Characterization of Saccharomyces cerevisiae Ras1p and chimaeric constructs of Ras proteins reveals the hypervariable region and farnesylation as critical elements in the adenylyl cyclase signaling pathway

Biochemistry. 2003 Dec 23;42(50):14903-12. doi: 10.1021/bi0349928.

Abstract

Ras1p and Ras2p, from Saccharomyces cerevisiae, are GTP-binding proteins that are essential elements in the signaling cascade leading to the activation of adenylyl cyclase. To overcome proteolytic activities that have hampered biochemical studies of Ras1p so far, its gene was genetically modified after which full-length Ras1p could be obtained. The interaction of farnesylated and unprenylated Ras1p with guanine nucleotides, guanine nucleotide exchange factors, GTPase activating proteins, and adenylyl cyclase was compared to Ras2p and human Ha-Ras interactions. Farnesylation of Ras proteins was demonstrated to be a prerequisite for membrane-bound guanine nucleotide exchange factor dependent formation of Ras-GTP complexes, and for efficient Ras-mediated adenylyl cyclase activation. To relate observed functional deviations with sequence differences between Ras1p and Ras2p, which reside almost exclusively within the hypervariable region, truncated versions and chimaeras of the Ras proteins were made. The characteristics of these constructs point to the presence of the hypervariable region of yeast Ras proteins for an efficient activation of adenylyl cyclase. The importance of the latter was confirmed as inhibition of the activation of adenylyl cyclase by an isolated farnesylated hypervariable region of Ras2p could be shown. This strongly suggests that the hypervariable region of Ras proteins can interact directly with adenylyl cyclase.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclase Inhibitors
  • Adenylyl Cyclases / metabolism
  • Adenylyl Cyclases / physiology*
  • Cell Cycle Proteins / metabolism
  • Cell Cycle Proteins / physiology
  • Cell Membrane / metabolism
  • Enzyme Activation / genetics
  • Fungal Proteins / metabolism
  • Fungal Proteins / physiology
  • Genes, ras
  • Genetic Variation*
  • Guanine Nucleotide Exchange Factors / metabolism
  • Guanine Nucleotide Exchange Factors / physiology
  • Humans
  • Oncogene Protein p21(ras) / chemistry
  • Oncogene Protein p21(ras) / genetics
  • Protein Binding / genetics
  • Protein Prenylation / genetics
  • Protein Structure, Tertiary / genetics
  • Recombinant Fusion Proteins / chemical synthesis*
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemical synthesis*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / isolation & purification
  • Saccharomyces cerevisiae Proteins / metabolism
  • Signal Transduction / genetics*
  • ras GTPase-Activating Proteins / physiology
  • ras Guanine Nucleotide Exchange Factors / physiology
  • ras Proteins / chemical synthesis*
  • ras Proteins / chemistry
  • ras Proteins / genetics
  • ras Proteins / isolation & purification
  • ras Proteins / metabolism
  • ras-GRF1 / metabolism
  • ras-GRF1 / physiology

Substances

  • Adenylyl Cyclase Inhibitors
  • CDC25 protein, S cerevisiae
  • Cell Cycle Proteins
  • Fungal Proteins
  • Guanine Nucleotide Exchange Factors
  • Recombinant Fusion Proteins
  • SDC25 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • ras GTPase-Activating Proteins
  • ras Guanine Nucleotide Exchange Factors
  • ras-GRF1
  • Oncogene Protein p21(ras)
  • ras Proteins
  • Adenylyl Cyclases