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Research article
Young Ho Chung
Korea Basic Science Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1779-0657
Sung-Gil Chi
Department of Life Science, Korea University
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Phosphorylation of NF-kappaB inhibitor alpha (IκBα) is key to regulation of NF-κB transcription factor activity in the cell. Several sites of IκBα phosphorylation by members of the IκB kinase family have been identified, but phosphorylation of the protein by other kinases remains poorly understood. We investigated a new phosphorylation site on IκBα and identified its biological function in breast cancer cells.
Methods: Previously, we observed that aurora kinase (AURK) binds IκBα in the cell. To identify the domains of IκBα essential for phosphorylation by AURK, we performed kinase assays with a series of IκBα truncation mutants. AURK significantly promoted activation of IκBα at serine 32 but not serine 36; by contrast, IκB kinase (IKK) family proteins activated both of these residues. We also confirmed phosphorylation of IκBα by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and nano-liquid chromatography hybrid quadrupole orbitrap mass spectrometer (nanoLC-MS/MS; Q-Exactive).
Results: We identified two novel sites of serine phosphorylation, S63 and S262. Alanine substitution of S63 and S262 (S63A and S262A) of IκBα inhibited proliferation and suppressed p65 transcription activity. In addition, S63A and/or S262A of IκBα regulated apoptotic and necroptotic effects in breast cancer cells.
Conclusions: Phosphorylation of IκBα by AURK at novel sites is related to the apoptosis and necroptosis pathways in breast cancer cells.
Keywords
Breast cancer, IκBα, new phosphor-site, necroptosis
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFiguresandTables.pdf
Supplementary Figure 1. Schematic diagram of phosphopeptide mapping Schematic diagram was constructed for phosphopeptide mapping. Supplementary Figure 2. Ion chromatograms of digested phosphopeptide including Serine 32. LC-quadrupole orbitrap MS/MS spectrum of precursor ion m/z 950.4053 (charge state 3+) representing the phosphorylated peptide of sequence 24-LLDDRHDpSGLDSMKDEEYEQMVK-48. The spectrum exhibits evidence of phosphorylation at serine 32, but not serine 36 Supplementary Figure 3. Induction of apoptosis and necroptosis in MCF7 and MDM-MB 468 expressing S63A and S262A mutant FACs-annexin V-staining was performed to measure apoptosis in MCF7 and MDA-MB 468. Compared with the control group, the distribution of early and late apoptosis was increased and the distribution of necrosis was increased in S63A and S262A Supplementary Figure 4. Expression level of IκBα and NFκB-p65 in MDA-MB 231 expressing S32A, S63A and S262 A Compared with the control group, expression of IκBα was increased in mutant groups, and expression of NFκB-p65 was decreased in mutant groups. There is no response in S156A, a mutant of S156 detected in LC/MS. Supplementary Table 1. Identification of a novel phosphorylation site by LC-MS New phosphorylation sites were identified between amino acids 54–67 and 246–264. Supplementary Table 2. Confirmation of novel phosphorylation site by MALDI-TOF MS New phosphorylation sites were confirmed using MALDI-TOF MS.
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View the published article at BMC Cancer.
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Editor invited
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Review #2 received
Received 27 May, 2020
Reviewer #2 agreed
On 07 May, 2020
Review #1 received
Received 17 Apr, 2020
Reviewer #1 agreed
On 02 Apr, 2020
Reviewers invited
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On 09 Jan, 2020
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A new preprint design is coming!
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See the published version of this preprint at BMC Cancer.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Young Ho Chung
Korea Basic Science Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1779-0657
Sung-Gil Chi
Department of Life Science, Korea University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at BMC Cancer.
