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.