Human Vaccinia-related Kinase 1 (VRK1) Activates the ATF2 Transcriptional Activity by Novel Phosphorylation on Thr-73 and Ser-62 and Cooperates with JNK

Ana Sevilla,Claudio R Santos,Francisco M Vega,Pedro A Lazo

doi:10.1074/jbc.m401009200

Abstract

In the human kinome, vaccinia-related kinase-1 (VRK1) is a new Ser-Thr kinase associated with proliferating tissues. VRK1 colocalizes with ATF2 in the nucleus and can form a stable complex. We have studied the phosphorylation of the transcription factor ATF2, which regulates gene expression by forming dimers with proteins with basic region-leucine zipper domains and recognizing cAMP-response element or AP1 sequences implicated in cellular responses to stress. VRK1 phosphorylates ATF2 mainly on Thr-73, stabilizing the ATF2 protein and increasing its intracellular level. Mutagenesis studies showed that Thr-73 and Ser-62 are implicated in ATF2 transcriptional activation by VRK1 detected in a functional assay based on ATF2 dimerization. VRK1 can activate the collagenase gene promoter that is regulated by ATF2 in a dose-dependent manner. Loss of kinase activity (K179E mutant) or the T73A substitution in ATF2 prevents both its accumulation and activation of transcription. VRK1 and JNK, which phosphorylates ATF2 in Thr-69 and Thr-71, have an additive effect on ATF2-dependent transcription at suboptimal doses. Therefore, two groups of amino acids in the ATF2 amino-terminal region can integrate different cellular signals mediated by at least five different kinases. VRK1 is an element of a novel signaling pathway that regulates gene transcription.

Highlights

Phosphorylation of transcription factors is a major regulatory mechanism of gene expression in the cellular response to many types of stress [1]
We have studied the phosphorylation of the transcription factor ATF2, which regulates gene expression by forming dimers with proteins with basic region-leucine zipper domains and recognizing cAMP-response element or AP1 sequences implicated in cellular responses to stress
ATF2 and vaccinia-related kinase-1 (VRK1) Colocalize in the Nucleus—The phosphorylation by the human VRK1 protein of p53 [37] suggested that it might phosphorylate other transcription factors that participate in the cellular response to stress mediated by other kinases, such as Jun NH2terminal kinase (JNK) and p38

Summary

Introduction

Phosphorylation of transcription factors is a major regulatory mechanism of gene expression in the cellular response to many types of stress [1]. The assay is based on the use of pSG-424-ATF2 [1–109] constructs with a GAL4 DNA binding domain, or its mutants and a p5ϫGAL4-Luc reporter were cotransfected in each sample in combination with phRL-tk Renilla luciferase as an internal control for transfection efficiency. ATF2 and VRK1 Colocalize in the Nucleus—The phosphorylation by the human VRK1 protein of p53 [37] suggested that it might phosphorylate other transcription factors that participate in the cellular response to stress mediated by other kinases, such as JNK and p38.

Results

Conclusion