Novel IκBα Proteolytic Pathway in WEHI231 Immature B Cells

ABSTRACT

The Rel/NF-κB family of transcription factors is sequestered in the cytoplasm of most mammalian cells by inhibitor proteins belonging to the IκB family. Degradation of IκB by a phosphorylation-dependent ubiquitin-proteasome (inducible) pathway is believed to allow nuclear transport of active Rel/NF-κB dimers. Rel/NF-κB (a p50–c-Rel dimer) is constitutively nuclear in murine B cells, such as WEHI231 cells. In these cells, p50, c-Rel, and IκBα are synthesized at high levels but only IκBα is rapidly degraded. We have examined the mechanism of IκBα degradation and its relation to constitutive p50–c-Rel activation. We demonstrate that all IκBα is found complexed with c-Rel protein in the cytoplasm. Additionally, rapid IκBα proteolysis is independent of but coexistent with the inducible pathway and can be inhibited by calcium chelators and some calpain inhibitors. Conditions that prevent degradation of IκBα also inhibit nuclear p50–c-Rel activity. Furthermore, the half-life of nuclear c-Rel is much shorter than that of the cytoplasmic form, underscoring the necessity for its continuous nuclear transport to maintain constitutive p50–c-Rel activity. We observed that IκBβ, another NF-κB inhibitor, is also complexed with c-Rel but slowly degraded by a proteasome-dependent process in WEHI231 cells. In addition, IκBβ is basally phosphorylated and cytoplasmic. We thus suggest that calcium-dependent IκBα proteolysis maintains nuclear transport of a p50–c-Rel heterodimer which in turn activates the synthesis of IκBα, p50, and c-Rel to sustain this dynamic process in WEHI231 B cells.

ACKNOWLEDGMENTS

We thank members of the Verma laboratory, M. Maki, and Z. J. Chen for many discussions. We appreciate R. Wisdom, E. Oltz, E. T. Alarid, and J. Stevenson for critical reading of the manuscript. We also acknowledge P. Chaio for murine IκBα cDNA, J. Stevenson for generating pLHL-CAHA and pLHL-CAHA-mIκBα clones, E. J. Corey for providing lactacystin, M. Maki for E64-d (through Taisho Pharmaceutical), F. Mercurio for ZLLF, R. Naviaux for pCLeco vector, C. Akazawa for CA promoter, and R. J. Ulevitch for 70Z/3-CD14 cells.

This work was funded by a Howard Hughes Medical Institute grant through the UW Medical School and a Shaw Scientist Award from Milwaukee Foundation to S.M. S.D.S. is a trainee under NIH predoctoral training grant T32GM07215.