Identification of novel ERK-mediated feedback phosphorylation sites at the C-terminus of B-Raf.
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.
Brummer
Tilman
T
Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck Institute for Immunobiology, Stuebeweg 51, Freiburg 79108, Germany.
Naegele
Heike
H
Reth
Michael
M
Misawa
Yukiko
Y
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D002645:Chickens / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D017353:Gene Deletion; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology
C482123:Braf protein, rat; D017346:Protein Serine-Threonine Kinases; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases