KLHL23

KLHL23 is a substrate-recognition adaptor of a CUL3-RING E3 ubiquitin ligase complex that selectively polyubiquitylates GTP-bound (active) CDC42 for proteasomal degradation, thereby spatiotemporally co-inactivating CDC42 in concert with RhoGDI-mediated sequestration of GDP-bound CDC42 to maintain membrane homeostasis during cell migration (PMID:40846997). KLHL23 directly binds actin and promotes stress fiber and focal adhesion assembly; its loss induces excessive filopodia and lamellipodia, enhances cell motility, and drives epithelial–mesenchymal transition in hepatocellular and urothelial carcinoma cells (PMID:29171033, PMID:34169920). The CDC42-Y64C germline variant found in Takenouchi–Kosaki Syndrome escapes KLHL23-mediated ubiquitylation, linking disruption of this pathway to a developmental disorder (PMID:40846997).

1. 2016 Medium

   Establishing that KLHL23 influences cancer cell proliferation and cyclin D1 levels placed it as a functionally relevant gene in gastric cancer, though the underlying mechanism remained undefined.

   Evidence Overexpression and siRNA knockdown of KLHL23 in gastric cancer cell lines with proliferation, drug resistance, and cyclin D1 immunoblotting readouts

   PMID:27833855

   Open questions at the time

   • No biochemical mechanism linking KLHL23 to cyclin D1 regulation was demonstrated
   • Whether the effect is through the PHOSPHO2-KLHL23 fusion transcript versus KLHL23 alone was not resolved
   • No in vivo validation
2. 2018 High

   Demonstrating that KLHL23 directly binds actin and suppresses actin polymerization revealed it as a negative regulator of membrane protrusions and EMT, connecting its loss to hypoxia signaling and metastasis in vivo.

   Evidence Subgenome-wide screen, KLHL23 knockdown with actin polymerization assays, filopodium/lamellipodium imaging, HIF/Notch signaling readouts, patient-derived xenograft mouse models in hepatocellular carcinoma cells

   PMID:29171033

   Open questions at the time

   • The molecular mechanism by which KLHL23 suppresses actin polymerization was not identified
   • Whether KLHL23's actin-regulatory function requires its kelch-repeat substrate-binding domain was unknown
   • The connection between actin binding and HIF/Notch signaling lacked mechanistic resolution
3. 2021 Medium

   Showing that KLHL23 localizes to actin stress fibers and is required for focal adhesion complex integrity extended its role beyond actin polymerization suppression to the structural organization of contractile and adhesive machinery in migrating cells.

   Evidence KLHL23 siRNA knockdown with wound healing assay, co-localization imaging of KLHL23/actin/vinculin in urothelial carcinoma cells

   PMID:34169920

   Open questions at the time

   • No biochemical reconstitution of the KLHL23–actin interaction at stress fibers was performed
   • Whether KLHL23's focal adhesion role depends on its CUL3 adaptor function was untested
   • Single cancer cell type studied
4. 2025 High

   Biochemical reconstitution of the KLHL23–CUL3 E3 ligase complex and identification of GTP-bound CDC42 as its specific substrate unified the actin and membrane-protrusion phenotypes under a single mechanism: KLHL23 degrades active CDC42, while RhoGDI sequesters inactive CDC42, providing coordinated spatiotemporal inactivation that maintains membrane homeostasis.

   Evidence Co-immunoprecipitation, in vitro and cellular ubiquitylation assays, FRET-based spatiotemporal CDC42 activity measurements, GTP/GDP-state-selective binding assays, switch II domain mutagenesis, CDC42-Y64C (Takenouchi–Kosaki Syndrome) variant functional analysis, KLHL23 knockout with membrane dynamics readout

   PMID:40846997

   Open questions at the time

   • Structural basis of the KLHL23 kelch domain–CDC42·GTP interaction is not yet resolved at atomic level
   • Whether KLHL23 ubiquitylates additional Rho GTPase substrates beyond CDC42 is unknown
   • The relative contributions of CUL3-dependent CDC42 degradation versus direct actin binding to KLHL23's anti-metastatic function have not been dissected

• Outstanding questions include whether KLHL23 has additional E3 ligase substrates beyond CDC42, how its direct actin-binding function is coordinated with its CUL3 adaptor activity, and what the structural determinants of GTP-state selectivity are.
• No structural model of the KLHL23–CDC42 complex exists
• The relationship between KLHL23's actin-binding and CUL3-adaptor functions has not been mechanistically separated
• Whether cyclin D1 regulation is a direct or indirect consequence of CDC42 degradation is unresolved