KLHL23

KLHL23 is a substrate-recognition adaptor that couples a CUL3 RING E3 ubiquitin ligase to the control of actin cytoskeleton dynamics, restraining cell migration, membrane protrusion, and EMT-driven metastasis (PMID:40846997, PMID:29171033). Mechanistically, KLHL23 selects the GTP-bound (active) form of CDC42 for polyubiquitylation and proteasomal degradation, engaging the CDC42 switch II region in competition with RhoGDI, which instead sequesters the GDP-bound form; together they achieve spatiotemporal co-inactivation of CDC42, and a germline CDC42-Y64C variant escapes this KLHL23-mediated turnover (PMID:40846997). Consistent with negative regulation of CDC42-driven actin remodeling, KLHL23 directly binds actin and suppresses its polymerization, and its loss induces filopodia and lamellipodia and activates hypoxic HIF/Notch signaling to drive EMT in hepatocellular carcinoma (PMID:29171033); KLHL23 also localizes to actin stress fibers in leading migratory cells and promotes focal adhesion formation, with its depletion abolishing stress fibers, redistributing vinculin, and enhancing cancer cell motility (PMID:34169920).

1. 2016 Low

   An initial functional link tied KLHL23 expression to cancer cell growth, raising the question of whether the protein had a defined molecular role beyond a proliferation correlate.

   Evidence Overexpression and siRNA silencing in gastric cancer cell lines with proliferation assays and cyclin D1 Western blot

   PMID:27833855

   Open questions at the time

   • Mechanistic link to cyclin D1 is correlative, not direct
   • Single lab and single expression system
   • KLHL23 derived from a PHOSPHO2-KLHL23 read-through transcript, leaving the native gene product's role ambiguous
2. 2018 Medium

   The question of KLHL23's biochemical action was addressed by showing it directly engages and restrains the actin cytoskeleton, establishing it as an invasion/EMT suppressor rather than a generic proliferation factor.

   Evidence Subgenome-wide invasion-suppressor screen, actin binding and polymerization assays, morphological readouts, HIF/Notch signaling measurements, and patient-derived xenograft validation in hepatocellular carcinoma

   PMID:29171033

   Open questions at the time

   • Direct actin-binding mechanism not reconstituted in vitro
   • Did not connect actin suppression to KLHL23's adaptor function
   • Single lab
3. 2021 Medium

   The cell-biological basis of migration control was refined by localizing KLHL23 to actin stress fibers and linking it to focal adhesion organization.

   Evidence Wound-healing assay, KLHL23 knockdown with vinculin localization imaging, and co-localization with actin fibers in urothelial carcinoma

   PMID:34169920

   Open questions at the time

   • Molecular basis of stress-fiber localization unresolved
   • Relationship to actin-binding versus E3 adaptor activity not defined
   • Single lab
4. 2025 High

   The unifying mechanism was established by showing KLHL23 is the CUL3 substrate adaptor that selectively ubiquitylates active CDC42 and co-inactivates it with RhoGDI, explaining how it restrains actin remodeling and metastasis.

   Evidence In vitro reconstitution of KLHL23-CUL3 E3 ligase activity toward CDC42•GTP, KLHL23/RhoGDI competitive binding assays at the switch II region, live-cell FRET, depletion with protrusion/metastasis readouts, and CDC42-Y64C escape variant

   PMID:40846997

   Open questions at the time

   • Whether KLHL23 ubiquitylates Rho-family GTPases beyond CDC42 is undefined
   • Mechanistic relationship between direct actin binding (#1) and CUL3-dependent CDC42 degradation not integrated
   • Upstream signals controlling KLHL23 activity unknown

• How KLHL23's two reported biochemical activities—direct actin binding and CUL3-mediated CDC42 ubiquitylation—are mechanistically reconciled, and what regulates KLHL23 itself, remain open.
• No structural model of the KLHL23-CUL3-CDC42 complex
• Substrate range beyond CDC42 unmapped
• Regulation of KLHL23 abundance and recruitment unknown