Phosphorylation of PLK3 Is Controlled by Protein Phosphatase 6

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.

CHEK2 Germline Variants in Cancer Predisposition: Stalemate Rather than Checkmate

“…The phosphorylation of CHK2 T68 has been frequently used as a marker of ATM activation [ 46 ], but T68 could be phosphorylated also by ATR in vitro [ 37 ] and by the DNA-dependent protein kinase catalytic subunit (PRKDC, alias DNA-PKcs) during mitosis [ 47 ]. Other CHK2 phosphorylation sites have been described as the targets of other kinases including polo-like kinase 3 (PLK3) [ 48 , 49 ] or PLK1 [ 50 , 51 ]. PLK3 phosphorylated S62 and S73 in vitro and was proposed to facilitate subsequent phosphorylation on T68 by ATM; however, this possibility has recently been challenged when no impact of PLK3 on checkpoint activation was found [ 48 ].…”

“…Other CHK2 phosphorylation sites have been described as the targets of other kinases including polo-like kinase 3 (PLK3) [ 48 , 49 ] or PLK1 [ 50 , 51 ]. PLK3 phosphorylated S62 and S73 in vitro and was proposed to facilitate subsequent phosphorylation on T68 by ATM; however, this possibility has recently been challenged when no impact of PLK3 on checkpoint activation was found [ 48 ]. PLK1 in a complex with TP53-binding protein 1 (53BP1) phosphorylates CHK2 on S164, T205, and S210 to prevent its activation in mitosis, with S164 phosphorylation showing the greatest effect.…”

Modelling the Functions of Polo-Like Kinases in Mice and Their Applications as Cancer Targets with a Special Focus on Ovarian Cancer

“…The protein is very stable (reviewed by [ 26 ]). PLK3 is enriched at the plasmalemma and at Golgi membranes [ 71 , 72 ], reviewed by [ 73 ], possibly involved in the modulation of cellular adhesion and in intracellular trafficking [ 71 ]. Importantly, PLK3 participates in the Fas ligand-induced pathway of apoptosis [ 74 ], reviewed by [ 72 ].…”

“…PLK3 is enriched at the plasmalemma and at Golgi membranes [ 71 , 72 ], reviewed by [ 73 ], possibly involved in the modulation of cellular adhesion and in intracellular trafficking [ 71 ]. Importantly, PLK3 participates in the Fas ligand-induced pathway of apoptosis [ 74 ], reviewed by [ 72 ]. Supposed roles for the progression of the cell cycle, namely entry into S phase are questioned [ 69 , 75 ] whereas PLK3 has been shown to participate in G2/M transition through interaction with cell division cycle Cdc25C protein phosphatase [ 76 ].…”

“…Supposed roles for the progression of the cell cycle, namely entry into S phase are questioned [ 69 , 75 ] whereas PLK3 has been shown to participate in G2/M transition through interaction with cell division cycle Cdc25C protein phosphatase [ 76 ]. PLK3 has been reported to function in the mediation of cellular response including cell cycle checkpoint activation [ 77 , 78 ] after various forms of stress including hypoxic, DNA damage and osmotic stress [ 79 , 80 ], for reviews see [ 47 , 72 ]. Results from Plk3 -knockout and its inactivation, however, argue against significant roles in cellular stress response [ 73 ].…”