Mutations in the v-Rel Transactivation Domain Indicate Altered Phosphorylation and Identify a Subset of NF-κB-Regulated Cell Death Inhibitors Important for v-Rel Transforming Activity

Abstract

Consistent with the constitutive activation of Rel/NF-κB in human hematopoietic tumors, the v-Rel oncoprotein induces aggressive leukemia/lymphomas in animal models. v-Rel is thus a valuable tool to characterize the role of Rel/NF-κB in cancer and the mechanisms involved. Prior studies by our group identified a serine-rich domain in v-Rel that was required for biological activity. Here, we investigated the molecular basis for the transformation defect of specific serine mutants. We show that the transforming efficiency of these mutants in primary lymphoid cells is correlated with their ability to mediate κB site-dependent transactivation and with specific changes in phosphorylation profiles. Interestingly, coexpression of the death antagonists Bcl-xL and Bcl-2 significantly increased their oncogenicity, whereas other NF-κB-regulated death inhibitors showed little or no effect. The fact that a subset of apoptosis inhibitors could rescue v-Rel transactivation mutants suggests that their reduced transcriptional activity may critically affect expression of defined death antagonists essential for oncogenesis. Consistent with this hypothesis, we observed selection for high endogenous expression of Bcl-2-related death antagonists in cells transformed by weakly transforming v-Rel mutants. These results emphasize the need for Rel/NF-κB to efficiently activate expression of a subset of antiapoptotic genes from the Bcl-2 family to manifest its oncogenic phenotype.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grants CA54999 and CA83937 from the National Cancer Institute (C.G.). B.R. is a Cure for Lymphoma Foundation Fellow (Alan & Berte Hirschfield Research Grant, CLF, New York) and was partially supported by the Foundation of the UMDNJ.

We are very grateful to M. Hannink (University of Missouri) and T. Gilmore (Boston University) for the pMH105 IRES and pMH105 IRES bcl-2 constructs and to J. Suh and A. Rabson for the IL-6-κB-luciferase plasmid. cDNAs for human a20, c-iap1, c-iap2, xiap, and bcl-xl were kind gifts from V. Dixit, D. W. Ballard, C. Duckett, and C. Labrie. We thank Jiuzhen Jin for advice on phosphoamino acid analysis and anonymous reviewers for insightful suggestions. We thank members of the Gélinas laboratory for fruitful discussions during the course of this work and are grateful to A. Rabson, J. Kucharczak, and M. Simmons for helpful comments on the manuscript.