The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
KLHL12 binding degron
Functional site description:
Early development and tissue homeostasis, as well as the growth of many human cancers are regulated by Wnt signalling. The Wnt signalling pathway is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation with KLHL12 being the first E3 ubiquitin ligase to be identified for DVL1-3. The degron recognized by the Kelch domain of KLHL12 is a circa 6 residues proline-rich peptide with consensus PPGxPP. This motif is also present in proteins involved in collagen trafficking from the endoplasmic reticulum, such as lunapark and peflin. Interestingly, neither lunapark nor peflin are targeted to proteasomal degradation after ubiquitination.
ELM Description:
KLHL12 (Q53G59) was the first E3 to be identified for the DVLs (Angers,2006). Its complex with the degron motif of DVL1 (O14640) was solved (6TTK) by crystallography, providing extra details into the recognition mechanism (Chen,2020).The same study also applied SPOT arrays, mutational analyses, stability and ubiquitination assays to refine the degron motif of DVL1, and also extended the research to two other paralogues, DVL2 (O14641) and DVL3 (Q92997) with additional SPOT arrays to experimentally confirm their binding to KLHL12. Based on a fluorescence polarization (FP) measurement performed on DVL1, the proline-rich degron motif of KLHL12 is expected to have a dissociation constant (Kd) of about 22 μM (Chen,2020). The authors defined the proline-rich degron as PGxPP and based on the crystal structure (6TTK) hypothesize that prolines at the first and fourth motif positions contribute most to the binding affinity. They also found that the PGxPP motif was enriched among KLHL12 interactors linked to the Wnt signalling and the vesicle coating pathways, although statistical overrepresentation tests or experimental validation was not performed. In these predicted instances the X position of the motif corresponds to small nonpolar amino acids [GA]. A second study (Zhao,2020) reported the same motif based on X-ray (6V7O), NMR and FP experiments for the DVL3 degron and several mutants. The motif in ELM is based on the structures and sequence alignments of the degron in DVL proteins and includes the residue preceding the first P of PGxPP, suggesting a consensus of PPGxPP. The motif was confirmed to be present in proteins involved in collagen trafficking from the endoplasmic reticulum, such as lunapark (Q9C0E8) and peflin (McGourty,2016) based on deletion experiments and mutant constructs for the substrate and/or KLHL12 in in vitro and in cellulo binding and ubiquitylation experiments (Yuniati,2020; McGourty,2016).
Pattern: [PGA]PG[AG]PP
Pattern Probability: 0.0000022
Present in taxon: Metazoa
Not represented in taxon: Arthropoda
Interaction Domains:
o See 11 Instances for DEG_Kelch_KLHL12_1
o Abstract
The GPCR Frizzled protein family (in humans there are 10 paralogues FZD1-FZD10) function as receptors for secreted Wnt ligands, which, upon binding, stimulate intracellular responses that ultimately lead to transcription factor β-catenin (P35222) stabilization (canonical Wnt signalling) or β-catenin-independent effects (non-canonical Wnt signaling). Dishevelled proteins DVL1-3 (O14640; O14641; Q92997) form principal components of both pathways and bind to the activated Frizzled receptors inside the cell via their central PDZ domain. The DIX domain of DVLs then mediates their self-polymerization and interaction with Axin (O15169) to facilitate the assembly of a Wnt signalosome (Schwarz-Romond,2007;Kishida,1999).
To date, several E3 ligases have been reported to regulate DVL protein degradation: the HECT-family E3 ligases ITCH (Q96J02) and NEDD4L (Q96PU5), and the Cullin-RING E3 ligase KLHL12 (Q53G59). ITCH specifically targets DVLs phosphorylated on the Tyr of the PPxY degron for proteasomal degradation (Wei,2012). NEDD4L recognises DVL2 but apparently not DVL1 or DVL3 for poly-ubiquitination and proteasomal degradation, and thereby negatively regulates canonical Wnt signalling (Novellasdemunt,2020). By contrast, the poly-ubiquitination of the three DVL paralogs, DVL1-3, by KLHL12 does not require a specific cell stimulus and appears to be constitutive (Angers,2006; Funato,2010). KLHL12 is also reported to mediate the polyubiquitination of the dopamine D4 receptor (P21917; Rondou,2010), the ubiquitination of KHSRP (Q92945), a protein that is involved in IRES translation, and also the ubiquitination of Sec31 (O94979), which is involved in endoplasmic reticulum−Golgi transport by regulating the size of COPII coats (Zhao,2020). KLHL12 also forms higher-order complexes with Sec31 (O94979), ALG2 (O75340) and PEF1 (Q9UBV8) a.k.a. peflin (McGourty,2016). Upon KLHL12 binding, Sec31 gets monoubiquitinated (via a yet uncharacterized degron) to drive the trafficking of collagen from the ER (McGourty,2016). It is hypothesized that peflin’s N-terminal PGxPP motif is bound by the partner subunit of the KLHL12 dimer for subsequent ubiquitylation (McGourty,2016) so that only active CUL3-KLHL12-ALG2-PEF1 is able to engage Sec31.
Furthermore, KLHL12 recognizes lunapark (Q9C0E8) by a PGxPP motif, however, this protein does not get degraded by the proteasome. Lunapark is involved in the formation of three-way junctions of the ER (Yuniati,2020), where it interacts with mTOR (P42345). Lunapark’s ubiquitination was shown to be crucial for mTOR recruitment to the membrane of lysosomes and late endosomes at three-way junctions (Yuniati,2020). It was found recently that lunapark interacts with KLHL12 but not with the active E3 ligase CUL3-KLHL12 to inhibit the CUL3-KLHL12-ALG2-PEF1 assembly (Akopian,2022). Upon successful formation of the assembly at higher ALG2-PEF1 concentration, substrates will get polyubiquitinated (including Sec31), and PEF1 will get monoubiquitinated in order to stabilize the complex for more efficient modification of the substrates.
PLEKHA4 also contains the PGxPP motif and was proven to be a pseudo-substrate of KLHL12, therefore it does not get polyubiquitinated and subsequently degraded, but its role is to sequester KLHL12 and thereby prevent ubiquitination of DVL3 (Shami Shah,2019).
KLHL12 was the first E3 ligase to be identified for the DVLs (Angers,2006), yet the molecular mechanisms determining its substrate interactions had remained unknown for years. More recently, the structures of KLHL12 in complex with the degron motifs of DVL1 and DVL3 (O14640; Q92997) were solved (6TTK; 6V7O). The crystal structure with DVL1 (6TTK) and its mutational analyses along with the SPOT assays of DLV1-3 enabled the definition of the PGxPP degron (Chen,2020). This is also supported by a second crystal structure (6V7O) and additional NMR and FP assays in a second study (Zhao,2020).
The PGxPP degron is bound by the Kelch domain of KLHL12. Kelch domains are 6 membered beta propeller domains that are present in a number of E3 ligases with quite different degron specificities including several ELM entries (DEG_Kelch_KLHL3; DEG_Kelch_Keap1_1; DEG_Kelch_Keap1_2; DEG_Kelch_actinfilin_1).
At time of entry preparation, in vitro determined binding constants were only available for DVL PGxPP degrons. The variant degron reported in the dopamine receptor D4, APGLPP, was observed to bind very weakly to the Kelch domain (Zhao,2020) and so is not currently included in the ELM motif. Other possible candidate proteins containing the degron sequence include PRR13, DAAM1, DAAM2, Sec23IP and CPSF6.
Insects appear to have lost the gene for KLHL12 as it is found in many other highly divergent animal lineages including chordates, mollusks, ciliates and polychaetes. The motif in ELM is based on data exclusively reported for vertebrates and might vary in other lineages: For example, alignments suggest that the first position in molluscs tolerates more alternatives to Pro.
o 4 selected references:

