91 Molecular and Cellular Biochemistry 227: 91–98, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. Identification and characterization of proteins that interact with Drosophila melanogaster protein kinase CK2 Regina L. Trott, 1 Madhavi Kalive, 1 Umesh Karandikar, 1 Rebecca Rummer, 1 Clifton P. Bishop 1 and Ashok P. Bidwai 1,2 1 Department of Biology and 2 Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV, USA Abstract D. melanogaster CK2 (DmCK2) is a highly conserved protein kinase that is composed of catalytic, α, and regulatory, β, subunits associated as an α 2 β 2 heterotetramer. In order to analyze the functions of CK2 in this metazoan model, we have used the two hybrid approach to identify interacting proteins. One of these cDNAs, DmA24, encodes a novel polypeptide with no homologs in GenBank, and is notable in that it contains a bipartite nuclear localization signal and two sites for phosphorylation by CK2. In situ hybridization to polytene chromosomes indicates that the DmA24 gene is located at the 61D interval of chromosome II a region that also harbors 3 additional genes with similar structure. DmA24p interacts with DmCK2α, but not with DmCK2β, demonstrating that this interaction is specific for the catalytic subunit of CK2. In addition, the protein is phosphorylated by the holoenzyme purified from Drosophila embryos. These studies identify DmA24p as a potentially new physiological partner of DmCK2. In addition, we also report the results of a large-scale screen that has identified a new set of DmCK2-interacting proteins. Most notable among these are Surf6, a nucleolar protein involved in RNA processing, and Spalt, a homeotic protein. (Mol Cell Biochem 227: 91–98, 2001) Key words: protein kinase, CK2, Drosophila, two hybrid Introduction CK2 is an ubiquitous protein kinase that is highly conserved among eukaryotes [1, 2], and is capable of functioning as an oncogene [3, 4]. In general, CK2 is composed of catalytic (α) and regulatory (β) subunits that combine to form an α 2 β 2 holoenzyme, and distinct isoforms of both subunits have been described in a wide variety of organisms. CK2 is unique in its site selectivity in that it preferentially phosphorylates Ser/ Thr residues that are followed by a stretch of acidic residues [2]. However, in at least one case, i.e. yeast Fpr3, the enzyme has been reported to phosphorylate Tyr residues [5]. Analy- sis of the phosphorylation of peptides suggests that the con- sensus site for CK2 can best be described as (S/T)xxD/E, and consistent with this, a number of proteins critical for tran- scription, cell-cycle regulation, and signal transduction con- tain such site(s) and are known to be phosphorylated in vitro and in vivo [6]. In vitro, CK2 activity is inhibited by poly- acidic compounds [7], and stimulated by polybasic com- pounds [8], but the in vivo relevance of these observations remains unclear. Comparisons between recombinant mono- meric α subunit and native or reconstituted α 2 β 2 holoenzyme have revealed that the β subunit plays a complex, yet at times paradoxical, role in regulating the basal activity of the α subunit [9–12]. On the one hand, the β subunit stabilizes the α subunit against proteolysis and thermal denaturation and stimulates its activity approximately 5-fold against most sub- strates [13]; on the other hand, it negatively regulates phos- phorylation of selected substrates, notably calmodulin [14, 15]. The β subunit also mediates stimulation of CK2 by poly- Address for offprints: A.P. Bidwai, Department of Biology, P.O. Box 6057, Brooks Hall, West Virginia University, Morgantown, WV 26506-60571, USA (E- mail: abidwai@wvu.edu)