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Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1

X F Zhang1, J Settleman, J M Kyriakis

  • 1Diabetes Unit, Harvard Medical School, Charlestown, Massachusetts.

Nature
|July 22, 1993
PubMed

Insights

Ras and Raf-1 proteins interact directly, with Raf-1 inhibiting Ras-GAP activity. Mutations affecting Ras binding also impair Ras transforming activity, revealing a key signaling pathway.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Oncogenesis

Background:

  • Ras and Raf-1 are proto-oncoproteins crucial for transducing growth and differentiation signals in higher eukaryotes.
  • These signals are typically initiated by tyrosine kinases, highlighting the importance of this pathway in cellular regulation.

Purpose of the Study:

  • To investigate the direct interaction between the Ras polypeptide and the amino-terminal regulatory domain of Raf-1 (residues 1-257).
  • To determine the binding preference of Raf-1 (1-257) for different Ras-GTP/GDP states and its effect on Ras-GAP activity.
  • To analyze the impact of mutations in the Ras effector domain on Raf-1 binding and Ras transforming activity.

Main Methods:

  • In vitro interaction assays to demonstrate direct binding between Ras and Raf-1 (1-257).
  • Yeast expression system utilized to confirm the interaction in a cellular context.
  • Assays to assess the binding affinity of Raf-1 (1-257) to GTP-Ras versus GDP-Ras.
  • Inhibition assays to evaluate the effect of Raf-1 (1-257) on Ras-GTPase-activating protein (Ras-GAP) activity.
  • Site-directed mutagenesis of the Ras effector domain to study the consequences of altered binding.

Main Results:

  • Direct interaction between the Ras polypeptide and the amino-terminal regulatory domain of Raf-1 (residues 1-257) was confirmed both in vitro and in a yeast expression system.
  • Raf-1 (1-257) demonstrated a preference for binding GTP-Ras over GDP-Ras.
  • The interaction with Raf-1 (1-257) resulted in the inhibition of Ras-GAP activity.
  • Mutations within or near the Ras effector domain concurrently impaired Ras binding to Raf-1 (1-257) and abolished Ras transforming activity.

Conclusions:

  • The Ras polypeptide and the amino-terminal regulatory domain of Raf-1 directly interact, forming a critical component of the Ras signaling pathway.
  • The binding preference for GTP-Ras and the inhibition of Ras-GAP activity by Raf-1 suggest a mechanism for signal amplification or regulation.
  • The parallel impairment of Raf-1 binding and transforming activity by effector domain mutations underscores the functional significance of this interaction in oncogenesis.

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