title: Coassociation of Rap1A and Ha-Ras with Raf-1 N-terminal region interferes with ras-dependent activation of Raf-1.
abstract: Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Ras-binding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes several Ras functions in vivo. Recently, we identified the cysteine-rich region (CRR) of Raf-1 as another Ras-binding domain. Ha-Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to activate Raf-1. Since these mutations replace Ras residues with those of Rap1A, we examined if Rap1A lacks the ability to bind to CRR. Contrary to the expectation, Rap1A exhibited a greatly enhanced binding to CRR compared with Ha-Ras. Enhanced CRR binding was also found with Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras-dependent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR.
authors.affiliation: Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
authors.forename: C D
authors.initials: CD
authors.lastname: Hu
authors.forename: K i
authors.initials: Ki
authors.lastname: Kariya
authors.forename: G
authors.initials: G
authors.lastname: Kotani
authors.forename: M
authors.initials: M
authors.lastname: Shirouzu
authors.forename: S
authors.initials: S
authors.lastname: Yokoyama
authors.forename: T
authors.initials: T
authors.lastname: Kataoka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003545:Cysteine; D017931:DNA Primers; D005347:Fibroblasts / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D008168:Lung / Q000378:metabolism; D008322:Mammals; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D020668:rap GTP-Binding Proteins
chemical_list: D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D019204:GTP-Binding Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D020668:rap GTP-Binding Proteins; D003545:Cysteine