Disruption of the 14-3-3 binding site within the B-Raf kinase domain uncouples catalytic activity from PC12 cell differentiation.
A number of Raf-associated proteins have recently been identified, including members of the 14-3-3 family of phosphoserine-binding proteins. Although both positive and negative regulatory functions have been ascribed for 14-3-3 interactions with Raf-1, the mechanisms by which 14-3-3 binding modulates Raf activity have not been fully established. We report that mutational disruption of 14-3-3 binding to the B-Raf catalytic domain inhibits B-Raf biological activity. Expression of the isolated B-Raf catalytic domain (B-Rafcat) induces PC12 cell differentiation in the absence of nerve growth factor. By contrast, the B-Rafcat 14-3-3 binding mutant, B-Rafcat S728A, was severely compromised for the induction of PC12 cell differentiation. Interestingly, the B-Rafcat 14-3-3 binding mutant retained significant in vitro catalytic activity. In Xenopus oocytes, the analogous full-length B-Raf 14-3-3 binding mutant blocked progesterone-stimulated maturation and the activation of endogenous mitogen-activated protein kinase kinase and mitogen-activated protein kinase. Similarly, the full-length B-Raf 14-3-3 binding mutant inhibited nerve growth factor-stimulated PC12 cell differentiation. We conclude that 14-3-3 interaction with the catalytic domain is not required for kinase activity per se but is essential to couple B-Raf catalytic activity to downstream effector activation.
MacNicol
M C
MC
Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA.
Muslin
A J
AJ
MacNicol
A M
AM
D048948:14-3-3 Proteins; D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation / Q000235:genetics; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D020932:Nerve Growth Factor / Q000494:pharmacology; D009865:Oocytes; D016716:PC12 Cells; D010766:Phosphorylation; D011374:Progesterone / Q000494:pharmacology; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014446:Tyrosine 3-Monooxygenase; D014981:Xenopus
D048948:14-3-3 Proteins; D011506:Proteins; D011374:Progesterone; D020932:Nerve Growth Factor; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases