Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6.

Cecilia Aquino Perez,Matous Palek,Libor Macurek,Lenka Stolarova,Patrick von Morgen

doi:10.3390/cells9061506

Cecilia Aquino Perez, Matous Palek + Show 3 more

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https://doi.org/10.3390/cells9061506

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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.

Highlights

Polo-like kinases (Plks) are evolutionary conserved Ser/Thr protein kinases that play critical roles in progression through the cell cycle and mitosis [1]
We find that to PLK1, PLK3 is phosphorylated in the T-loop and that inhibition of protein phosphatases increases the level of PLK3 modification
Two independent clones of RPE-PLK3-KO showed no differences in stabilization of HIF1 in hypoxia conditions, in phosphorylation of c-Jun after exposure of cells to UV irradiation and in activation of DNA damage response after exposure of cells to ionizing radiation

Summary

Introduction

Polo-like kinases (Plks) are evolutionary conserved Ser/Thr protein kinases that play critical roles in progression through the cell cycle and mitosis [1]. Polo-like kinase family comprises of five members including PLK1 that is essential for formation of a bipolar mitotic spindle and for cytokinesis [3,4]; PLK2 and PLK4 that are involved in centriole biogenesis and duplication [5]; and PLK5 that lacks the kinase activity and plays a structural role in neurons [6]. Out of the Plks, the function of PLK3 is the least explored. The function of PLK3 in the cell cycle is likely not essential as PLK3 knock-out mice are viable and fertile [11]

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