{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,2]],"date-time":"2026-07-02T09:44:25Z","timestamp":1782985465385,"version":"3.54.5"},"reference-count":52,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2020,6,20]],"date-time":"2020-06-20T00:00:00Z","timestamp":1592611200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001824","name":"Grantov\u00e1 Agentura \u010cesk\u00e9 Republiky","doi-asserted-by":"publisher","award":["17-04742S"],"award-info":[{"award-number":["17-04742S"]}],"id":[{"id":"10.13039\/501100001824","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>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 HIF1\u03b1 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.<\/jats:p>","DOI":"10.3390\/cells9061506","type":"journal-article","created":{"date-parts":[[2020,6,24]],"date-time":"2020-06-24T07:14:19Z","timestamp":1592982859000},"page":"1506","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5305-4454","authenticated-orcid":false,"given":"Cecilia","family":"Aquino Perez","sequence":"first","affiliation":[{"name":"Cancer Cell Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, CZ14220 Prague, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matous","family":"Palek","sequence":"additional","affiliation":[{"name":"Cancer Cell Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, CZ14220 Prague, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0047-1502","authenticated-orcid":false,"given":"Lenka","family":"Stolarova","sequence":"additional","affiliation":[{"name":"Cancer Cell Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, CZ14220 Prague, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0614-6292","authenticated-orcid":false,"given":"Patrick","family":"von Morgen","sequence":"additional","affiliation":[{"name":"Cancer Cell Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, CZ14220 Prague, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0987-1238","authenticated-orcid":false,"given":"Libor","family":"Macurek","sequence":"additional","affiliation":[{"name":"Cancer Cell Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, CZ14220 Prague, Czech Republic"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,6,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1038\/nrm3819","article-title":"Polo-like kinases: Structural variations lead to multiple functions","volume":"15","author":"Zitouni","year":"2014","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/S0092-8674(03)00725-6","article-title":"The Molecular Basis for Phosphodependent Substrate Targeting and Regulation of Plks by the Polo-Box Domain","volume":"115","author":"Elia","year":"2003","journal-title":"Cell"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"825","DOI":"10.1038\/nrc2964","article-title":"Shared and separate functions of polo-like kinases and aurora kinases in cancer","volume":"10","author":"Lens","year":"2010","journal-title":"Nat. Rev. Cancer"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"646","DOI":"10.1016\/j.devcel.2008.04.014","article-title":"Polo on the Rise-from Mitotic Entry to Cytokinesis with Plk1","volume":"14","author":"Petronczki","year":"2008","journal-title":"Dev. Cell"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1140","DOI":"10.1038\/ncb1320","article-title":"The Polo kinase Plk4 functions in centriole duplication","volume":"7","author":"Habedanck","year":"2005","journal-title":"Nat. Cell Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1225","DOI":"10.1128\/MCB.00607-10","article-title":"Plk5, a Polo Box Domain-Only Protein with Specific Roles in Neuron Differentiation and Glioblastoma Suppression","volume":"31","author":"Escobar","year":"2011","journal-title":"Mol. Cell. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"4832","DOI":"10.1038\/sj.onc.1203845","article-title":"Adhesion induced expression of the serine\/threonine kinase Fnk in human macrophages","volume":"19","author":"Holtrich","year":"2000","journal-title":"Oncogene"},{"key":"ref_8","first-page":"376","article-title":"The serum-inducible protein kinase Snk is a G1 phase polo-like kinase that is inhibited by the calcium- and integrin-binding protein CIB","volume":"1","author":"Ma","year":"2003","journal-title":"Mol. Cancer Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1847","DOI":"10.1073\/pnas.0610856104","article-title":"Polo-like kinase 3 is required for entry into S phase","volume":"104","author":"Zimmerman","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2658","DOI":"10.1038\/sj.onc.1207425","article-title":"Cdc25C phosphorylation on serine 191 by Plk3 promotes its nuclear translocation","volume":"23","author":"Bahassi","year":"2004","journal-title":"Oncogene"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.mrfmmm.2011.02.006","article-title":"Absence of polo-like kinase 3 in mice stabilizes Cdc25A after DNA damage but is not sufficient to produce tumors","volume":"714","author":"Myer","year":"2011","journal-title":"Mutat. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"6633","DOI":"10.1038\/sj.onc.1205850","article-title":"Mammalian Polo-like kinase 3 (Plk3) is a multifunctional protein involved in stress response pathways","volume":"21","author":"Bahassi","year":"2002","journal-title":"Oncogene"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1016\/j.mrfmmm.2005.12.002","article-title":"Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage","volume":"596","author":"Bahassi","year":"2006","journal-title":"Mutat. Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"43305","DOI":"10.1074\/jbc.M106050200","article-title":"Plk3 Functionally Links DNA Damage to Cell Cycle Arrest and Apoptosis at Least in Part via the p53 Pathway","volume":"276","author":"Xie","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"877","DOI":"10.1083\/jcb.201401146","article-title":"Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1","volume":"206","author":"Barton","year":"2014","journal-title":"J. Cell Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"25928","DOI":"10.1074\/jbc.M801326200","article-title":"Activation of Polo-like Kinase 3 by Hypoxic Stresses","volume":"283","author":"Wang","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"38944","DOI":"10.1074\/jbc.M110.160325","article-title":"Plk3 Functions as an Essential Component of the Hypoxia Regulatory Pathway by Direct Phosphorylation of HIF-1\u03b1","volume":"285","author":"Xu","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4077","DOI":"10.1158\/0008-5472.CAN-07-6182","article-title":"Polo-like Kinase 3 Functions as a Tumor Suppressor and Is a Negative Regulator of Hypoxia-Inducible Factor-1\u03b1 under Hypoxic Conditions","volume":"68","author":"Yang","year":"2008","journal-title":"Cancer Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"29827","DOI":"10.1074\/jbc.M114.597161","article-title":"Osmotic Stress-induced Phosphorylation of H2AX by Polo-like Kinase 3 Affects Cell Cycle Progression in Human Corneal Epithelial Cells","volume":"289","author":"Wang","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1951","DOI":"10.1074\/jbc.M110.166009","article-title":"Hyperosmotic Stress-induced ATF-2 Activation through Polo-like Kinase 3 in Human Corneal Epithelial Cells","volume":"286","author":"Wang","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1083\/jcb.200208099","article-title":"RAF1-activated MEK1 is found on the Golgi apparatus in late prophase and is required for Golgi complex fragmentation in mitosis","volume":"161","author":"Colanzi","year":"2003","journal-title":"J. Cell Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3822","DOI":"10.1038\/sj.onc.1207479","article-title":"MEK1-induced Golgi dynamics during cell cycle progression is partly mediated by Polo-like kinase-3","volume":"23","author":"Xie","year":"2004","journal-title":"Oncogene"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.yexcr.2003.10.022","article-title":"Polo-like kinase 3 is Golgi localized and involved in regulating Golgi fragmentation during the cell cycle","volume":"294","author":"Ruan","year":"2004","journal-title":"Exp. Cell Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1442","DOI":"10.1021\/bi7022877","article-title":"Protein Phosphatase 6 Regulatory Subunits Composed of Ankyrin Repeat Domains","volume":"47","author":"Stefansson","year":"2008","journal-title":"Biochemistry"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/j.bbamcr.2018.07.015","article-title":"The multiple functions of protein phosphatase 6","volume":"1866","author":"Ohama","year":"2019","journal-title":"Biochimi. Biophys. Acta (BBA) Mol. Cell Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"rs12","DOI":"10.1126\/scisignal.aab3138","article-title":"Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells","volume":"8","author":"Rusin","year":"2015","journal-title":"Sci. Signal."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1315","DOI":"10.1083\/jcb.201008106","article-title":"Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2","volume":"191","author":"Zeng","year":"2010","journal-title":"J. Cell Biol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Mi, J., Dziegielewski, J., Bolesta, E., Brautigan, D.L., and Larner, J.M. (2009). Activation of DNA-PK by Ionizing Radiation Is Mediated by Protein Phosphatase 6. PLoS ONE, 4.","DOI":"10.1371\/journal.pone.0004395"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e241","DOI":"10.1038\/cddis.2011.126","article-title":"Serine\/threonine protein phosphatase 6 modulates the radiation sensitivity of glioblastoma","volume":"2","author":"Shen","year":"2011","journal-title":"Cell Death Dis."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4647","DOI":"10.1038\/onc.2014.398","article-title":"Abrogation of protein phosphatase 6 promotes skin carcinogenesis induced by DMBA","volume":"34","author":"Hayashi","year":"2015","journal-title":"Oncogene"},{"key":"ref_31","first-page":"3429","article-title":"Melanoma-associated mutations in protein phosphatase 6 cause chromosome instability and DNA damage owing to dysregulated Aurora-A","volume":"126","author":"Hammond","year":"2013","journal-title":"J. Cell Sci."