o 9 GO-Terms:

o 11 Instances for DEG_Kelch_KLHL12_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
198 203 GPPKDSSAPGGPPERTVTPA TP 4 Homo sapiens (Human)
176 181 QPGEGPGGPGGPPEVSRGEE TP 5 Homo sapiens (Human)
O94979 SEC31A
957 962 GASFQHGGPGAPPSSSAYAL TP 7 Homo sapiens (Human)
17 22 CPGAAGQAPGAPPGSYYPGP TP 7 Homo sapiens (Human)
Q16630 CPSF6
222 227 DRFPGPAGPGGPPPPFPAGQ U 1 Homo sapiens (Human)
O14640 DVL1
657 662 AYTVVGGPPGGPPVRELAAV TP 10 Homo sapiens (Human)
O14641 DVL2
698 703 PVPPAVQPPGAPPVRDLGSV TP 5 Homo sapiens (Human)
Q92997 DVL3
678 683 PPPAAMGPPGAPPGRDLASV TP 16 Homo sapiens (Human)
Q16630 CPSF6
347 352 PPHLPGPPPGAPPPAPHVNP U 1 Homo sapiens (Human)
Q86T65 DAAM2
568 573 PPGGPPTPPGAPPCLGMGLP U 1 Homo sapiens (Human)
570 575 PPPPPPLPPGGPPPPPGPPP U 1 Homo sapiens (Human)
Please cite: The Eukaryotic Linear Motif resource: 2022 release. (PMID:34718738)

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