Mutant p53-induced Up-regulation of Mitogen-activated Protein Kinase Kinase 3 Contributes to Gain of Function

Aymone Gurtner,Giuseppe Starace,Giuseppe Norelli,Giulia Piaggio,Ada Sacchi,Gianluca Bossi

doi:10.1074/jbc.m109.094813

Aymone Gurtner, Giuseppe Starace + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.094813

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Abstract

Mitogen-activated protein kinase kinase 3 (MAP2K3) is a member of the dual specificity kinase group. Growing evidence links MAP2K3 to invasion and tumor progression. Here, we identify MAP2K3 as a transcriptional target of endogenous gain-of-function p53 mutants R273H, R175H, and R280K. We show that MAP2K3 modulation occurred at the mRNA and protein levels and that endogenous mutant p53 proteins are capable of binding to and activate the MAP2K3 promoter. In addition, we found that the studied p53 mutants regulate MAP2K3 gene expression through the involvement of the transcriptional cofactors NF-Y and NF-kappaB. Finally, functional studies showed that endogenous MAP2K3 knockdown inhibits proliferation and survival of human tumor cells, whereas the ectopic expression of MAP2K3 can rescue the proliferative defect induced by mutant p53 knockdown. Taken together, our findings define a novel player through which mutant p53 exerts its gain-of-function activity in cancer cells.

Highlights

Variety of commonly used anti-cancer drugs [5, 6]
To unveil a possible role of MAP2K3 up-regulation in mutp53 GOF activity, we studied the molecular mechanisms through which different mutp53 proteins up-regulate MAP2K3 expression and the biologic effects of its up-regulation in different cell lines
Reported data indicate that the MAP2K3 up-regulation is involved in invasion and progression of glioma and breast tumors

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—Non-small cell lung carcinoma H1299 (p53null), colon adenocarcinoma HT29 (mutp53R273H), breast carcinoma SKBR3 (mutp53R175H) [10], breast adenocarcinoma MDA-MB468 (mutp53R273H) To mutant induced significant increases in MAP2K3 promoter activity confirm these results in a more relevant cellular context, similar (Fig. 2B) These data indicate that the analyzed cancer-associated p53 experiments were performed with a panel of human cancer cell mutants can up-regulate MAP2K3 expression via transactivation of lines naturally harboring mutations in the TP53 locus. Experiments were performed with p53-null H1299 cells where the transcriptional activity of mutp proteins on the MAP2K3 promoter was explored by exogenously expressing different p53 mutants. We asked whether mutp proteins contribute directly to MAP2K3 up-regulation via physical recruitment onto the MAP2K3 promoter To this end, chromatin immunoprecipitation assays were performed on chromatin isolated from our human cancer cell lines infected with either the sh/p53 or the sh/scr lentivirus. Our findings indicate that mutp up-regulates MAP2K3 expression through the involvement of NF-Y and NF-␬B transcriptional cofactors

Knockdown of Endogenous

Can Rescue the Proliferative Defect

DISCUSSION