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Abstract
Bob1 (Obf-1 or OCA-B) is a 34-kDa transcriptional coactivator encoded by the Pou2af1 gene that is essential for normal B-cell development and immune responses in mice. During lymphocyte activation, Bob1 protein levels dramatically increase independently of mRNA levels, suggesting that the stability of Bob1 is regulated. We used a fluorescent protein-based reporter system to analyze protein stability in response to genetic and physiological perturbations and show that, while Bob1 degradation is proteasome mediated, it does not require ubiquitination of Bob1. Furthermore, degradation of Bob1 in B cells appears to be largely independent of the E3 ubiquitin ligase Siah. We propose a novel mechanism of Bob1 turnover in B cells, whereby an acidic region in the C terminus of Bob1 regulates the activity of degron signals elsewhere in the protein. Changes that make the C terminus more acidic, including tyrosine phosphorylation-mimetic mutations, stabilize the instable murine Bob1 protein, indicating that B cells may regulate Bob1 stability and activity via signaling pathways. Finally, we show that expressing a stable Bob1 mutant in B cells suppresses cell proliferation and induces changes in surface marker expression commonly seen during B-cell differentiation.
ACKNOWLEDGMENTS
We thank Elias Hobeika and Patrick Heun for cells and antibodies, Michael Mitterer, Sabine Sané, Luzia Ballmer, and Osric Forrest for technical assistance, Marinus Lamers for critical reading of the manuscript, Patrick Matthias for providing the human Siah1 construct and comments regarding the manuscript, Gregory Warr for the catfish Bob1 DNA construct, Brad Magor for the catfish B-cell lines, Georg Riegger for fish serum, members of the group of Wolfgang Schamel for help with the PhosTag system, Verdon Taylor for the Hes3 cDNA, Thomas Wirth for the anti-Bob1 hybridoma, the lab of Rudolf Grosschedl for the Ebf1 and Pax5 cDNA clones, and the lab of Robert Schneider for qPCR reagents.
J.M.L. is supported by an IMPRS-MCB fellowship from the Max Planck Society, and A.M. is supported by a fellowship and grants from the National Breast Cancer Foundation and Cancer Council Queensland.