Necdin is a nuclear protein expressed in virtually all postmitotic neurons, and ectopic expression of this protein strongly suppresses the proliferation of NIH3T3 cells. Simian virus 40 large T antigen targets both p53 and the retinoblastoma protein (Rb) 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 COS-1 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 Rb-binding domain. Moreover, necdin bound to adenovirus E1A, another viral oncoprotein that forms a specific complex with Rb. We then examined the ability of necdin to bind to the transcription factor E2F1, a cellular Rb-binding factor involved in cell-cycle progression. Intriguingly, necdin, like Rb, bound to a carboxyl-terminal domain of E2F1, and repressed E2F-dependent transactivation in vivo. In addition, necdin suppressed the colony formation of Rb-deficient SAOS-2 osteosarcoma cells. These results suggest that necdin is a postmitotic neuron-specific growth suppressor that is functionally similar to Rb.
We have examined the transcriptional activity of four cis-elements, Ad1(CRE), Ad2, Ad3, and Ad4, that are present in the promoter of the bovine CYP11B (11 beta-hydroxylase P-450) gene using beta-globin reporter gene constructs and transient transfection into steroidogenic and nonsteroidogenic cell types. Only Ad1(CRE), a CRE homolog, showed forskolin-dependent transcriptional activity in adrenal tumor Y-1 cells, whereas the other elements were not able to stimulate transcription by themselves. As Ad3 and Ad4 had previously been identified as the cis-elements required for full cAMP-dependent transcription of this gene, we examined the effect of combinations of different cis-elements on the transcription of the reporter gene. In Y-1 cells, Ad1(CRE) and four tandem copies of any one of the other cis-elements substantially activated transcription in response to forskolin treatment. The template carrying Ad1(CRE) and Ad4 was also active in testicular Leydig cells, I-10, whereas it was inactive in nonsteroidogenic PC-12 cells. Transcriptional activation by the 4xAd4/Ad1(CRE) combination presumably depended on the presence of Ad4-binding protein (Ad4BP), which is absent in PC-12 cells, as shown by immunoblot analysis. This was confirmed by cotransfecting an expression vector for Ad4BP into PC-12 cells, which caused forskolin-dependent transcription to increase in proportion to the amount of expression vector. In Y-1 cells, transcriptional activation by forskolin was mimicked by cotransfection of an expression vector for the catalytic subunit of protein kinase-A.(ABSTRACT TRUNCATED AT 250 WORDS)
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