| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1998: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Necdin is a nuclear protein, whose gene is expressed in postmitotic neurons from early embryonal stages. Ectopic expression of necdin suppresses the growth of proliferative NIH3T3 cells. SV40 large T antigen targets both p53 and pRb for cellular transformation. By analogy with the interactions of the large T antigen with these nuclear growth suppressors, we examined the ability of necdin to bind to the large T antigen. Necdin was co-immunoprecipitated with the large T antigen from the nuclear extract of necdin cDNA-transfected COS1 cells. Yeast two-hybrid and in vitro binding analyses revealed that necdin bound to an amino- terminal region of the large T antigen, which encompasses the pRb-binding domain. We then examined the ability of necdin to bind to the transcription factor E2F1, cellular pRb-binding factor involved in cell-cycle progression. Intriguingly, necdin bound to a carboxyl-terminal domain of E2Fl, and repressed E2F-dependent transactivation in vivo. In addition, necdin su
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ppressed the colony formation of Rb-deficient SAOS2 cells. These results suggest that necdin is a postmitotic neuron-specific growth suppressor that is functionally similar to pRb. There is a substantial degree of homology between the E2F1 and p53 activation domains, suggesting a conservation of binding sites for specific proteins. The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to a narrow region located between the MDM2-binding site and the proline-rich region in the amino-terminal domain of p53. The electrophoretic mobility shift assay showed that necdin supershifted a complex between p53 and its binding DNA, implying that the p53-necdin complex is competent for DNA binding. In p53-deficient SAOS2 cells, necdin markedly suppressed p53-dependent activation of the p21 promoter. Necdin and p53 suppressed cell growth without apoptosis as assessed by the colony formation of SAOS2 cells. On the other hand, necdin inhibited p53-induced apoptosis of U2OS cells. Thus, necdin can be a growth suppressor that targets p53 and modulates its biological functions in postmitotic neurons. Less
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