogaster (Fruit fly)
2 
P56524 HDAC4
HDAC4_HUMAN
629 634 GGHRPLSRAQSSPASATFPV TP 3 Homo sapiens (Human)
1 
P43565 RIM15
RIM15_YEAST
1072 1076 FSLLDISRSSTPPLANPTNS TP 1 Saccharomyces cerevisiae (Baker"s yeast)
1 
P26045 PTPN3
PTN3_HUMAN
356 361 VWNPAMRRSLSVEHLETKSL TP 1 Homo sapiens (Human)
1 
O43524 FOXO3
FOXO3_HUMAN
250 255 SGKAPRRRAVSMDNSNKYTK TP 1 Homo sapiens (Human)
1 
O43524 FOXO3
FOXO3_HUMAN
29 34 FEPQSRPRSCTWPLQRPELQ TP 1 Homo sapiens (Human)
1 
Q9P0K1 ADAM22
ADA22_HUMAN
854 863 KRFRPRSNSTETLSPAKSPS TP 4 Homo sapiens (Human)
2 
Q9P0K1 ADAM22
ADA22_HUMAN
831 836 ICENGRPRSNSWQGNLGGNK TP 2 Homo sapiens (Human)
2 
Q02156 PRKCE
KPCE_HUMAN
364 370 ELENNIRKALSFDNRGEEHR TP 3 Homo sapiens (Human)
1 
Q02156 PRKCE
KPCE_HUMAN
343 348 SSPSEEDRSKSAPTSPCDQE TP 3 Homo sapiens (Human)
1 
P56524 HDAC4
HDAC4_HUMAN
242 248 KDDFPLRKTASEPNLKLRSR TP 4 Homo sapiens (Human)
Q9UQL6 HDAC5
HDAC5_HUMAN
255 261 RDDFPLRKTASEPNLKVRSR TP 2 Homo sapiens (Human)
Q14432 PDE3A
PDE3A_HUMAN
426 430 LAIPKRLRRSLPPGLLRRVS TP 2 Homo sapiens (Human)
1 
P98177 FOXO4
FOXO4_HUMAN
194 199 SGKAPRRRAASMDSSSKLLR TP 2 Homo sapiens (Human)
2 
Q63572 Tesk1
TESK1_RAT
436 441 QPETPVRRCRSLPSSPELPR TP 2 Rattus norvegicus (Norway rat)
1 
A2ALK8 Ptpn3
PTPN3_MOUSE
356 361 VWNPVMRRSLSVERLETKSL TP 2 Mus musculus (House mouse)
2 
P32418 SLC8A1
NAC1_HUMAN
388 394 HAADQARKAVSMHEVNTEVT TP 2 Homo sapiens (Human)
2 
P08965 mei2
MEI2_SCHPO
524 529 TGSLGMRRSLTVGANASCSN TP 4 Schizosaccharomyces pombe 972h-
2 
O35147 Bad
BAD_RAT
134 139 ELSPFRGRSRSAPPNLWAAQ TP 1 Rattus norvegicus (Norway rat)
1 
Q99683 MAP3K5
M3K5_HUMAN
963 968 AGSNEYLRSISLPVPVLVED TP 2 Homo sapiens (Human)
1 
P04049 RAF1
RAF1_HUMAN
618 623 HSLPKINRSASEPSLHRAAH TP 2 Homo sapiens (Human)
1 
P03076 Middle T anti
MT_POVM3
254 259 NPTYSVMRSHSYPPTRVLQQ TP 2 Mouse polyomavirus (strain 3)
1 
P54253 ATXN1
ATX1_HUMAN
773 777 PAATRKRRWSAPESRKLEKS TP 4 Homo sapiens (Human)
2 
P46937-2 YAP1
YAP1_HUMAN
124 129 ALTPQHVRAHSSPASLQLGA TP 3 Homo sapiens (Human)
Q9EPK5 Wwtr1
WWTR1_MOUSE
86 91 AGGAQHVRSHSSPASLQLGT TP 3 Mus musculus (House mouse)
P46937 YAP1
YAP1_HUMAN
124 129 ALTPQHVRAHSSPASLQLGA TP 3 Homo sapiens (Human)
O15151 MDM4
MDM4_HUMAN
364 369 NDVPDCRRTISAPVVRPKDA TP 3 Homo sapiens (Human)
1 
P98177 FOXO4
FOXO4_HUMAN
29 34 FEPQSRPRSCTWPLPRPEIA TP 3 Homo sapiens (Human)
1 
P04049 RAF1
RAF1_HUMAN
254 263 GSLSQRQRSTSTPNVHMVST TP 6 Homo sapiens (Human)
2 
Q61337 Bad
BAD_MOUSE
109 114 GAMETRSRHSSYPAGTEEDE TP 3 Mus musculus (House mouse)
1 
Q61337 Bad
BAD_MOUSE
