A central role for CK1 in catalyzing phosphorylation of the p53 transactivation domain at serine 20 after HHV-6B viral infection
Authors: Bettina Bundgaard
Ted R Hupp
Nicola J MacLaine
David W Meek
Pedro A Lazo
Per Höllsberg
Bodil Oster
Carolyn Buckner
Jennifer A Fraser
Journal: Journal of Biological Chemistry (2008)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: p53; Ser20; kinase; CK1; virus-induced phosphorylation
Affiliations: University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR, United Kingdom.

Abstract:  The tumor suppressor protein p53 is activated by distinct cellular stresses including radiation, hypoxia, type I interferon, and DNA/RNA virus infection. The transactivation domain of p53 contains a phosphorylation site at Ser20 whose modification stabilizes the binding of the transcriptional co-activator p300 and whose mutation in murine transgenics induces B-cell lymphoma. Although the checkpoint kinase CHK2 is implicated in promoting Ser20 site phosphorylation after irradiation, the enzyme that triggers this phosphorylation after DNA viral infection is undefined. Using human herpesvirus 6B (HHV-6B) as a virus that induces Ser20 site phosphorylation of p53 in T-cells, we sought to identify the kinase responsible for this virus-induced p53 modification. The p53 Ser20 kinase was fractionated and purified using cation, anion, and dye-ligand exchange chromatography. Mass spectrometry identified casein kinase 1 (CK1) and vaccinia-related kinase 1 (VRK1) as enzymes that coeluted with virus-induced Ser20 site kinase activity. Immunodepletion of CK1 but not VRK1 removed the kinase activity from the peak fraction, and bacterially expressed CK1 exhibited Ser20 site kinase activity equivalent to that of the virus-induced native CK1. CK1 modified p53 in a docking-dependent manner, which is similar to other known Ser20 site p53 kinases. Low levels of the CK1 inhibitor D4476 selectively inhibited HHV-6B-induced Ser20 site phosphorylation of p53. However, x-ray-induced Ser20 site phosphorylation of p53 was not blocked by D4476. These data highlight a central role for CK1 as the Ser20 site kinase for p53 in DNA virus-infected cells but also suggest that distinct stresses may selectively trigger different protein kinases to modify the transactivation domain of p53 at Ser20. 

Retracted: No

PubPeer comments:
 Figure 6. Much more similar than expected. ![file] ![file] , 
 The authors agree that this data look very similar. The original blots are no longer available for resolving this issue so the original data from the paper was downloaded for fine analysis. The aim of this figure 6 was to measure the elution of three different protein kinases from a column. All three of the different protein kinases elute in: fraction 29 for ck1 (Fig 6A), fraction 29 for ck1alpha (Fig 6b), and fractions 29-33 for VRK1 (Fig 6C). There would be no kinases present in fractions 45-97 (this is the second panel of blots which has the apparently duplicated images) so that these latter data are not necessary to support the conclusion of the figure: that VRK1 elutes differently than CK1 or ck1alpha. Neverthless, we think these blots are not duplicated images. These blots most likely represent an overlay blot. After immunoblotting with the antibody in figure 6A, because there were no bands, the same blot was then likely washed and then overlayed with the antibody used in Fig 6B. It is known that overlay blots carry u201cshadowsu2019 from one exposure to the next exposure after the next antibody overlay. However, the background features in these two appear different in different regions of the blots (highlighted attached) which is more consistent with another exposure of the same blot to X-ray film rather than the image being just being identical. ![image] ,