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Updated: Feb 4, 2026
In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
ANKRD9 is associated with tumor suppression as a substrate receptor subunit of ubiquitin ligase
Yejin Lee1, Byungho Lim2, Seon Woo Lee2
1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea; Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon 34113, Republic of Korea.
Background:
Human ANKRD9 (ankyrin repeat domain 9) expression is altered in some cancers.
Methods:
We tested genetic association of ANKRD9 with gastric cancer susceptibility and examined functional association of ANKRD9 with altered proliferation of MKN45 gastric cancer cells. We then identified ANKRD9-binding partners in HEK 293 embryonic kidney cells using quantitative proteomics, western blotting and complex reconstitution assays. We finally demonstrated ANKRD9's role of recognizing substrates for ubiquitination using in vitro ubiquitylation assay.
Results:
ANKRD9 is associated with cancer susceptibility in a comparison of single-nucleotide polymorphisms between 1092 gastric cancer patients and 1206 healthy controls. ANKRD9 depletion accelerates tumor progression by increasing cellular proliferation, piling up, and anchorage-independent growth of MKN45 cells. We discovered that ANKRD9 is a ubiquitin ligase substrate receptor subunit and has an anti-proliferative activity. ANKRD9 associates with CUL5 (not CUL2), ELOB, ELOC, and presumably RNF7 subunits, which together assemble into a cullin-RING superfamily E3 ligase complex. ANKRD9 belongs to the ASB family of proteins, which are characterized by the presence of ankyrin repeats and a SOCS box. In addition to its interactions with the other E3 ligase subunits, ANKRD9 interacts with two isoforms of inosine monophosphate dehydrogenase (IMPDH). These IMPDH isoforms are cognate substrates of the ANKRD9-containing E3 enzyme, which ubiquitinates them for proteasomal degradation. Their ubiquitination and turnover require the presence of ANKRD9.
Conclusion:
ANKRD9, a previously unidentified E3 substrate receptor subunit, functions in tumor suppression by recognizing the oncoprotein IMPDH isoforms for E3 ubiquitination and proteasomal degradation.
Insights
Ankyrin repeat domain 9 (ANKRD9) is a tumor suppressor that targets oncoproteins for degradation. This E3 ligase subunit recognizes IMPDH isoforms, leading to their ubiquitination and proteasomal destruction, inhibiting cancer cell proliferation.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Altered expression of human ankyrin repeat domain 9 (ANKRD9) is observed in various cancers.
- ANKRD9's role in cancer development and its molecular mechanisms remain largely unexplored.
Purpose of the Study:
- To investigate the genetic association between ANKRD9 and gastric cancer susceptibility.
- To elucidate the functional role of ANKRD9 in regulating gastric cancer cell proliferation.
- To identify ANKRD9-binding partners and its function within the E3 ubiquitin ligase complex.
Main Methods:
- Genetic association study comparing single-nucleotide polymorphisms in 1092 gastric cancer patients and 1206 healthy controls.
- Cell proliferation assays using MKN45 gastric cancer cells with and without ANKRD9.
- Quantitative proteomics and western blotting to identify ANKRD9-interacting proteins in HEK 293 cells.
- In vitro ubiquitylation assays to confirm ANKRD9's role in substrate recognition.
Main Results:
- ANKRD9 is genetically associated with gastric cancer susceptibility.
- ANKRD9 depletion enhances gastric cancer cell proliferation, migration, and anchorage-independent growth.
- ANKRD9 functions as a substrate receptor subunit of a cullin-RING E3 ligase, associating with CUL5, ELOB, ELOC, and RNF7.
- ANKRD9 specifically recognizes and targets two isoforms of inosine monophosphate dehydrogenase (IMPDH) for ubiquitination and proteasomal degradation.
Conclusions:
- ANKRD9 acts as a tumor suppressor by mediating the ubiquitination and degradation of oncoprotein IMPDH isoforms.
- The ANKRD9-containing E3 ligase complex plays a critical role in inhibiting cancer cell proliferation through substrate targeting.
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