title: Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation.
abstract: Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1). Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain. Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site. On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently. Thus, the strength of interaction at the second Ras-binding site appears to be a critical determinant of CYR1 regulation by Ras: too-weak and too-strong interactions are both detrimental to CYR1 activation. These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Ras-binding site in effector regulation.
authors.affiliation: Department of Physiology II, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan.
authors.forename: F
authors.initials: F
authors.lastname: Shima
authors.forename: T
authors.initials: T
authors.lastname: Okada
authors.forename: M
authors.initials: M
authors.lastname: Kido
authors.forename: H
authors.initials: H
authors.lastname: Sen
authors.forename: Y
authors.initials: Y
authors.lastname: Tanaka
authors.forename: M
authors.initials: M
authors.lastname: Tamada
authors.forename: C D
authors.initials: CD
authors.lastname: Hu
authors.forename: Y
authors.initials: Y
authors.lastname: Yamawaki-Kataoka
authors.forename: K
authors.initials: K
authors.lastname: Kariya
authors.forename: T
authors.initials: T
authors.lastname: Kataoka
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D004789:Enzyme Activation; D008840:Microfilament Proteins; D009154:Mutation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C410502:Capt protein, Drosophila; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D008840:Microfilament Proteins; C073751:SRV2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D018631:ras Proteins; D000262:Adenylyl Cyclases