DEG_Kelch_KLHL12_1

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.