| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Casein kinase 2(CK2) is a ubiquitous eukaryotic Ser/Thr protein kinase that plays an important role in cell cycle progression. Whilst its function in this process remains unclear, it is known to be required for the G_1 and G_2/M transitions in yeast. Here, we show that CK2 activity changes notably during cell-cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, elF5. Using mass spectrometry we show that Ser389 and Ser390 of elF5 are major sites of phosphorylation by CK2. This is confirmed using elF5 mutants that lack CK2 sites ; the phosphorylation levels of mutant elF5 proteins are significantly reduced, relative to wild-type elF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous elF5, and that they perturb synchronous progression of cells through the S phase to the M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature elF5/elF2/elF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of wild type elF5 was almost abolished in a GFP-tagged elF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy (FCS). These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation.
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