| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Embryonic stem (ES) cells have the ability to grow indefinitely while maintaining pluripotency. These properties lead to expectation that ES cell might be useful to treat patients of various diseases and injuries. Oct-4(also known as Oc-3 orOct-3/4) is essential to maintaining these ES cell properties. Oct-4 usually functions in ES cells as a complex. Especially, Sox-2 is the best known partner of Oct-4 and this Oct-4/Sox-2 complex regulates many genes. Oct-4 is a member of octamer family and other octamer factors, i. e., Oct-1 and Oct-6 are also expressed in ES cells. However, these octamer factors can not substitute for Oct-4, in other word, only Oct-4 can maintain these ES cell properties. This unique function of Oct-4 leads us to speculate that Oct-4 has a specific biochemical character that other octamer factors do not have. To explore the specific biochemical property of Oct-4, I try to identify the essential portion of Oct-4 to function in ES cells by means of phenotypic complementation assay developed by Niwa and colleagues. Namely, the Oct-4/Oct-6 chimeric protein is expressed in ES cells instead of Oct-4 and I monitor whether this chimeric protein can substitute for Oct-4 or not. In this approach, I identified the essential amino acid residue, i.e., threonine residue in the POU-specific domain. By means of the simulation of three dimensional structure of Oct-4/Sox-2/DNA complex, this threonine residue appears to be positioned close to basic amino residue of Sox-2. From this simulation, I hypothesize the phosphorylation of this threonine residue because the interaction of phosphorylated threonine and basic region of Sox-2 is stronger than that of native threonine. In this report, I demonstrate the possibility of phophorylation of threonine residue by means of in vivo labeling of Oct-4 with orthophosphate and mass-spectrometric analysis.
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