Raf-1 activation disrupts its binding to keratins during cell stress.
Keratins 8 and 18 (K8/18) heteropolymers may regulate cell signaling via the known K18 association with 14-3-3 proteins and 14-3-3 association with Raf-1 kinase. We characterized Raf-keratin-14-3-3 associations and show that Raf associates directly with K8, independent of Raf kinase activity or Ras-Raf interaction, and that K18 is a Raf physiologic substrate. Raf activation during oxidative and toxin exposure in cultured cells and animals disrupt keratin-Raf association in a phosphorylation-dependent manner. Mutational analysis showed that 14-3-3 residues that are essential for Raf binding also regulate 14-3-3-keratin association. Similarly, Raf phosphorylation sites that are important for binding to 14-3-3 are also essential for Raf binding to K8/18. Therefore, keratins may modulate some aspects of Raf signaling under basal conditions via sequestration by K8, akin to Raf-14-3-3 binding. Keratin-bound Raf kinase is released upon Raf hyperphosphorylation and activation during oxidative and other stresses.
Department of Medicine, VA Palo Alto Medical Center, 3801 Miranda Ave., 154J, Palo Alto, CA 94304, USA.
Nam-On
NO
Ku
Haian
H
Fu
M Bishr
MB
Omary
D048948:14-3-3 Proteins; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007633:Keratins / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D011108:Polymers / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology*; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D048948:14-3-3 Proteins; D018076:DNA, Complementary; D011108:Polymers; D007633:Keratins; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010100:Oxygen