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Updated: Apr 28, 2026
Assays for Validating Histone Acetyltransferase Inhibitors
Published on: August 6, 2020
Inflammation Drives Phosphorylation and Acetylation of MutS Homolog 3 and Interaction with Cytosolic HDAC6
Stephanie S Tseng-Rogenski1, Minoru Koi1,2, John M Carethers1,2,3,4
1Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
Background:
MutS Homolog 3 (MSH3), part of the MutSβ DNA mismatch repair complex with MSH2, can reversibly translocate from the nucleus to cytosol via IL-6 signaling, abrogating nuclear MutSβ function and is associated with metastasis and poor patient survival. A polymorphism consisting of deletion of 27-bp proximate to the nuclear localization signal (NLS) (Δ27bpMSH3) allows MSH3 cytosolic retention with IL-6 or oxidative stress. Here, we examined for IL-6-induced post-translational modifications associated with MSH3 cytosolic translocation.
Methods:
We utilized MSH3-genotyped colon cancer cell lines after IL-6 treatment to assess post-translational modification of MSH3 via Western blots (WB). We modified sequences within the MSH3-NLS-EGFP reporter construct to assess MSH3 localization via immunofluorescent microscopy and WB after nuclear-cytosolic fractionation. Immunoprecipitation (IP) followed by WB was used to study post-IL-6-induced interactions with MSH3.
Results:
MSH3 and Δ27bpMSH3 increased serine phosphorylation after 2 hours followed by tyrosine phosphorylation 18 hours post IL-6 treatment, with Δ27bpMSH3 showing more robust phosphorylation than MSH3 likely due to increased cytosolic translocation. MSH3 cytosolic localization was enhanced by acetylation of lysine residues within MSH3's NLS, specifically at residues K99, K100 and K103. With the observed acetylation control for MSH3 cytosolic localization, IP experiments demonstrate binding of cytosolic-located histone deacetylase 6 (HDAC6) to acetylated Δ27bpMSH3.
Conclusions:
Polymorphic MSH3 undergoes serine/tyrosine phosphorylation and NLS acetylation upon IL-6 signaling for its nuclear-cytosolic shift and binds HDAC6 in the cytosol which may contribute to anticipated deacetylation and MSH3 protein stability when separated from MSH2. These modifications might be targeted to regulate MSH3's intracellular localization.
Insights
MutS Homolog 3 (MSH3) undergoes phosphorylation and acetylation upon IL-6 signaling, promoting its shift to the cytosol. This cytosolic retention, particularly in polymorphic MSH3, involves binding to histone deacetylase 6 (HDAC6).
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- MutS Homolog 3 (MSH3), a component of the MutSβ DNA mismatch repair complex with MSH2, can translocate between the nucleus and cytosol.
- IL-6 signaling can induce MSH3 nuclear-cytosolic translocation, disrupting nuclear MSH3 function and correlating with metastasis and poor patient survival.
- A specific MSH3 polymorphism (Δ27bpMSH3) facilitates enhanced cytosolic retention upon IL-6 stimulation or oxidative stress.
Purpose of the Study:
- To investigate the IL-6-induced post-translational modifications of MSH3.
- To elucidate the mechanisms governing MSH3 cytosolic translocation.
- To explore the interaction of MSH3 with other proteins during translocation.
Main Methods:
- Utilized MSH3-genotyped colon cancer cell lines treated with IL-6.
- Assessed MSH3 post-translational modifications using Western blots (WB).
- Employed immunofluorescent microscopy and WB after nuclear-cytosolic fractionation to analyze MSH3 localization using a modified MSH3-NLS-EGFP reporter construct.
- Conducted immunoprecipitation (IP) followed by WB to study post-IL-6-induced interactions with MSH3.
Main Results:
- MSH3 and Δ27bpMSH3 exhibited increased serine phosphorylation (2 hours) and tyrosine phosphorylation (18 hours) post-IL-6 treatment.
- Δ27bpMSH3 showed more robust phosphorylation, linked to increased cytosolic translocation.
- MSH3 cytosolic localization was enhanced by acetylation of lysine residues (K99, K100, K103) within its nuclear localization signal (NLS).
- Immunoprecipitation revealed binding of cytosolic histone deacetylase 6 (HDAC6) to acetylated Δ27bpMSH3.
Conclusions:
- Polymorphic MSH3 undergoes IL-6-induced serine/tyrosine phosphorylation and NLS acetylation, facilitating its nuclear-to-cytosolic shift.
- The binding of MSH3 to HDAC6 in the cytosol may influence MSH3 deacetylation and protein stability.
- These post-translational modifications offer potential targets for regulating MSH3's intracellular localization and function.
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