The Ras signalling pathway

Ras is an oncogene. Mutations that cause overexpression or constitutive activity of Ras are found in many different forms of cancer. In colon cancer Ras mutations are involved in the progression from benign to malignant adenomas - once Ras is mutant it only requires mutations in one more gene for the tumour to become malignant. The Ras signalling pathway has been analysed using a combination of biochemical studies of proteins that interact with Ras in mammalian cells and genetic studies of Drosophila eye development or vulva induction in C.elegans.

Initial biochemical and molecular genetic (cloning) studies showed that Ras is a membrane bound G-protein which is inactive when bound to GDP and active when bound to GTP. Oncogenic forms of Ras bind GTP permanently. These studies also indicated a link between Ras activation and growth factor-receptor binding.

The receptors for growth factors such as EGF, which can stimulate Ras activity belong to a group called the receptor tyrosine kinases or RTKs. RTKs are transmembrane proteins the extracellular domain of which binds a ligand such as EGF or TGFa while the intracellular domain has tyrosine kinase activity. Ligand binding induces dimerisation and autophosphorylation of tyrosine residues.

Biochemical studies implicate a number of cellular proteins, which were found to be tyrosine phosphorylated following RTK activation or that formed complexes with activated RTKs as possible links between RTK phosphorylation and Ras activation. These studies were unable to identify which of these candidate molecules interacted with RTKs or Ras in vivo or to determine the order in which they might act. Answers to these questions were obtained by genetic studies of Ras signalling during vulva induction in C. elegans and the development of the R7 photoreceptor in Drosophila.

R7 development and Ras

A Drosophila homologue of Ras (Dras) was uncovered in a screen for dominant enhancers of the sevenless phenotype (E(sev)). Dras mutations were homozygous lethal. Induced mitotic recombination was used to show that clones that were homozygous for Dras in the eye lack R7 cells.

A second E(sev)mutant called Son of sevenless (Sos) was also homozygous lethal and as for Dras clones homozygous for Sos in the eye lacked R7 cells. Oncogenic forms of Dras expressed in the eye could rescue the Sos phenotype demonstrating that Sos acts upstream of Dras

Sos shows homology with Guanadine Nucleotide Exchange Factors. It does not bind SEV directly and its activity is unaffected by RTK signalling, however, SOS translocates to membrane when SEV is active which brings it into association with membrane bound Dras.

C.elegans vulva induction and Ras

In C. elegans we have already seen that three genes involved in vulva induction are homologues of EGF (lin-3), the EGF receptor (let-23) and Ras (let-60). Another gene with a Vul mutant phenotype sem-5 was found to act inbetween let-23 and let-60 by epistasis analysis. Sem-5 contains an SH2 domain flanked by SH3 domains. SH2 domains had previously been shown in biochemical studies to bind phosphorylated tyrosine residues on RTKs and SH3 domains to bind proline rich sequences in the SOS GNEF. Sem-5 is homologous to the mammalian adapter protein Grb2 and Drosophila Drk

So the molecular mechanism by which RTK activation induces Ras activity is as follows:-

• Ligand binds RTK inducing dimerisation and autophosphorylation
• Adaptor protein SH2 domains then bind phosphotyrosine.
• The SH3 domains of the adaptor protein are bound to GNEF in the cytosol and the whole Adaptor-GNEF complex translocates to the membrane where GNEF activates Ras

References

Molecular Cell Biology 6th  Edition

Ras signalling pp 684-688

Invertebrate Signal transduction

Dickson and Hafen (1994)

Current Opinion in Genetics and Development 4 64-70.