Cell type-specific importance of ras-c-raf complex association rate constants for MAPK signaling.
We generated 17 c-Raf (RAF proto-oncogene serine-threonine protein kinase) mutants with altered Ras-Raf association and dissociation rates to investigate the role of electrostatically driven Ras-Raf association rates on epidermal growth factor (EGF)-activated mitogen-activated protein kinase (MAPK) signal transduction. Some of these mutants had compensating changes in association and dissociation rates, enabling the effects of changes in association rate to be distinguished from those of changes in affinity. In rabbit kidney (RK13) cells, these mutants affected downstream signaling, with changes in Ras-c-Raf association rates having a greater effect on MAPK signaling than did similar changes in dissociation rates. Mutants with compensating decreases in both association and dissociation rates stimulated less extracellular signal-regulated kinase (ERK)-dependent reporter activity than did wild-type c-Raf, whereas the converse was true for mutants with increased association and dissociation rates. In marked contrast, the mutants had little or no effect on signaling in human embryonic kidney (HEK) 293 cells. These two cell lines also showed distinct patterns of EGF-dependent ERK phosphorylation and signaling: ERK activation and signaling were transient in HEK293 cells and sustained in RK13 cells, with the difference resulting from the lack of negative feedback from ERK to Sos (Son of Sevenless) in the latter. Computer simulation revealed that, in the presence of negative feedback, changes in the rate of Ras-c-Raf binding have little effect on ERK activation. Thus, EGF-MAPK activation kinetics and feedback regulation is cell type specific and depends on the network topology.
Kiel
Christina
C
European molecular biology laboratory-centre for genomic regulation (CRG), systems biology unit, university pompeu fabra (UPF), Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain. christina.kiel@crg.es
Serrano
Luis
L
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003198:Computer Simulation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D000090063:Proto-Oncogene Mas; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D020837:SOS1 Protein / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013997:Time Factors; D014162:Transfection
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D020837:SOS1 Protein; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras)