Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras.
The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the "activator domain" (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.
Department of Physiology II, Kobe University School of Medicine, Japan.
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Hu
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Kariya
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Tamada
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Akasaka
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Shirouzu
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Yokoyama
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Kataoka
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine; D017931:DNA Primers; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D003545:Cysteine