Cloning of the mouse insulin receptor substrate-3 (mirs-3) promoter, and its regulation by p53.

The insulin receptor susbtrate-3 (IRS-3) is a member of a family of intermediate adapter proteins that function as major intracellular targets for phosphorylation by the activated insulin and IGF-I receptors. Among the 4 IRS proteins identified so far, IRS-3 exhibits a rather peculiar expression pattern during both the embryonic development and adult life, suggesting a different mechanism of regulation of its expression. In this study, we cloned the 5’ flanking region of the mIRS-3 gene and analyzed its promoter activity. The mIRS-3 promoter is inhibited by wt p53 and this effect is completely abolished by co-transfection of a dominant negative p53. Tumor-derived p53 mutants show variable, but lower suppressing capability than wt p53. In addition, treatment with Doxorubicin inhibits endogenous expression of mIRS-3 mRNA in C2C12 and 3T3-L1 cells. The DNA region spanning from nt –287 and –178 in the mIRS-3 promoter is responsible for a 32.2% reduction of the MDM2 promoter activity, suggesting its involvement in the p53-mediated inhibitory effect. In conclusion, our study demonstrates that the mIRS-3 promoter is regulated by p53 at the transcriptional level. The inhibition of mIRS-3 promoter by wt p53, and its de-repression by tumor-derived p53 mutants, appears to be similar to that previously reported for the IGF-I receptor promoter, suggesting a common role of these two genes in p53-mediated cell growth and differentiation.