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KLHL39 suppresses colon cancer metastasis by blocking KLHL20-mediated PML and DAPK ubiquitination
H Y Chen1,2, J Y Hu2,3, T H Chen2,3
1Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
Abstract:
Cullin 3 (Cul3)-family ubiquitin ligases use the BTB-domain-containing proteins for the recruitment of substrates, but the regulation of this family of ubiquitin ligases has not been completely understood. KLHL20 is a BTB-family protein and targets tumor suppressor promyelocytic leukemia protein (PML) and death-associated protein kinase (DAPK) to its kelch-repeat domain for ubiquitination and degradation. Here, we show that another BTB-kelch protein KLHL39 is recruited to the substrate-binding domain of KLHL20 but is not a substrate of Cul3-KLHL20 complex. Interestingly, KLHL39 does not bind Cul3 because of the absence of certain conserved residues in the BTB domain. Instead, KLHL39 blocks KLHL20-mediated ubiquitination of PML and DAPK by disrupting the binding of these substrates to KLHL20 as well as the binding of KLHL20 to Cul3. Through the two mechanisms, KLHL39 increases the stability of PML and DAPK. In human colon cancers, downregulations of KLHL39, PML and DAPK are associated with metastatic progression. Furthermore, preclinical data indicate that KLHL39 promotes colon cancer migration, invasion and survival in vitro and metastasis in vivo through a PML- and DAPK-dependent mechanism. Our study identifies KLHL39 as a negative regulator of Cul3-KLHL20 ubiquitin ligase and reveals a role of KLHL39-mediated PML and DAPK stabilization in colon cancer metastasis.
Insights
KLHL39 inhibits the Cul3-KLHL20 ubiquitin ligase by blocking substrate and Cul3 binding, stabilizing tumor suppressors PML and DAPK. KLHL39 downregulation correlates with colon cancer metastasis.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Cullin 3 (Cul3)-family ubiquitin ligases utilize BTB-domain proteins for substrate recruitment, but their regulation remains incompletely understood.
- KLHL20, a BTB-family protein, targets tumor suppressors like promyelocytic leukemia protein (PML) and death-associated protein kinase (DAPK) for ubiquitination and degradation.
- The precise regulatory mechanisms governing Cul3-KLHL20 ligase activity require further elucidation.
Purpose of the Study:
- To investigate the regulatory role of the BTB-kelch protein KLHL39 in the Cul3-KLHL20 ubiquitin ligase complex.
- To determine how KLHL39 affects the ubiquitination and stability of substrates PML and DAPK.
- To explore the association of KLHL39, PML, and DAPK with colon cancer progression and metastasis.
Main Methods:
- Biochemical assays to assess protein-protein interactions between KLHL20, KLHL39, Cul3, PML, and DAPK.
- Ubiquitination assays to quantify the effect of KLHL39 on PML and DAPK ubiquitination by the Cul3-KLHL20 complex.
- Analysis of KLHL39, PML, and DAPK expression levels in human colon cancer tissues and correlation with metastatic status.
- In vitro and in vivo experiments to evaluate the role of KLHL39 in colon cancer cell migration, invasion, survival, and metastasis.
Main Results:
- KLHL39 interacts with KLHL20 but does not bind Cul3 due to specific residue differences in its BTB domain.
- KLHL39 inhibits KLHL20-mediated ubiquitination of PML and DAPK by disrupting substrate binding to KLHL20 and KLHL20 binding to Cul3.
- KLHL39 enhances the stability of PML and DAPK, and its downregulation in colon cancers correlates with metastatic progression.
- KLHL39 promotes colon cancer cell migration, invasion, survival, and metastasis in a PML- and DAPK-dependent manner.
Conclusions:
- KLHL39 acts as a novel negative regulator of the Cul3-KLHL20 ubiquitin ligase.
- Stabilization of PML and DAPK by KLHL39 plays a critical role in suppressing colon cancer metastasis.
- KLHL39 represents a potential therapeutic target for mitigating colon cancer progression.
