Studies of the interactions of Ras protein with its regulators.
Item
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Title
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Studies of the interactions of Ras protein with its regulators.
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Identifier
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AAI9618108
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identifier
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9618108
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Creator
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Sung, Ying-Ju.
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Contributor
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Adviser: Yu Wen Hwang
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Date
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1995
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Cell | Chemistry, Biochemistry
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Abstract
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Ras proteins structural elements required for interacting with their regulators were determined. Dominant negative ras mutants were used to achieve this goal. Specifically, two types of mutants were employed: (1) H-ras(N116Y), (N116I), (K117E); (2) N-ras(G60A). These two classes of mutants affect two different functional domains in ras. Mutants v-H-ras(N116I) and v-H-ras(K117E) form stable complexes with guanine-nucleotide exchange factor (GEF), thereby inhibiting guanine-nucleotide exchange. In contrast, the G60A mutant primarily affects the interactions with effectors.;In the first part of this thesis Sdc25p-C, SDC25 C domain gene product, a ras GEF, was used to differentiate H-ras and K-ras guanine-nucleotide exchange activities and binding affinities. H-ras and K-ras differ extensively in their C-termini. Therefore, the interaction of H-ras and K-ras' C-terminal domain with Sdc25p-C was investigated. Studies using ras chimeras showed that H-ras and K-ras interact with Sdc25p-C in a highly selective manner. Ras p21{dollar}\sp\prime{dollar} hypervariable C-terminal domain is the main determinant for ras{dollar}\bullet{dollar}Sdc25p-C complex formation. These studies represent the first functional assignment of the C-terminal domain.;The second part of this thesis focused on characterizing H-ras-(G60A). This residue is conserved in all regulatory GTPases, and is critical for GTP-induced conformational change. Mutating Gly-60 to Ala hindered the GTP-induced conformational change of ras, abolished ras{dollar}\sp\prime{dollar} biological activity, and reduced the GTPase (both intrinsic and GAP-stimulated) of ras. This mutation also substantially decreased the ras-Raf interaction. Interestingly, rasGAP interaction was not affected by the mutation. Ras(G60A) is a dominant negative mutant against viral (oncogenic) H-ras. The inhibition was found to be due to the limitation of Raf. These studies established that Raf is likely to be the direct cellular target of ras protein.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
