KLHL22

KLHL22 is a substrate-adaptor subunit of the CUL3-RBX1 RING E3 ubiquitin ligase that channels amino-acid signaling into mTORC1 activation and controls the turnover of diverse regulatory proteins. In response to amino acids, CUL3-KLHL22 catalyzes K48-linked polyubiquitination and proteasomal degradation of the GATOR1 subunit DEPDC5, relieving GATOR1-mediated inhibition of Rag GTPases and thereby activating mTORC1; KLHL22 itself is targeted for degradation by the E3/E4 ligase UBE4B, creating a regulatory circuit that governs neural precursor proliferation and differentiation (PMID:29769719, PMID:36440598). Beyond nutrient sensing, CUL3-KLHL22 ubiquitinates PD-1 before it reaches the T-cell surface, limiting immune checkpoint expression and sustaining antitumor T-cell responses, and also ubiquitinates the mitotic kinase PLK1 and the metabolic enzyme GDH1, for which a cryo-EM structure reveals a W-shaped E3–substrate architecture (PMID:33109719, PMID:24067371, PMID:37788672). KLHL22 additionally mediates K48-linked ubiquitination and degradation of SARS-CoV-2 NSP6, restoring calcium homeostasis and counteracting virus-induced autophagic cell death (PMID:40373961).

1. 2013 Medium

   Establishing KLHL22 as a CUL3 E3 adaptor: the first substrate identified was the mitotic kinase PLK1, demonstrating that KLHL22 functions as a BTB-Kelch specificity subunit that recruits substrates via two distinct contact motifs independently of substrate kinase activity.

   Evidence Co-immunoprecipitation, PLK1 mutant binding analysis, and in vitro ubiquitination assay in human cells

   PMID:24067371

   Open questions at the time

   • No in vivo or structural validation of the KLHL22–PLK1 interaction
   • Physiological consequence of PLK1 ubiquitination by CUL3-KLHL22 on mitotic progression not determined
   • Binding motifs on KLHL22 Kelch domain not mapped
2. 2018 High

   Linking KLHL22 to nutrient sensing: amino-acid-stimulated CUL3-KLHL22 ubiquitinates DEPDC5 (GATOR1 subunit) for proteasomal degradation, thereby activating mTORC1 via Rag GTPases — establishing KLHL22 as a critical node in the amino-acid–mTORC1 pathway conserved from C. elegans to mammals.

   Evidence Co-IP, K48-linked ubiquitination assays, KLHL22 knockdown/overexpression, in vivo xenograft tumor model, and genetic epistasis with the C. elegans orthologue MEL-26

   PMID:29769719

   Open questions at the time

   • Amino-acid-dependent regulation of KLHL22 activity or DEPDC5 recognition mechanism unknown
   • Whether KLHL22-mediated DEPDC5 degradation operates at the lysosomal surface not directly shown
3. 2020 High

   Expanding substrate repertoire to immune regulation: CUL3-KLHL22 ubiquitinates PD-1 before surface delivery, controlling the abundance of this immune checkpoint; KLHL22 deficiency elevates PD-1, suppressing antitumor T-cell immunity.

   Evidence Reciprocal Co-IP identifying KLHL22 as a major PD-1 interactor, K48-linked ubiquitination assay, KLHL22 knockdown with PD-1 surface expression readout, in vivo tumor models

   PMID:33109719

   Open questions at the time

   • Signal or context that modulates KLHL22-PD-1 binding in T cells not defined
   • Relative contribution of KLHL22-mediated degradation versus other PD-1 turnover pathways not quantified
4. 2022 High

   Identifying the upstream control of KLHL22 abundance: UBE4B polyubiquitylates and degrades KLHL22, forming a regulatory cascade (UBE4B → KLHL22 → DEPDC5 → mTORC1) essential for neural precursor proliferation and differentiation.

   Evidence Conditional UBE4B knockout mouse, in vivo ubiquitination assay, KLHL22 knockdown rescue, rapamycin rescue in neural precursors

   PMID:36440598

   Open questions at the time

   • How UBE4B activity toward KLHL22 is itself regulated (e.g., by amino acids or growth factors) is unknown
   • Whether UBE4B–KLHL22 axis operates outside neural tissue in vivo not established
5. 2023 High

   Providing the first structural model: cryo-EM of CUL3-RBX1-KLHL22 bound to GDH1 hexamer at 3.06 Å reveals a W-shaped dimeric architecture in which three E3 dimers dynamically engage one substrate hexamer, illuminating how KLHL22 Kelch domains present substrates for polyubiquitination.

   Evidence Cryo-EM structure determination combined with in vitro polyubiquitination reconstitution

   PMID:37788672

   Open questions at the time

   • Structural basis for KLHL22 discrimination among its multiple substrates (DEPDC5, PD-1, PLK1, GDH1) not resolved
   • Neddylation state and E2 identity in the reconstituted complex not fully characterized
6. 2025 Medium

   Extending KLHL22 function to host defense and pharmacological resistance: KLHL22 degrades SARS-CoV-2 NSP6 to restore calcium homeostasis, and its stabilization upon UBE4B loss drives BET-inhibitor resistance through a JAK2-PIM1 axis in hepatocellular carcinoma.

   Evidence Co-IP and ubiquitination assays for NSP6; sequential siRNA knockdown with drug sensitivity readouts and Western blotting for JAK2/PIM1/LNK in HCC cells

   PMID:40228637 PMID:40373961

   Open questions at the time

   • NSP6 degradation study from a single lab; independent replication needed
   • Direct biochemical link between KLHL22 and JAK2 regulation (direct ubiquitination vs. indirect) not established
   • Whether KLHL22-mediated NSP6 degradation is relevant during live SARS-CoV-2 infection not tested

• It remains unknown how amino-acid signals or other upstream cues regulate KLHL22 substrate selectivity and whether a common recognition degron unifies its diverse substrates (DEPDC5, PD-1, PLK1, GDH1, NSP6).
• No degron motif shared across substrates has been identified
• Post-translational modifications of KLHL22 that switch substrate preference are uncharacterized
• Tissue-specific roles beyond neural precursors, T cells, and tumor contexts are largely unexplored