The upstream stimulatory factor USF1 is regulated by protein kinase CK2 phosphorylation Sarah Lupp a , Claudia Götz a, ⁎, Sunia Khadouma a , Tina Horbach b , Elitsa Y. Dimova b , Anna-Maria Bohrer a , Thomas Kietzmann b , Mathias Montenarh a a Medical Biochemistry and Molecular Biology, Saarland University, Homburg, Germany b Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, Oulu, Finland abstract article info Article history: Received 7 August 2014 Accepted 29 August 2014 Available online 4 September 2014 Keywords: Protein kinase CK2 Phosphorylation Hetero-dimerization Transcription factor Transactivation The upstream stimulatory factors 1 (USF1) and 2 (USF2) are transcription factors which bind to E-box motifs of various promoters regulating a variety of different cellular processes. Only little is known about the regulation of USFs. Here, we identified protein kinase CK2 as an enzyme that phosphorylates USF1 but not USF2. Using deletion mutants and point mutants we were able to identify threonine 100 as the major phosphorylation site for CK2. It is well known that USF1 and USF2 form hetero-dimers. Binding studies revealed that the inhibition of CK2 kinase activity by a specific inhibitor enhanced binding of USF1 to USF2. Furthermore, transactivation studies showed that the inhibition of CK2 phosphorylation of USF1 stimulated transcription from the glucokinase promoter as well as the fatty acid synthetase promoter but not from the heme oxygenase-1 promoter. Thus, we have shown for the first time that CK2 phosphorylation of USF1 modulates two functionally important properties of USF1, namely hetero-dimerization and transactivation. © 2014 Elsevier Inc. All rights reserved. 1. Introduction The upstream stimulatory factors 1 (USF1) and 2 (USF2) participate in a large number of gene regulating networks affecting stress reactions, immune responses, the cell cycle, proliferation and metabolism [1]. USF1 and USF2 are encoded by different genes. They are ubiquitously expressed although the abundance of the USF proteins varies between different cell types [2,3]. The USF1 gene codes for a 43 kDa protein whereas USF2 codes for a 44 kDa protein. Both, USF1 and USF2 were first purified from HeLa cells [4,5]. In vivo the proteins exist mainly as USF1/USF2 hetero-dimers [2]. Experiments with USF knock-out mice revealed an asymmetrical regulation of the expression of the two isotypes, i.e. USF1 -/- mice displayed an enhanced USF2 expression, whereas USF2 -/- mice express less USF1 protein compared to wild- type mice [6]. Like many other transcription factors, USF1 is a phospho- protein [7] where the degree of phosphorylation seems to vary between primary cells and cells in tissue culture. USF2 is also a phosphoprotein and in particular protein kinase A (PKA) and glycogen synthase kinase-3 (GSK3) are known to phosphorylate USF2 and to modulate its DNA binding activity [8]. The cellular processes regulated by USFs, like cell cycle, proliferation, carbohydrate metabolism, and embryonic development are also strongly regulated by protein kinase CK2 [9–13]. CK2 is a tetrameric enzyme consisting of two catalytic α- and α′-subunits and two non-catalytic β- subunits. Interestingly, the majority of CK2 targets are proteins involved in signalling, protein synthesis and transcriptional regulation [14–16]. According to the fact that we previously identified several transcription factors as interaction partners or as substrates for CK2, and taking into account known and overlapping functions between CK2 and USF proteins, we asked whether there is a direct or indirect functional link between USF proteins and CK2. In the present study, we demonstrate that in contrast to USF2, USF1 is phosphorylated by CK2 at threonine 100. As a consequence, this phosphorylation influences the interaction between USF1 and USF2 as well as the transcription factor activity of USF1. 2. Materials and methods 2.1. Chemicals and biological reagents Tissue culture media were purchased from GIBCO. The foetal calf serum (FCS) was from PAA (Pasching, Austria) and [ 32 P]γATP and [ 32 P] orthophosphate were purchased from Hartmann Analytic (Braunschweig, Germany). The CK2 specific inhibitor CX-4945 was purchased from Selleckchem (Munich, Germany). CX-4945, 4,5,6,7- tetrabromobenzotriazole (TBB, VWR, Darmstadt, Germany) [17] and quinalizarin (Labotest OHG, Niederschöna, Germany) [18] were dissolved in dimethyl sulfoxide (DMSO) to 10 mM stock solutions. Cellular Signalling 26 (2014) 2809–2817 ⁎ Corresponding author at: Medical Biochemistry and Molecular Biology, Saarland University, Building 44, D-66424 Homburg, Germany. Tel.: +49 6841 1626502; fax: +49 6841 1626027. E-mail address: claudia.goetz@uks.eu (C. Götz). http://dx.doi.org/10.1016/j.cellsig.2014.08.028 0898-6568/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig