Abstract
Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which regulates the transcription elongation of RNA polymerase II and controls 60-70% of mRNA synthesis. Our previous studies show that HEXIM1 interacts with two key p53 regulators, nucleophosmin and human double minute-2 protein (HDM2), implying a possible connection between HEXIM1 and the p53 signaling pathway. Here we report the interaction between p53 and HEXIM1 in breast cancer, acute myeloid leukemia, and colorectal carcinoma cells. The C-terminal regions of p53 and HEXIM1 are required for the protein-protein interaction. Overexpression of HEXIM1 prevents the ubiquitination of p53 by HDM2 and enhances the protein stability of p53, resulting in up-regulation of p53 target genes, such as Puma and p21. Induction of p53 can be achieved by several means, such as UV radiation and treatment with anti-cancer agents (including doxorubicin, etoposide, roscovitine, flavopiridol, and nutlin-3). Under all the conditions examined, elevated protein levels of p53 are found to associate with the increased p53-HEXIM1 interaction. In addition, knockdown of HEXIM1 significantly inhibits the induction of p53 and releases the cell cycle arrest caused by p53. Finally, the transcription of the p53 target genes is regulated by HEXIM1 in a p53-dependent fashion. Our results not only identify HEXIM1 as a positive regulator of p53, but also propose a novel molecular mechanism of p53 activation caused by the anti-cancer drugs and compounds.
Highlights
To investigate the involvement of Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) in the p53 signaling pathway, we examine the functional connection between HEXIM1 and p53
Reciprocal IP was carried out using an anti-p53 antibody and HEXIM1 was detected in the immunoprecipitated complexes, confirming the interaction between p53 and HEXIM1 (Fig. 1B)
Besides binding to cyclin-dependent kinase 9 (CDK9)/cyclin T1, HEXIM1 has been shown to associate with ER␣, MyoD, histone deacetylases, importin ␣, human double minute-2 protein (HDM2), NPM, NF-B, and glucocorticoid receptors (17, 21, 25, 26, 50 –52)
Summary
To investigate the involvement of HEXIM1 in the p53 signaling pathway, we examine the functional connection between HEXIM1 and p53. We previously identified two novel HEXIM1-binding proteins, nucleophosmin (NPM) and human double minute-2 protein (HDM2), two key regulators of the p53 signaling pathway. Fusion of ubiquitin to HEXIM1 exhibits stronger inhibition on P-TEFb-dependent transcription, indicating a role for HDM2 in regulation of P-TEFb activity [26] These findings show functional interactions between HEXIM1 and p53 regulators, and indicate a possible involvement of HEXIM1 in the p53 signaling pathway. We identify HEXIM1 as a p53-binding protein and our results suggest a novel role for HEXIM1 in the regulation of p53 induction
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