229 Molecular and Cellular Biochemistry 191: 229–234, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. Multiple forms of protein kinase CK2 present in leukemic cells: In vitro study of its origin by proteolysis Joan Roig, 1 Andreas Krehan, 2 Dolors Colomer, 3 Walter Pyerin, 2 Emilio Itarte 1 and Maria Plana 1 1 Departament de Bioquímica i Biologia Molecular, Unitat de Ciències, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola; 2 Biochemische Zellphysiologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 3 Servei d’Hematologia Biològica, Hospital Clinic, Barcelona, Spain Abstract Human recombinant CK2 subunits were incubated for different times with the two main cytosolic proteases m-calpain and 20 S proteasome. Both, m-calpain in a calcium dependent manner and the 20 S proteasome, were able to degrade CK2 subunits in vitro. In both cases, CK2α′ was more resistant to these proteases than CK2α. When these proteases were assayed on the reconstituted (α 2 β 2 holoenzyme, a 37 kDa α-band, analogous to that observed in AML extracts, was generated which was resistant to further degradation. No degradation was observed when the 26 S proteasome was assayed on free subunits. Studies with CK2α deletion mutants showed that m-calpain and the 20 S proteasome acted on the C-terminus end of CK2α. These results pointed to cytosolic proteases as agents involved in the control of the amount of free CK2 subunits within the cell, which becomes evident when CK2 is overexpressed as in AML cells. (Mol Cell Biochem 191: 229–234, 1999) Key words: protein kinase CK2, leukemia, calcium activated protease, m-calpain, proteasome Introduction Protein kinase CK2 is a ubiquitous serine/threonine kinase present in all eukaryotic organisms (reviewed in [1–3]). It has a tetrameric structure with two catalytic (CK2α or CK2α′, of 44 kDa and 38 kDa respectively) and two regulatory (CK2β, of 28 kDa) subunits. CK2β subunit enhances CK2α/ α′ activity [4], and also changes substrate specificity and stabilizes the catalytic subunits [5]; it is synthesized in excess of the catalytic subunit α, and the newly synthesized fraction that is not incorporated to the holoenzyme is degraded through a non-lysosomal proteolytic system with a very low ATP requirement [6]. CK2 phosphorylates a broad number of proteins, some of them implicated in the control of cell growth and division (reviewed in [7]). Although its function is vital for the cell, Address for offprints: M. Plana, Departament de Bioquímica i Biologia Molecular, Unitat de Ciències, Universitat Autònoma de Barcelona, Edifici C, Campus de Bellaterra, 08193 -Cerdanyola del Vallès, Spain the exact role of protein kinase CK2 and its control is not fully understood. CK2 has been implicated in signal transduction pathways, and increased kinase activity has been observed after serum or growth factor stimulation in different cell types (reconsidered in [8]). Also, mitogenic stimulation can be reduced or eliminated by antisense oligonucleotides com- plementary to CK2 mRNAs or by microinjection of anti-CK2 subunit antibodies, indicating that CK2 is necessary for cell cycle progression [9–11]. CK2 activity has been found elevated in rapidly proliferating tissues and cell lines, in solid tumors, in virus-transformed cells, and in a lympho-proli- ferative disease induced by intracellular parasites (see [12– 18]). The α subunit of CK2 has also been shown to act as an oncogene when expressed in lymphocytes of transgenic mice [19]. All this data suggested a relationship between the enzyme and the control of the processes of cell growth and