A Central Role for CK1 in Catalyzing Phosphorylation of the p53 Transactivation Domain at Serine 20 after HHV-6B Viral Infection

Nicola J Maclaine,Bodil Øster,Bettina Bundgaard,Jennifer A Fraser,Carolyn Buckner,Pedro A Lazo,David W Meek,Per Höllsberg,Ted R Hupp

doi:10.1074/jbc.m804433200

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.

Highlights

The generation of transgenic mice with phosphoacceptor site mutations at the key regulatory phosphoacceptor sites of Ser20 and Ser392 equivalents in murine p53 results in elevated cancer incidence
In order to identify the p53 Ser20 kinase, we subjected crude lysates to chromatographic fractionation to determine whether the kinase activity correlates with the known Ser20 site kinases, including CHK1/2 and death-associated protein kinase 1 (DAPK-1)
human herpesvirus 6B (HHV-6B)-infected and mock-infected MOLT-3 whole-cell lysates were applied onto a Q-Sepharose Fast Flow anion exchanger column, proteins were eluted in a linear gradient of 0 –1 M KCl, and fractions were collected and assayed for kinase activity toward the Ser20 and Ser15 sites of FLp53 (Fig. 2)

Summary

Introduction

The generation of transgenic mice with phosphoacceptor site mutations (to alanine) at the key regulatory phosphoacceptor sites of Ser and Ser392 equivalents in murine p53 results in elevated cancer incidence. Mutation of Ser results in enhanced spontaneous B-cell lymphoma and attenuated damage-induced apoptosis in B-cells [7], whereas mutation of Ser392 results in enhanced UV-induced skin cancer or carcinogen-induced bladder cancer [8, 9] These biochemical and genetic results highlight the critical role that phosphorylation of p53 can play in modulating its tumor suppressor function and the likelihood that these phosphorylation events are “stress-” and/or cell type-specific. We show that the enzyme responsible for this virus-induced phosphorylation of p53 is the DNA damage-regulatory enzyme CK1 These data suggest a dominant role for CK1 in p53 activation after HHV-6B virus infection and highlight a specific kinase pathway for further integration into the antiviral sensing machinery of the cell

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Conclusion