Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation.
Raf kinase inhibitory protein (RKIP; also known as phosphatidylethanolamine-binding protein or PEBP) is a modulator of the Raf/MAPK signaling cascade and a suppressor of metastatic cancer. Here, we show that RKIP inhibits MAPK by regulating Raf-1 activation; specifically, RKIP acts subsequent to Raf-1 membrane recruitment, prevents association of Raf-1 and p21-activated kinase (PAK), and blocks phosphorylation of the Raf-1 kinase domain by PAK and Src family kinases. Mutation of the PAK and Src phosphorylation sites on Raf-1 to aspartate, a phosphate mimic, prevented RKIP association with or inhibition of Raf-1 signaling. Interestingly, although RKIP can interact with B-Raf, RKIP depletion had no effect on activation of B-Raf. Because c-Raf-1 and B-Raf are both required for maximal MAPK stimulation by epidermal growth factor in neuronal and epithelial cell lines, we determined whether RKIP significantly affects MAPK signaling. In fact, RKIP depletion increased not only the amplitude but also the sensitivity of MAPK and DNA synthesis to epidermal growth factor stimulation by up to an order of magnitude. These results indicate that selective modulation of c-Raf-1 but not B-Raf activation by RKIP can limit the dynamic range of the MAPK signaling response to growth factors and may play a critical role in growth and development.
Trakul
Nicholas
N
Committee on Cancer Biology, University of Chicago, Chicago, Illinois 60637, USA.
Menard
Raymond E
RE
Schade
George R
GR
Qian
Zhijian
Z
Rosner
Marsha Rich
MR
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D047468:Immunoprecipitation; D020935:MAP Kinase Signaling System; D008954:Models, Biological; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D049488:Phosphatidylethanolamine Binding Protein / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012313:RNA / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
D006133:Growth Substances; D049488:Phosphatidylethanolamine Binding Protein; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D001224:Aspartic Acid; D014443:Tyrosine; D012694:Serine; D004815:Epidermal Growth Factor; D012313:RNA; D004247:DNA; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf