Protein kinase CK2 catalyzes tyrosine phosphorylation in mammalian cells Greg Vilk a , Jane E. Weber a , Jacob P. Turowec a , James S. Duncan a , Chenggang Wu a , D. Richard Derksen a , Piotr Zien a , Stefania Sarno b , Arianna Donella-Deana b , Gilles Lajoie a , Lorenzo A. Pinna b , Shawn S.C. Li a , David W. Litchfield a, ⁎ a Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1 b Venetian Institute for Molecular Medicine and Department of Biological Chemistry, University of Padova, Padova, Italy ABSTRACT ARTICLE INFO Article history: Received 24 June 2008 Accepted 3 July 2008 Available online 6 July 2008 Keywords: Protein kinase CK2 Tyrosine phosphorylation Dual-specificity kinase Peptide arrays Protein kinase Protein kinase CK2 exhibits oncogenic activity in mice and is over-expressed in a number of tumors or leukemic cells. On the basis of its amino acid sequence and a wealth of experimental information, CK2 has traditionally been classified as a protein serine/threonine kinase. In contrast to this traditional view of CK2, recent evidence has shown that CK2 can also phosphorylate tyrosine residues under some circumstances in vitro and in yeast. In this study, we provide definitive evidence demonstrating that CK2 also exhibits tyrosine kinase activity in mammalian cells. Tyrosine phosphorylation of CK2 in cells and in CK2 immunoprecipitates is dependent on CK2 activity and is inhibited by the CK2 selective inhibitor 4,5,6,7-tetrabromobenzotriazole. Examination of phosphotyrosine profiles in cells reveals a number of proteins, including CK2 itself, which exhibit increased tyrosine phosphorylation when CK2 levels are increased. Peptide arrays to evaluate the specificity determinants for tyrosine phosphorylation by CK2 reveal that its specificity for tyrosine phosphorylation is distinct from its specificity for serine/threonine phosphorylation. Of particular note is the requirement for an aspartic acid immediately C-terminal to the phosphorylatable tyrosine residue. Collectively, these data provide conclusive evidence that CK2 catalyzes the phosphorylation of tyrosine residues in mammalian cells, a finding that adds a new level of complexity to the challenge of elucidating its cellular functions. Furthermore, these results raise the possibility that increased CK2 levels that frequently accompany transformation may contribute to the increased tyrosine phosphorylation that occurs in transformed cells. © 2008 Elsevier Inc. All rights reserved. 1. Introduction Protein kinase CK2 is a ubiquitously expressed protein kinase that is essential for viability and has apparent functions associated with pathways involved in the control of cell proliferation and survival [1–9]. CK2 has also been shown to exhibit oncogenic activity in transgenic mice and to promote transformation in cultured mam- malian cells [10–14]. In this regard, it is intriguing that the list of likely physiological targets of CK2 contains a myriad of regula- tory proteins including proto-oncogene products such as c-Myc [15], c-Myb [15,16], c-Jun [17], Tal-1 [18] and tumor suppressor gene products such as p53 [19], BRCA-1 [20] and PML [21]. However, a detailed mechanistic understanding of how CK2 participates in transformation and tumorigenesis remains far from complete [22]. Furthermore, since systematic studies to identify its cellular substrates have been hindered by its high constitutive activity and enigmatic mode of intracellular modulation, it is certain that many CK2 targets remain to be identified. CK2 has traditionally been viewed as a protein serine/threonine kinase with a preference for substrates with serine/threonine residues embedded within a stretch of acidic amino acids that conform to a rather well defined substrate consensus [1,7]. On the basis of numerous studies primarily performed with synthetic peptides, this consensus has been defined as S/T-X-X-D/E/pS/pY where X can be any amino acid except proline. While the delineation of this consensus has been invaluable in the identification of many potential physiological CK2 substrates, it is evident from the examination of the sites within proteins that are phosphorylated by CK2 that there are CK2 phosphorylation sites that do not conform to this canonical CK2 consensus. For example, one of the DNA damage-inducible phosphor- ylation sites on p53, S392 in human p53, is efficiently phosphorylated by CK2 although it does not match the CK2 recognition consensus [19]. In addition, although CK2 has traditionally been viewed as a serine/ threonine kinase, studies in yeast have raised the specter that CK2 can also phosphorylate tyrosine residues. Notably, in S. cerevisiae, tyrosine phosphorylation of the immunophilin Fpr3 is dependent upon CK2 Cellular Signalling 20 (2008) 1942–1951 ⁎ Corresponding author. Fax: +519 661 3175. E-mail address: litchfi@uwo.ca (D.W. Litchfield). 0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.07.002 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig