Mapping of the interaction sites between Wee1 kinase and the regulatory β-subunit of protein kinase CK2

Abstract

Abstract. Somatic Wee1 is a protein kinase that plays a key role in cell cycle progression at the onset of mitosis by phosphorylating CDK1 at the inhibitory Tyr15 amino acid residue. Wee1 is regulated at multiple levels, i.e., phosphorylation, protein-protein association and proteasome-mediated degradation. We have recently shown that the regulatory ß-subunit of protein kinase CK2 participates in PLK1-Wee1 complex formation interacting directly with Wee1 and thereby contributing to the regulation of G2-M cell cycle transition. Here, we show that Wee1 binds CK2ß via two domains comprising amino acids 59-71 and 232-332. By employing deletion mutants of the CK2ß-subunit we also show that two regions between residues 1-5 and 155-170 are necessary for binding Wee1. Furthermore, we demonstrate that the interaction between CK2ß and Wee1 does not modify the kinase activity of the latter, instead CK2ß directly upregulates CDK1 kinase activity by reversing the inhibitory effect which follows Wee1-mediated phosphorylation. Taken together, our findings reinforce the notion that CK2ß is a modulator of protein kinases implicated in cell cycle regulation and exerts functions that are independent of CK2 tetramers. IntroductionProtein kinase CK2 is a highly conserved enzyme ubiquitously expressed among all the eukaryotic organisms investigated so far. It is composed of two catalytic subunits · and/or ·' and two regulatory ß-subunits. CK2 was originally described as an enzyme characterized by a tetrameric structure but evidence suggests that the individual subunits can exist in association with various intracellular proteins (1,2). Because of the broad spectrum of proteins that interact with or are phosphorylated by CK2, it is not surprising that this enzyme is implicated in various intracellular processes ranging from regulation of transcription, DNA replication and repair to survival, proliferation and differentiation (reviewed in refs. 2,3). Numerous studies have also suggested that CK2 exerts a vital function. Indeed it was shown by gene knockout experiments in mammals that both the ·-and ß-subunits are essential for the development of mice (4,5) while lack of expression of the ·'-subunit is not lethal but leads to infertility (6). CK2 has also been linked to the regulation of cell cycle progression in lower as well as higher eukaryotes (reviewed in refs. 7,8).Cell cycle progression is controlled by numerous proteins which act in order to preserve the integrity of the genetic information crucial for the survival of a cell. Entry into mitosis is mainly regulated by CDK1 whose optimal activation is achieved following the association with cyclin B and phosphorylation in the conserved T-loop domain. During interphase, CDK1 activity is maintained low by phosphorylation of two amino acids residues (i.e., Thr14 and Tyr15) catalyzed by Myt1 and Wee1 protein kinases (9,10). Entry into mitosis requires the intervention of the CDC25 dual specificity protein phosphatase family members that catalyze the activating dephosphorylation of CDK...