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Identification of novel ERK-mediated feedback phosphorylation sites at the C-terminus of B-Raf

Oncogene volume 22, pages 8823–8834 (2003)Cite this article

Abstract

The extracellular signal-regulated kinase (ERK) pathway plays an important role during the development and activation of B lymphocytes. We have recently shown that B-Raf is a dominant ERK activator in B-cell antigen receptor signalling. We now show that B-Raf is hyperphosphorylated upon BCR engagement and undergoes a prominent electrophoretic mobility shift. This shift correlates with ERK activation and is prevented by the MEK inhibitor U0126. Syk-deficient DT40 B cells display neither dual ERK phosphorylation nor a mobility shift of B-Raf upon BCR engagement. The inducible expression of a constitutively active B-Raf in this mutant line restores dual ERK phosphorylation and the mobility shift of endogenous B-Raf, indicating that these two events are connected to each other. By site-directed mutagenesis studies, we demonstrate that the shift is due to an ERK2-mediated feedback phosphorylation of serine/threonine residues within an evolutionary conserved SPKTP motif at the C-terminus of B-Raf. Replacement of these residues by negatively charged amino acids causes a constitutive mobility shift and a reduction of PC12 cell differentiation. We discuss a model in which ERK-mediated phosphorylation of the SPKTP motif is involved in negative feedback regulation of B-Raf.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft through SFB 388 and the Leibniz Prize to MR. We would like to thank Dr Tomohiro Kurosaki for providing DT40 cells, Dr Jean-Marie Buerstedde for the bursal cDNA library, Dr Helge Steen for the pAloxPΔB-Raf-neo plasmid and Ms Careen Pollmer for technical assistance. We also thank Ms Jianfen Shu (Department of Health Evaluation Science, University of Virgina Health System, Charlottesville, USA) for the statistical analysis. We are grateful to Drs Peter J Nielsen, Michael Huber and Florian Losch for helpful discussions.

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  1. Yukiko Misawa

    Present address: Department of Pharmacology, University of Virginia Health System, P.O. BOX 800735, 1300 Jefferson Park Avenue, Charlottesville, VA, 22908, USA

  2. Tilman Brummer

    Present address: Cancer Research Program, The Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia

Authors and Affiliations

  1. Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck Institute for Immunobiology, Stuebeweg 51, Freiburg, 79108, Germany

    Tilman Brummer, Heike Naegele, Michael Reth & Yukiko Misawa

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Correspondence to Yukiko Misawa.

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Brummer, T., Naegele, H., Reth, M. et al. Identification of novel ERK-mediated feedback phosphorylation sites at the C-terminus of B-Raf. Oncogene 22, 8823–8834 (2003). https://doi.org/10.1038/sj.onc.1207185

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