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Updated: Dec 17, 2025
A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors
Published on: April 29, 2022
Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6
Cecilia Aquino Perez1, Matous Palek1, Lenka Stolarova1
1Cancer Cell Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, CZ14220 Prague, Czech Republic.
Abstract:
Polo-like kinases play essential roles in cell cycle control and mitosis. In contrast to other members of this kinase family, PLK3 has been reported to be activated upon cellular stress including DNA damage, hypoxia and osmotic stress. Here we knocked out PLK3 in human non-transformed RPE cells using CRISPR/Cas9-mediated gene editing. Surprisingly, we find that loss of PLK3 does not impair stabilization of HIF1α after hypoxia, phosphorylation of the c-Jun after osmotic stress and dynamics of DNA damage response after exposure to ionizing radiation. Similarly, RNAi-mediated depletion of PLK3 did not impair stress response in human transformed cell lines. Exposure of cells to various forms of stress also did not affect kinase activity of purified EGFP-PLK3. We conclude that PLK3 is largely dispensable for stress response in human cells. Using mass spectrometry, we identify protein phosphatase 6 as a new interacting partner of PLK3. Polo box domain of PLK3 mediates the interaction with the PP6 complex. Finally, we find that PLK3 is phosphorylated at Thr219 in the T-loop and that PP6 constantly dephosphorylates this residue. However, in contrast to PLK1, phosphorylation of Thr219 does not upregulate enzymatic activity of PLK3, suggesting that activation of both kinases is regulated by distinct mechanisms.
Insights
Polo-like kinase 3 (PLK3) is not essential for human cell stress responses like hypoxia or DNA damage. New research shows PLK3 interacts with protein phosphatase 6, but its phosphorylation does not affect kinase activity.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Polo-like kinases (PLKs) are crucial for cell cycle regulation and mitosis.
- PLK3 is distinct from other PLKs, reportedly activated by cellular stress (DNA damage, hypoxia, osmotic stress).
Purpose of the Study:
- To investigate the role of PLK3 in cellular stress responses.
- To identify PLK3 interacting partners and understand its regulation.
Main Methods:
- CRISPR/Cas9 gene editing to knockout PLK3 in RPE cells.
- RNA interference (RNAi) to deplete PLK3 in transformed cell lines.
- Mass spectrometry to identify interacting proteins.
- Biochemical assays to assess kinase activity and phosphorylation.
Main Results:
- Loss of PLK3 did not impair hypoxia-induced HIF1α stabilization, osmotic stress-induced c-Jun phosphorylation, or DNA damage response dynamics.
- PLK3 kinase activity was unaffected by various cellular stresses.
- Protein phosphatase 6 (PP6) was identified as a PLK3 interacting partner via the PLK3 polo box domain.
- PP6 dephosphorylates PLK3 at Thr219, but this phosphorylation does not upregulate PLK3 activity, unlike PLK1.
Conclusions:
- PLK3 is largely dispensable for stress response in human cells.
- PLK3 interacts with PP6, and its T-loop phosphorylation is regulated by PP6 but does not control kinase activity.
- PLK3 and PLK1 activation mechanisms differ significantly.
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