Abstract
Cells are constantly exposed to diverse factors that cause DNA damage, which may lead to development of genome instability and tumor development. To avoid these deleterious consequences after DNA damage, cells developed the DNA damage response signalling pathway (DDR).
Wip1 phosphatase (also known as PPM1D) plays very important role in terminating this pathway predominantly through p53 dephosphorylation. Apart from p53 Wip1 was reported to dephosphorylate also other targets including KAP1, histone H2AX and p38.
In this study we describe a novel method to detect targets of phosphatases using nuclear extracts and recombinantly expressed and purified proteins, which allows better simulation of the situation in the cell than in vitro phosphatase assays with phosphopeptides. Using nuclear extracts we aimed to validate putative targets of Wip1 phosphatase and possibly identify novel potential targets.
We confirmed some of the previously described targets. On the other hand, we provide evidence that p38 is not a physiological substrate of Wip1 phosphatase, despite previous reports.
We confirmed that deletion of Wip1 using CRISPR/Cas9 or inhibition of Wip1 by a small molecule inhibitor GSK 2830371 did not affect the activation of p38/MK2 pathway after various forms of stress. We also identified novel potential substrates of Wip1 phosphatase such as DBC1 (deleted in breast cancer 1) and MRE11 (meiotic recombination 11).
DBC1 is negatively regulating SIRT1 deacetylase which among others deacetylates p53. Dephosphorylation of DBC1 by Wip1 might therefore affect p53 activity through SIRT1.
DBC1 also inhibits some other enzymes like HDAC3 histone deacetylase and Suv39H1 histone methyltransferase, which plays important role in heterochromatin formation. We will discuss plausible functions of new Wip1 targets in double-stranded break repair (DSB) and chromatin organization.