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. 2021 Feb 16;22(4):1936.
doi: 10.3390/ijms22041936.

OTUD6A Is an Aurora Kinase A-Specific Deubiquitinase

Hyo Jin Kim  1 , Jongchan Kim  1
Affiliations

OTUD6A Is an Aurora Kinase A-Specific Deubiquitinase

Hyo Jin Kim et al. Int J Mol Sci. .

Abstract

Aurora kinases are serine/threonine kinases required for cell proliferation and are overexpressed in many human cancers. Targeting Aurora kinases has been a therapeutic strategy in cancer treatment. Here, we attempted to identify a deubiquitinase (DUB) that regulates Aurora kinase A (Aurora-A) protein stability and/or kinase activity as a potential cancer therapeutic target. Through pull-down assays with the human DUB library, we identified OTUD6A as an Aurora-A-specific DUB. OTUD6A interacts with Aurora-A through OTU and kinase domains, respectively, and deubiquitinates Aurora-A. Notably, OTUD6A promotes the protein half-life of Aurora-A and activates Aurora-A by increasing phosphorylation at threonine 288 of Aurora-A. From qPCR screening, we identified and validated that the cancer gene CKS2 encoding Cyclin-dependent kinases regulatory subunit 2 is the most upregulated cell cycle regulator when OTUD6A is overexpressed. The results suggest that OTUD6A may serve as a therapeutic target in human cancers.

Keywords: Aurora kinase A; OTUD6A; cancer; deubiquitinase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Pull-down assays to identify Aurora-A-binding DUBs. (a) Each SFB-tagged DUB was co-transfected with MYC-tagged Aurora-A into HEK293T cells, followed by pulling down DUBs with S-protein beads and immunoblotting with antibodies against FLAG (to detect DUBs) and MYC (to detect Aurora-A). The first screening identified 31 binding DUBs (highlighted in red). (b) Pull-down assay was repeated with 31 binding DUBs identified from the first screening. 13 DUBs (highlighted in red) which display a relatively strong binding affinity to Aurora-A were selected in this second screening.
Figure 2
Figure 2
OTUD1 and OTUD6A deubiquitinate Aurora-A. (a) SFB-tagged 13 candidate DUBs were co-transfected with MYC-tagged Aurora-A and HA-tagged ubiquitin into HEK293T cells. After treating MG132 for 6 h, cells were lysed, followed by pulling down Aurora-A with MYC-beads and immunoblotting with antibodies against HA (to detect polyubiquitinated Aurora-A) and MYC (to detect Aurora-A). DUB assay identified OTUD1 and OTUD6A as Aurora-A-deubiquitinating DUBs. (b) DUB assay was repeated with OTUD1 and OTUD6A and a similar degree of deubiquitination of Aurora-A was observed.
Figure 3
Figure 3
OTUD6A interacts with Aurora-A through the OTU and kinase domains, respectively. (a) SFB-tagged OTUD1 and OTUD6A were transfected into HEK293T cells, followed by pulling down DUBs with S-protein beads and immunoblotting with antibodies against FLAG (to detect DUBs) and Aurora-A. (b) SFB-tagged OTUD6A was co-transfected with MYC-tagged Aurora-A into HEK293T cells, followed by pulling down Aurora-A with MYC-beads and immunoblotting with antibodies against FLAG (to detect OTUD6A) and MYC (to detect Aurora-A). (c) Schematic representation of full-length (FL) and truncated mutants of Aurora-A. (d) SFB-tagged OTUD6A was co-transfected with MYC-tagged full length or each mutant Aurora-A into HEK293T cells, followed by pulling down OTUD6A with S-protein beads and immunoblotting with antibodies against MYC (to detect Aurora-A (FL and mutants)) and FLAG (to detect OTUD6A). (e) Schematic representation of full-length (FL) and truncated mutants of OTUD6A. (f) MYC-tagged Aurora-A was co-transfected with SFB-tagged full-length or each mutant OTUD6A into HEK293T cells, followed by pulling down OTUD6A (FL and mutants) with S-protein beads and immunoblotting with antibodies against MYC (to detect Aurora-A) and FLAG (to detect OTUD6A (FL and mutants)).
Figure 4
Figure 4
OTUD6A induces protein stability and enzymatic activity of Aurora-A. (a) SFB-tagged OTUD6A or GFP was transfected into HEK293T cells and translation inhibitor cycloheximide (50 μg/mL) was treated for the indicated period. The protein stability of endogenous Aurora-A was examined with a specific antibody. (b) Relative protein stability of Aurora-A tested in (a) was quantitated by normalizing with the expression levels of loading control HSP90. (c) The levels of phosphorylated Aurora-A at threonine 288 were induced by OTUD6A expression, which in turn increased phospho-PLK1 levels (at threonine 210). (d) Silencing OTUD6A with siRNAs suppressed Aurora-A phosphorylation at threonine 288.
Figure 5
Figure 5
qPCR screening to identify cell cycle-regulating genes regulated by OTUD6A. (a) Scatterplot describes the relative expression of cell cycle-regulating genes in OTUD6A-overexpressing cells following qPCR screening. CCNG1 and CKS2 are indicated as two of the most upregulated genes. (b) qPCR validates CKS2 expression is significantly upregulated by OTUD6A. Statistical significance in (b) was determined by an unpaired t-test. Error bars are s.e.m.
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