CBLL1

CBLL1 (Hakai) is a RING-type E3 ubiquitin ligase with dual roles in cell adhesion remodeling and epitranscriptomic regulation. Its HYB domain forms a zinc-coordinated homodimer that recognizes phosphotyrosine motifs on E-cadherin and other Src substrates, ubiquitinating E-cadherin at juxtamembrane lysines K5 and K83 to drive endocytosis and lysosomal/proteasomal degradation, thereby disrupting cell-cell contacts and promoting epithelial-mesenchymal transition (PMID:11836526, PMID:22693575, PMID:25074933). Beyond E-cadherin, Hakai ubiquitinates FASN, Smurf2, and LRP4, and it also performs ligase-independent functions including binding estrogen receptor-α to inhibit its transcriptional activity and interacting with PSF to modulate oncogenic RNA binding (PMID:36266428, PMID:39034451, PMID:20608937, PMID:19535458). Hakai is a conserved subunit of the m6A mRNA methyltransferase writer complex: loss of Hakai in Arabidopsis and Drosophila reduces m6A levels, impairs alternative splicing, and destabilizes other writer components, establishing it as essential for epitranscriptomic modification (PMID:28503769, PMID:33846330).

1. 2002 High

   The fundamental question of how E-cadherin turnover is regulated post-translationally was answered by identification of Hakai as a phosphotyrosine-dependent E3 ubiquitin ligase that ubiquitinates E-cadherin, drives its endocytosis, and promotes cell motility — establishing Hakai as the first E3 ligase targeting the adherens junction complex.

   Evidence Yeast two-hybrid screen, co-immunoprecipitation, ubiquitination and endocytosis assays in epithelial cells

   PMID:11836526

   Open questions at the time

   • Precise ubiquitination sites on E-cadherin were not identified
   • In vivo relevance in mammalian tissues was not shown
   • How Hakai is recruited to adherens junctions in response to upstream signals was unknown
2. 2009 Medium

   Two parallel discoveries expanded Hakai's functional scope beyond E-cadherin: interaction with PSF revealed an E-cadherin-independent role in oncogenic RNA binding and proliferation, while Drosophila genetic nulls showed Hakai regulates multiple targets (including aPKC) essential for embryonic morphogenesis.

   Evidence Co-immunoprecipitation and RNA-binding assays in mammalian cells; genetic null mutants and in vivo phenotyping in Drosophila

   PMID:19535458 PMID:19682089

   Open questions at the time

   • The RNA targets downstream of PSF–Hakai interaction were not comprehensively identified
   • Relationship between Drosophila Hakai phenotypes and E-cadherin ubiquitination differed from mammals, mechanism unclear
   • No structural basis for Hakai–PSF interaction
3. 2010 Medium

   Hakai was shown to inhibit ERα transcriptional activity by binding its DNA-binding domain and competing with coactivators SRC-1 and GRIP-1, establishing a ligase-independent transcriptional regulatory function.

   Evidence Co-immunoprecipitation, domain mapping, and transcriptional reporter assays in breast cancer cells

   PMID:20608937

   Open questions at the time

   • Whether this interaction occurs in vivo and affects estrogen-responsive gene programs is untested
   • The physiological context triggering Hakai–ERα interaction is unknown
   • Not independently confirmed
4. 2011 Medium

   The upstream pathway recruiting Hakai to E-cadherin was elucidated: Slit2–Robo1 signaling recruits Hakai to ubiquitinate E-cadherin for lysosomal degradation, linking Hakai to an established axon guidance pathway repurposed for EMT.

   Evidence siRNA rescue, ubiquitination assays, lysosomal inhibition, and in vivo tumor/metastasis models

   PMID:21283129

   Open questions at the time

   • Direct physical mechanism by which Robo1 recruits Hakai was not resolved
   • Whether other guidance cues similarly activate Hakai is unknown
5. 2012 High

   Two studies resolved key regulatory details: site-directed mutagenesis identified K5 and K83 on the E-cadherin juxtamembrane domain as Hakai ubiquitination sites that inhibit p120-catenin binding, while miR-203 was identified as a direct negative regulator of Hakai mRNA, linking Hakai abundance to microRNA control of proliferation.

   Evidence Lysine-to-arginine mutagenesis with mitochondrial targeting assays; luciferase 3′-UTR reporter with miRNA overexpression/inhibition and epistasis via siRNA

   PMID:22693575 PMID:23285092

   Open questions at the time

   • Whether additional E-cadherin lysines are modified in vivo is untested
   • The regulatory hierarchy between miR-203 and Hakai in tumor contexts lacks in vivo validation
6. 2014 High

   NMR structural determination of the HYB domain revealed the molecular basis for substrate recognition: an atypical zinc-coordinated homodimer that binds phosphotyrosine motifs, with a truncated form undergoing ligand-induced dimerization, explaining how Hakai selectively targets Src-phosphorylated substrates.

   Evidence NMR structure, isothermal titration calorimetry, analytical ultracentrifugation, size-exclusion chromatography

   PMID:25074933

   Open questions at the time

   • No full-length Hakai structure including the RING domain was solved
   • Structure of Hakai in complex with intact E-cadherin or other substrates is lacking
7. 2017 Medium

   Two studies revealed functions beyond canonical E3 ligase activity: Hakai was identified as a conserved subunit of the m6A mRNA writer complex in Arabidopsis, and separately was shown to stabilize δ-catenin through a ligase-independent Src-dependent mechanism that inhibits GSK-3β.

   Evidence Proteomics-based co-purification and m6A quantification in Arabidopsis; co-immunoprecipitation with kinase inhibitor treatment in mammalian cells

   PMID:28069439 PMID:28503769

   Open questions at the time

   • Whether Hakai's m6A writer function requires its E3 ligase activity was unknown
   • The mammalian Hakai–m6A writer connection had not yet been confirmed
   • The δ-catenin stabilization mechanism was from a single lab
8. 2018 Medium

   Hakai was found to interact with the LIM-domain protein Ajuba and induce its neddylation rather than ubiquitination, expanding the repertoire of post-translational modifications Hakai can promote beyond ubiquitin.

   Evidence Co-immunoprecipitation, neddylation inhibitor (MLN4924) treatment, and siRNA knockdown in hepatocellular carcinoma cells

   PMID:30041665

   Open questions at the time

   • Whether Hakai directly catalyzes neddylation or recruits a neddylation enzyme is unresolved
   • Not independently confirmed
   • Functional consequence of Ajuba neddylation on downstream signaling is unclear
9. 2020 Medium

   Hakai was identified as an Hsp90 client protein, establishing that its own stability is regulated by chaperone-dependent quality control; Hsp90 inhibition causes lysosomal degradation of Hakai and consequent E-cadherin stabilization.

   Evidence Co-immunoprecipitation, pharmacological Hsp90 inhibition with geldanamycin, cell motility assays

   PMID:31952268

   Open questions at the time

   • The specific Hsp90 cochaperones involved are not identified
   • In vivo relevance of Hsp90-dependent Hakai regulation is untested
10. 2021 High

    Genetic and biochemical analysis in Drosophila confirmed that Hakai is an integral, mutually stabilizing subunit of the m6A writer complex; loss of Hakai reduces transcriptome-wide m6A and causes aberrant Sxl alternative splicing, firmly establishing the epitranscriptomic function as conserved across metazoa.

    Evidence Genetic mutants, MeRIP-seq, co-immunoprecipitation, and alternative splicing assays in Drosophila

    PMID:33846330

    Open questions at the time

    • Whether Hakai's RING domain or E3 ligase activity contributes to m6A writer function is unknown
    • Mammalian Hakai–m6A writer interaction had not yet been confirmed by equivalent genetic experiments
    • Whether Hakai regulates specific mRNA targets within the m6A program is unresolved
11. 2022 Medium

    Interactome analysis and ubiquitination assays identified FASN as a direct Hakai substrate targeted for lysosomal degradation, linking Hakai to lipid metabolism.

    Evidence Proteomics, co-immunoprecipitation, ubiquitination assay, lysosomal inhibition in mammalian cells

    PMID:36266428

    Open questions at the time

    • The specific ubiquitination sites on FASN are not mapped
    • In vivo metabolic consequences of Hakai-mediated FASN degradation are uncharacterized
    • Not independently confirmed
12. 2024 Medium

    Hakai was shown to protect Runx2 from Smurf2-mediated degradation by ubiquitinating and degrading Smurf2 itself; a catalytically dead mutant (C109A) lost this activity, demonstrating that Hakai's E3 ligase activity can regulate osteoblast differentiation by targeting another E3 ligase.

    Evidence Affinity pulldown proteomics, co-immunoprecipitation, C109A catalytic mutant, in vitro osteoblast differentiation assay

    PMID:39034451

    Open questions at the time

    • In vivo bone phenotypes of Hakai loss have not been characterized
    • Whether Hakai ubiquitinates Smurf2 directly or requires cofactors is unclear
13. 2025 Medium

    Recent studies connected Hakai to Wnt signaling via LRP4 ubiquitination and degradation, to keratinocyte differentiation via steroid sulfatase-regulated Hakai expression, and identified UBE3C as an upstream E3 ligase that ubiquitinates Hakai to modulate m6A levels during cortical neurogenesis.

    Evidence Co-IP and ubiquitination assays with Wnt reporter; CRISPR STS knockout in keratinocytes; proteomic profiling of UBE3C-deficient forebrains and organoids with genetic/pharmacological rescue (preprint)

    PMID:39764917 PMID:bio_10.1101_2025.04.09.646620 PMID:bio_10.1101_2025.11.20.689197

    Open questions at the time

    • LRP4 and UBE3C findings are from preprints awaiting peer review
    • How Hakai balances its E-cadherin ubiquitination and m6A writer roles in the same cell is unknown
    • No mammalian genetic model (conditional knockout) characterizing Hakai loss-of-function exists

• Key unresolved questions include: whether Hakai's RING-domain E3 ligase activity is mechanistically required for its role in the m6A writer complex; the full-length structure of Hakai in complex with writer subunits or E-cadherin; and the phenotypic consequences of mammalian Hakai loss-of-function in vivo.
• No mammalian conditional knockout phenotype reported
• No full-length structural model of Hakai
• Relationship between ligase activity and m6A writer function untested in mammals