KLHL21

KLHL21 is a BTB-Kelch family substrate adaptor for Cullin-3-based E3 ubiquitin ligase complexes that controls mitotic progression, cell migration, and inflammatory signaling. During anaphase and cytokinesis, KLHL21 localizes to midzone microtubules and the midbody—facilitated by SCCRO/DCUN1D1-dependent Cul3 neddylation—where it directly ubiquitinates Aurora B kinase to drive chromosomal passenger complex translocation and abscission (PMID:19995937, PMID:28620047). In interphase, CRL3-KLHL21 ubiquitylates the microtubule plus-end tracking protein EB1 at focal adhesions to regulate cortical microtubule dynamics, lamellipodia formation, and cell migration, and independently suppresses NF-κB signaling both by sequestering IKKβ through its Kelch domain (in a ligase-independent manner) and by stabilizing the deubiquitinase CYLD to restrain p65 activation (PMID:27641145, PMID:27387502, PMID:36538852). In gastric epithelium, KLHL21 loss stabilizes the PABPC1–eIF4G translational complex to upregulate PIK3CB mRNA translation, activating STAT3 and promoting metaplastic stemness and tumorigenesis (PMID:38969490).

1. 2009 High

   Establishing that KLHL21 is the Cul3 adaptor for Aurora B ubiquitination resolved how the chromosomal passenger complex relocates from chromosomes to the spindle midzone during anaphase, linking a BTB-Kelch protein to mitotic regulation for the first time.

   Evidence Co-immunoprecipitation, in vitro ubiquitination assay, siRNA knockdown with cytokinesis phenotype, and fluorescence microscopy in human cells

   PMID:19995937

   Open questions at the time

   • How KLHL21 itself is recruited to midzone microtubules was not determined
   • Whether KLHL21-mediated Aurora B ubiquitination triggers degradation versus non-degradative signaling was unclear
   • No structural basis for KLHL21–Aurora B recognition
2. 2016 High

   Discovery that CRL3-KLHL21 ubiquitylates EB1 at focal adhesions extended its function beyond mitosis, revealing a mechanism by which cortical microtubule dynamics and actin-based cell migration are coordinated through substrate-specific ubiquitination.

   Evidence In vitro ubiquitination of EB1, non-ubiquitylatable EB1 mutant phenocopy, live-cell imaging of focal adhesion dynamics and migration

   PMID:27641145

   Open questions at the time

   • The ubiquitin chain type attached to EB1 and whether it is degradative or regulatory was not resolved
   • How KLHL21 is targeted specifically to leading-edge focal adhesions is unknown
3. 2016 Medium

   Demonstrating that KLHL21 suppresses NF-κB by sequestering IKKβ through a ligase-independent Kelch-domain interaction revealed a non-catalytic adaptor function, separating its ubiquitin ligase and scaffolding roles.

   Evidence Co-immunoprecipitation domain mapping, overexpression and siRNA with IKKβ activation and IκBα degradation readouts upon TNFα stimulation

   PMID:27387502

   Open questions at the time

   • The study relies on overexpression/knockdown without genetic knockout confirmation
   • Whether the sequestration model operates in vivo under physiological expression levels is untested
   • Structural basis of the Kelch–IKKβ kinase domain interaction is lacking
4. 2017 High

   Placing SCCRO/DCUN1D1-mediated Cul3 neddylation upstream of KLHL21-dependent Aurora B ubiquitination at the midbody defined the activating step of this E3 ligase during abscission, explaining why neddylation-deficient cells fail cytokinesis.

   Evidence siRNA/shRNA of SCCRO and KLHL21, immunofluorescence at midbody, epistasis with Aurora B inhibitor rescuing abscission defects

   PMID:28620047

   Open questions at the time

   • Whether other BTB adaptors compete with KLHL21 for Cul3 binding at the midbody is unknown
   • Direct measurement of Cul3 neddylation status in complex with KLHL21 at the midbody was not performed
5. 2022 Medium

   Identification of CYLD as a KLHL21-stabilized partner that mediates NF-κB suppression in bladder cancer provided a second, ligase-relevant arm of NF-κB regulation distinct from IKKβ sequestration, and showed that KLHL21's tumor-suppressive effect depends on CYLD.

   Evidence Co-immunoprecipitation, CYLD ubiquitination assay, epistasis (CYLD deletion abolishes KLHL21 anti-tumor effect), xenograft model

   PMID:36538852

   Open questions at the time

   • Whether KLHL21 protects CYLD through direct deubiquitination competition or by an indirect mechanism is unresolved
   • Single-lab finding without independent replication
   • The relationship between the IKKβ-sequestration and CYLD-stabilization mechanisms is unclear
6. 2024 High

   Conditional knockout of Klhl21 in gastric epithelium revealed a translation-regulatory role: KLHL21 loss stabilizes the PABPC1–eIF4G complex to enhance PIK3CB mRNA translation, activating STAT3 and promoting metaplastic stemness, connecting KLHL21 to gastric tumorigenesis.

   Evidence Conditional Klhl21 knockout mouse, ribosome sequencing, mass spectrometry proteomics, single-cell RNA-seq, pharmacological STAT3 inhibitor rescue

   PMID:38969490

   Open questions at the time

   • Whether KLHL21 directly ubiquitinates PABPC1 or eIF4G, or acts indirectly, was not established
   • The connection between known E3 substrates (Aurora B, EB1) and the translational regulation axis is unexplained
   • Whether this gastric phenotype extends to other epithelial tissues is untested

• How KLHL21 coordinates its multiple substrate-specific activities across cell-cycle stages and tissue contexts—and whether a unifying regulatory switch governs substrate selection—remains an open question.
• No structural model of KLHL21 in complex with any substrate exists
• Post-translational modifications or binding partners that switch KLHL21 between mitotic and interphase substrates are unknown
• Whether KLHL21 ubiquitin ligase activity toward PABPC1/eIF4G is direct has not been tested biochemically