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REGULATORY ROLE OF CK2 DURING THE PROGRESSION OF CELL CYCLE Miwako K. Homma* and Yoshimi Homma Department of Biomolecular Science, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan *[email protected] INTRODUCTION Protein kinase CK2 is a ubiquitous eukaryotic Ser/Thr kinase. Genetic, biochemical and cell biological studies indicate that the enzyme is involved in the control of cell division and in signal transduction. We have characterized potent roles for CK2 in the progression of cell cycle. METHOD For cell synchronization, logarithmically growing cells were starved in 0.2% FBS for 48h and were collected in G0 or released into fresh medium containing 10% FBS to obtain cell populations synchronized at S phase or pro-metaphase. CK2, APC and eIF5 were immunoprecipitated separately from synchronized cells. CK2 activity was determined by a p81 filter method. Proteins associated with CK2 were identified by mass spectrometric analysis. RESULTS We found that CK2 directly interacts with the tumor suppressor protein adenomatous polyposis coli (APC) in a cell cycle-dependent manner. The C-terminal region of APC suppressed the kinase activity of CK2, although APC-CK2 interactions involved the N-terminal region of APC. The inhibitory region localized between amino acid residues 2086-2394, and over-expression of this fragment in cultured colorectal carcinoma cells suppressed cell proliferation rates as well as colony formation on soft agar. We further identified eukaryotic translational initiation factor 5 (eIF5) as a downstream target for CK2, that was phosphorylated by CK2 in vivo and in vitro. The phosphorylation levels of eIF5 and its association with CK2 changed dramatically during the progression of cell cycle. We determined the phosphorylation sites in eIF5 produced by CK2 and eIF5 mutants that lack those phosphorylation sites perturbed synchronous progression of cells through the S to M phase. DISCUSSION These findings provide insight into understanding the molecular link between APC and the regulation of phosphorylation-dependent signal transduction. In colorectal carcinoma cells, truncated APC mutants lacking the C-terminal domain bind to CK2 but fail to effectively suppress CK2 activity. The results indicate that hetero-complex formation between CK2 and full-length APC regulates CK2 activity in vivo and has regulatory effects on cell cycle progression, which seems to be defective in truncated APC mutants that are frequently observed in colorectal carcinomas. These results suggest that growth inhibitory effects of APC may be regulated by CK2. Also, CK2 carries out its growth promoting effect through eIF5 and the results suggest the critical role for its phosphorylation by CK2 on the progression of cell cycle. ACKNOWLEDGMENT This work was supported in part by Yamada Science Foundation (Osaka) and by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. REFERENCES 1. Homma, M.K., and Homma, Y. (2002) Proc. Natl. Acad. Sci. USA 99, 5959-5964 2. Litchfield, D.W. (2003) Biochem. J. 369, 1-15 3. Ahmed, K., Gerber, D.A., and Cochet, C.,(2002) Trends Cell Biol. 12, 226-230