The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch |
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| Authors: | Amardeep S Dhillon Yan Yan Yip G Joan Grindlay Julian L Pakay Marc Dangers Meike Hillmann William Clark Andrew Pitt Harald Mischak Walter Kolch |
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| Institution: | 1. Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Bioscience and Biotechnology, University of Melbourne, 30 Flemington Road, Parkville, VIC 3010, Australia;2. The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK;3. Department of Nephrology, Medizinische Hochschule Hannover, 30625 Hannover, Germany;4. Mosaiques Diagnostics and Therapeutics AG, Feodor-Lynen Sts. 21, 30625 Hannover, Germany;5. Sir Henry Wellcome Functional Genomics Facility, University of Glasgow, Glasgow G12 8QQ, UK;1. Department of Biological Science and Technology, Tokushima University Graduate School, Tokushima, Japan;2. Department of Infectious Medicine, Division of Eukaryotic Microbiology, Kurume University School of Medicine, Kurume, Japan;1. UCL Cancer Institute, University College London, London WC1E 6BT, UK;2. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA;3. European Molecular Biology Laboratory, 69117 Heidelberg, Germany;4. Randall Division of Cell and Molecular Biophysics, King’s College London, London SE1 1UL, UK;5. UCL Centre of Advanced Biomedical Imaging, Division of Medicine and Institute of Child Health, University College London, London WC1E 6BT, UK;1. Laboratory of Cell and Developmental Signaling, NCI-Frederick, Frederick, MD 21702, USA;2. NCI-Ras Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Frederick, MD 21702, USA;1. Novartis Institutes for Biomedical Research, Emeryville, CA 94608, USA;2. Novartis Institutes for Biomedical Research, Cambridge, CA 02139, USA;3. Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA;4. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA;1. Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA;2. Institute for Cancer Genetics, Columbia University Medical Center, New York, NY 10032, USA;3. Department of Dermatology, Columbia University Medical Center, New York, NY 10032, USA;4. Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY 10032, USA;5. Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA;6. Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA;7. Department of Medicine, Harvard Medical School, Boston, MA 02115, USA;8. Department of Medicine, Weill Cornell Medical College, New York, NY 10065, USA |
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| Abstract: | The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein–protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation. |
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| Keywords: | Raf-1 Phosphorylation 14-3-3 Serine 621 |
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