Version 3
Posted 21 Oct, 2020
No community comments so far
Review #1 received
Received 17 Feb, 2021
Reviewers invited
Invitations sent on 27 Oct, 2020
Reviewer #1 agreed
On 27 Oct, 2020
Editor assigned
On 13 Oct, 2020
Submission checks complete
On 12 Oct, 2020
Editor invited
On 12 Oct, 2020
No comments provided
Editorial decision: Major revision
On 07 Aug, 2020
Review #2 received
Received 06 Aug, 2020
Review #1 received
Received 21 Jul, 2020
Reviewer #2 agreed
On 17 Jul, 2020
Editor assigned
On 14 Jul, 2020
Reviewers invited
Invitations sent on 14 Jul, 2020
Reviewer #1 agreed
On 14 Jul, 2020
Submission checks complete
On 13 Jul, 2020
Editor invited
On 13 Jul, 2020
No comments provided
Editorial decision: Major revision
On 05 Jun, 2020
Review #2 received
Received 27 May, 2020
Reviewer #2 agreed
On 07 May, 2020
Review #1 received
Received 17 Apr, 2020
Reviewer #1 agreed
On 02 Apr, 2020
Reviewers invited
Invitations sent on 03 Feb, 2020
Editor assigned
On 31 Jan, 2020
Submission checks complete
On 09 Jan, 2020
Editor invited
On 08 Jan, 2020
First submitted
On 17 Dec, 2019
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Cancer.
Background: Phosphorylation of NF-kappaB inhibitor alpha (IκBα) is key to regulation of NF-κB transcription factor activity in the cell. Several sites of IκBα phosphorylation by members of the IκB kinase family have been identified, but phosphorylation of the protein by other kinases remains poorly understood. We investigated a new phosphorylation site on IκBα and identified its biological function in breast cancer cells.
Methods: Previously, we observed that aurora kinase (AURK) binds IκBα in the cell. To identify the domains of IκBα essential for phosphorylation by AURK, we performed kinase assays with a series of IκBα truncation mutants. AURK significantly promoted activation of IκBα at serine 32 but not serine 36; by contrast, IκB kinase (IKK) family proteins activated both of these residues. We also confirmed phosphorylation of IκBα by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and nano-liquid chromatography hybrid quadrupole orbitrap mass spectrometer (nanoLC-MS/MS; Q-Exactive).
Results: We identified two novel sites of serine phosphorylation, S63 and S262. Alanine substitution of S63 and S262 (S63A and S262A) of IκBα inhibited proliferation and suppressed p65 transcription activity. In addition, S63A and/or S262A of IκBα regulated apoptotic and necroptotic effects in breast cancer cells.
Conclusions: Phosphorylation of IκBα by AURK at novel sites is related to the apoptosis and necroptosis pathways in breast cancer cells.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFiguresandTables.pdf
Supplementary Figure 1. Schematic diagram of phosphopeptide mapping Schematic diagram was constructed for phosphopeptide mapping. Supplementary Figure 2. Ion chromatograms of digested phosphopeptide including Serine 32. LC-quadrupole orbitrap MS/MS spectrum of precursor ion m/z 950.4053 (charge state 3+) representing the phosphorylated peptide of sequence 24-LLDDRHDpSGLDSMKDEEYEQMVK-48. The spectrum exhibits evidence of phosphorylation at serine 32, but not serine 36 Supplementary Figure 3. Induction of apoptosis and necroptosis in MCF7 and MDM-MB 468 expressing S63A and S262A mutant FACs-annexin V-staining was performed to measure apoptosis in MCF7 and MDA-MB 468. Compared with the control group, the distribution of early and late apoptosis was increased and the distribution of necrosis was increased in S63A and S262A Supplementary Figure 4. Expression level of IκBα and NFκB-p65 in MDA-MB 231 expressing S32A, S63A and S262 A Compared with the control group, expression of IκBα was increased in mutant groups, and expression of NFκB-p65 was decreased in mutant groups. There is no response in S156A, a mutant of S156 detected in LC/MS. Supplementary Table 1. Identification of a novel phosphorylation site by LC-MS New phosphorylation sites were identified between amino acids 54–67 and 246–264. Supplementary Table 2. Confirmation of novel phosphorylation site by MALDI-TOF MS New phosphorylation sites were confirmed using MALDI-TOF MS.
- Westernblotfullsizeimage.pdf
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