ZBP-89 Promotes Growth Arrest through Stabilization of p53

Abstract

Transcription factor p53 can induce growth arrest and/or apoptosis in cells through activation or repression of downstream target genes. Recently, we reported that ZBP-89 cooperates with histone acetyltransferase coactivator p300 in the regulation of p21^waf1, a cyclin-dependent kinase inhibitor whose associated gene is a target gene of p53. Therefore, we examined whether ZBP-89 might also inhibit cell growth by activating p53. In the present study, we demonstrate that elevated levels of ZBP-89 induce growth arrest and apoptosis in human gastrointestinal cell lines. The ZBP-89 protein accumulated within 4 h, and the p53 protein accumulated within 16 h, of serum starvation without changes in p14ARF levels, demonstrating a physiological increase in the cellular levels of these two proteins. Overexpression of ZBP-89 stabilized the p53 protein and enhanced its transcriptional activity through direct protein-protein interactions. The DNA binding and C-terminal domains of p53 and the zinc finger domain of ZBP-89 mediated the interaction. A point mutation in the p53 DNA binding domain, R273H, greatly reduced ZBP-89-mediated stabilization but not their physical interaction. Furthermore, ZBP-89 formed a complex with p53 and MDM2 and therefore did not prevent the MDM2-p53 interaction. However, heterokaryon assays demonstrated that ZBP-89 retained p53 in the nucleus. Collectively, these data indicate that ZBP-89 regulates cell proliferation in part through its ability to directly bind the p53 protein and retard its nuclear export. Our findings further our understanding of how ZBP-89 modulates cell proliferation and reveals a novel mechanism by which the p53 protein is stabilized.

ACKNOWLEDGMENTS

J. L. Merchant is an assistant investigator of the Howard Hughes Medical Institute. The work was supported by Public Health Service NIH grant DK 55732 and the Robert and Sally Funderburg Award from the American Digestive Health Foundation.

We thank the University of Michigan Cancer Center flow cytometry and Vector Cores (NIH grant 5P30 CA46592–13). We thank Bert Vogelstein (Johns Hopkins University) for the generous gifts of HCT 116 p53 wild-type and null cell lines, p53 wild-type and mutant expression vectors, and the p21^waf1-Luc reporter construct. Also we thank Thomas Shenk (Princeton University), Ken-ichi Yamamoto (Kanazawa University, Japan), and Moshe Oren (Weizmann Institute of Science, Israel) for providing the p53 GST constructs and human p53 luciferase reporter constructs, respectively.