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Burdova, K., Storchova, R., Palek, M., and Macurek, L. (2019). WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors. Cells, 8.","DOI":"10.3390\/cells8101258"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"8795","DOI":"10.1074\/jbc.M211392200","article-title":"NFBD1\/KIAA0170 Is a Chromatin-associated Protein Involved in DNA Damage Signaling Pathways","volume":"278","author":"Xu","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2281","DOI":"10.1038\/onc.2009.501","article-title":"Wip1 phosphatase is associated with chromatin and dephosphorylates \u03b3H2AX to promote checkpoint inhibition","volume":"29","author":"Lindqvist","year":"2010","journal-title":"Oncogene"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"678","DOI":"10.4161\/cc.3.5.865","article-title":"Murine FGF-inducible kinase is rapidly degraded via the nuclear ubiquitin-proteosome system when overexpressed in NIH 3T3 cells","volume":"3","author":"Alberts","year":"2004","journal-title":"Cell Cycle"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"914","DOI":"10.1038\/cr.2016.78","article-title":"Ligand stimulation of CD95 induces activation of Plk3 followed by phosphorylation of caspase-8","volume":"26","author":"Helmke","year":"2016","journal-title":"Cell Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"10577","DOI":"10.1074\/jbc.M513156200","article-title":"Polo Box Domain of Plk3 Functions as a Centrosome Localization Signal, Overexpression of Which Causes Mitotic Arrest, Cytokinesis Defects, and Apoptosis","volume":"281","author":"Jiang","year":"2006","journal-title":"J. Biolo. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"32121","DOI":"10.1074\/jbc.M702791200","article-title":"Stress-induced c-Jun Activation Mediated by Polo-like Kinase 3 in Corneal Epithelial Cells","volume":"282","author":"Wang","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/ncb1446","article-title":"Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway","volume":"8","author":"Ziv","year":"2006","journal-title":"Nat. Cell Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1782","DOI":"10.1002\/ijc.32385","article-title":"Identification of deleterious germline CHEK2 mutations and their association with breast and ovarian cancer","volume":"145","author":"Kleiblova","year":"2019","journal-title":"Int. J. Cancer"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"41656","DOI":"10.1074\/jbc.M101831200","article-title":"Inhibition of Polo-like Kinase-1 by DNA Damage Occurs in an ATM- or ATR-dependent Fashion","volume":"276","author":"Smits","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"eaaq1441","DOI":"10.1126\/scisignal.aaq1441","article-title":"Global assessment of its network dynamics reveals that the kinase Plk1 inhibits the phosphatase PP6 to promote Aurora A activity","volume":"11","author":"Kettenbach","year":"2018","journal-title":"Sci. Signal."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1038\/nature07185","article-title":"Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery","volume":"455","author":"Macurek","year":"2008","journal-title":"Nature"},{"key":"ref_44","first-page":"801","article-title":"Bora and Aurora-A continue to activate Plk1 in mitosis","volume":"127","author":"Bruinsma","year":"2014","journal-title":"J. Cell Sci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1007\/s00412-008-0165-5","article-title":"Plk1 regulates mitotic Aurora A function through \u03b2TrCP-dependent degradation of hBora","volume":"117","author":"Chan","year":"2008","journal-title":"Chromosom. Biol. Nucl."},{"key":"ref_46","first-page":"23","article-title":"Small-molecule inhibitors of ser\/thr protein phosphatases: Specificity, use and common forms of abuse","volume":"365","author":"Swingle","year":"2007","journal-title":"Methods Mol. Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"2809","DOI":"10.1016\/j.celrep.2018.02.045","article-title":"A PP6-ASK3 Module Coordinates the Bidirectional Cell Volume Regulation under Osmotic Stress","volume":"22","author":"Watanabe","year":"2018","journal-title":"Cell Rep."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2727","DOI":"10.1091\/mbc.e13-02-0114","article-title":"Sit4p\/PP6 Regulates ER-to-Golgi Traffic by Controlling the Dephosphorylation of COPII Coat Subunits","volume":"24","author":"Bhandari","year":"2013","journal-title":"Mol. Biol. Cell"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Ohama, T., Wang, L., Griner, E., and Brautigan, D. (2013). Protein Ser\/Thr phosphatase-6 is required for maintenance of E-cadherin at adherens junctions. BMC Cell Biol., 14.","DOI":"10.1186\/1471-2121-14-42"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1517","DOI":"10.2147\/CMAR.S176762","article-title":"Polo-like kinase 3 is associated with poor prognosis and regulates proliferation and metastasis in prostate cancer","volume":"11","author":"Lin","year":"2019","journal-title":"Cancer Manag. Res."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1002\/mc.23123","article-title":"The role of polo-like kinase 3 in the response of BRAF-mutant cells to targeted anticancer therapies","volume":"59","author":"Babagana","year":"2020","journal-title":"Mol. Carcinog."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"426","DOI":"10.1186\/s13046-019-1418-2","article-title":"Polo-like kinase 3 inhibits glucose metabolism in colorectal cancer by targeting HSP90\/STAT3\/HK2 signaling","volume":"38","author":"Ou","year":"2019","journal-title":"J. Exp. Clin. Cancer Res. CR"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/9\/6\/1506\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:41:13Z","timestamp":1760175673000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/9\/6\/1506"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,6,20]]},"references-count":52,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2020,6]]}},"alternative-id":["cells9061506"],"URL":"https:\/\/doi.org\/10.3390\/cells9061506","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,6,20]]}}}