title: A central role for CK1 in catalyzing phosphorylation of the p53 transactivation domain at serine 20 after HHV-6B viral infection.
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.
authors.affiliation: University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR, United Kingdom.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: MacLaine
authors.forename: Bodil
authors.initials: B
authors.lastname: Oster
authors.forename: Bettina
authors.initials: B
authors.lastname: Bundgaard
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Fraser
authors.forename: Carolyn
authors.initials: C
authors.lastname: Buckner
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Lazo
authors.forename: David W
authors.initials: DW
authors.lastname: Meek
authors.forename: Per
authors.initials: P
authors.lastname: Höllsberg
authors.forename: Ted R
authors.initials: TR
authors.lastname: Hupp
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000494:pharmacology; D047389:Casein Kinase I / Q000737:chemistry / Q000502:physiology*; D002384:Catalysis; D045744:Cell Line, Tumor; D015654:Herpesvirus 6, Human / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D019349:Roseolovirus Infections / Q000821:virology*; D012694:Serine / Q000737:chemistry*; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*
chemical_list: D000998:Antiviral Agents; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D012694:Serine; D047389:Casein Kinase I