133 138 ELSPFRGRSRSAPPNLWAAQ TP 5 Mus musculus (House mouse)
1 
P46938 Yap1
YAP1_MOUSE
109 114 ALTPQHVRAHSSPASLQLGA TP 4 Mus musculus (House mouse)
1 
Q9GZV5 WWTR1
WWTR1_HUMAN
86 91 AGGAQHVRSHSSPASLQLGT TP 8 Homo sapiens (Human)
1 
Q96PU5 NEDD4L
NED4L_HUMAN
339 344 REPSSRLRSCSVTDAVAEQG TP 2 Homo sapiens (Human)
O15553-2 MEFV
MEFV_HUMAN
239 244 YLPSGKMRPRSLEVTISTGE TP 4 Homo sapiens (Human)
P78314 SH3BP2
3BP2_HUMAN
222 227 PAFSDMPRAHSFTSKGPGPL TP 5 Homo sapiens (Human)
Q6TGC4 PADI6
PADI6_HUMAN
7 12 MVSVEGRAMSFQSIIHLSLD TP 1 Homo sapiens (Human)
Q6P597 KLC3
KLC3_HUMAN
463 468 AGAAGMKRAMSLNTLNVDAP TP 1 Homo sapiens (Human)
Q9H0B6 KLC2
KLC2_HUMAN
579 584 PPNPRMKRASSLNFLNKSVE TP 1 Homo sapiens (Human)
Q9Y3M2 CBY1
CBY1_HUMAN
16 22 PKKTPPRKSASLSNLHSLDR TP 4 Homo sapiens (Human)
P13569 CFTR
CFTR_HUMAN
792 797 RKTTASTRKVSLAPQANLTE TP 3 Homo sapiens (Human)
P13569 CFTR
CFTR_HUMAN
751 755 QGEAILPRISVISTGPTLQA TP 3 Homo sapiens (Human)
P13569 CFTR
CFTR_HUMAN
766 770 PTLQARRRQSVLNLMTHSVN TP 3 Homo sapiens (Human)
Q86TI0 TBC1D1
TBCD1_HUMAN
593 598 PQQAFRRRANTLSHFPIECQ TP 1 Homo sapiens (Human)
Q9UQC2 GAB2
GAB2_HUMAN
207 212 SRRAENARSASFSQGTRASF TP 2 Homo sapiens (Human)
Q86VP3 PACS2
PACS2_HUMAN
434 439 KQAGRRGRSTSLKERQAARP TP 4 Homo sapiens (Human)
O15553-2 MEFV
MEFV_HUMAN
205 213 EVRLRRNASSAGRLQGLAGG TP 4 Homo sapiens (Human)
P49796-3 RGS3
RGS3_HUMAN
940 948 NSLRRRTHSEGSLLQEPRGP TP 5 Homo sapiens (Human)
P14136 GFAP
GFAP_HUMAN
5 14 MERRRITSAARRSYVSSGEM TP 4 Homo sapiens (Human)
P68431 HIST1H3A
H31_HUMAN
9 17 TKQTARKSTGGKAPRKQLAT TP 3 Homo sapiens (Human)
Q9UQC2 GAB2
GAB2_HUMAN
389 393 VAATIPRRNTLPAMDNSRLH TP 2 Homo sapiens (Human)
Q86TI0 TBC1D1
TBCD1_HUMAN
233 239 PVRRPMRKSFSQPGLRSLAF TP 1 Homo sapiens (Human)
P78314 SH3BP2
3BP2_HUMAN
276 280 RVPATPRRMSDPPLSTMPTA TP 5 Homo sapiens (Human)
Q96PU5 NEDD4L
NED4L_HUMAN
443 448 LIEPQIRRPRSLSSPTVTLS TP 3 Homo sapiens (Human)
P68431 HIST1H3A
H31_HUMAN
27 31 QLATKAARKSAPATGGVKKP TP 2 Homo sapiens (Human)
P04049 RAF1
RAF1_HUMAN
231 235 MPVSSQHRYSTPHAFTFNTS TP 2 Homo sapiens (Human)
Q29495 AANAT
SNAT_SHEEP
29 33 GSPGRQRRHTLPANEFRCLT TP 1 Ovis aries (Sheep)
P58340 MLF1
MLF1_HUMAN
31 36 ENMRQMIRSFSEPFGRDLLS TP 2 Homo sapiens (Human)
O43896 KIF1C
KIF1C_HUMAN
1089 1094 TTPPRMRRQRSAPDLKESGA TP 3 Homo sapiens (Human)
P56524 HDAC4
HDAC4_HUMAN
464 469 RQHRPLGRTQSAPLPQNAQA TP 2 Homo sapiens (Human)
Please cite: ELM 2016-data update and new functionality of the eukaryotic linear motif resource. (PMID:26615199)

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