Molecular BIology of Cancer Topics             

Ras Oncogenes

Ras oncogenes are the most common oncogenes in human tumors (30% of human tumors have a ras oncogene). Ras proto-oncogenes are expressed in all human cell types and are highly conserved among eukaryotes (Table 1). H-, K-, and N-Ras proteins are signal transducing GTPases, farnecylated at a carboxy-terminus cysteine. Ras proteins cycle between a GDP-bound and a GTP-bound states: GDP-Ras is inactive, GTP-Ras is active.

Ras-GDP is activated by a GTP-GDP exchange factors (GEF, e.g. SOS) that releases GDP from Ras and allows GTP to bind in its place (because GTP is more abundant than GDP in the cell). Ras-GTP is inactivated by its own intrinsic GTPase activity, where Ras hydrolyzes GTP to form GDP + PO. Ras GTPase activity is accelerated 100 fold by a GTPase activating protein (GAP, e.g. GAP or NF1).

Activated Ras bind proteins like Raf and PI3 kinase through its effector domain (Loop 2). In addition to this effector domain and the farnesylation domain responsible for membrane attachement, Ras contains domains for GTP/GDP binding, and GAP binding (see figure). L2 swings out when Ras binds GTP and then it can bind a Ras effector protein (see figure).

Post-translational modifications of Ras include farnesylation at Cys 186, cleavage of amino acids 187-189, carboxymethylation of Cys186, and palmitylation at one or more additional Cys residues. MVA is converted to farnesylpyrophosphate (FPP), then FPP is attached to Ras.

Ras functions in signal transduction pathways for most mammalian growth factors and cytokines. Signals that increase the levels of Ras-GTP include PDGF, EGF, TGFb, Src, Abl and Neu. In normal cells, addition of a growth factor increases the ratio of Ras-GTP to Ras-GDP within minutes (Satoh et. al. Proc Natl Acad Sci USA 87: 7926-7929, 1990).

Signaling Pathways

After a receptor tyrosine kinase is activated GRB-2 binds to the activated growth factor receptor (through its SH2 domain) and brings SOS close to Ras. The GTP-GDP exchange factor SOS activates Ras, and Ras-GTP can now bind its effector, for example Raf. Ras brings Raf near the plasma membrane, were other plasma membrane proteins or lipids activate Raf.

A true Ras effector should interact preferentially with GTP-bound Ras (and not GDP-bound Ras) and continue the signal transduction cascade, bind oncogenic Ras proteins and not bind Ras effector domain mutants. While Ras has many effector molecules, only Raf and PI3K have a known role in transformation.

Raf phosphorylates and activates MEK, and then MEK phosphorylates and activates MAP kinase. The Raf, MEK, and MAP kinases are downstream of Ras in signal transduction pathways. Microinjection of Ras protein into cells activates MAP kinase. Dominant negative Ras mutations block activation of Raf and MAP kinase by tyrosine kinases. Ras effector domain mutants block MAP kinase activation.

The Akt/PKB (protein kinase B) has a pleckstrin homology (PH) domain, a kinase domain, and a tail. The PH domain of PKB/Akt binds to the membrane phospholipids PtdIns (3,4,5)P3 or PtdIns (3,4)P2, both products of PI3 kinase. Thus PKB/Akt is translocated from the cytosol to the inner face of the plasma membrane by the action of PI3. Once at the membrane, other kinases phosphorylate PKB/Akt. Akt is encoded by a proto-oncogene.

To end the signaling, Ras hydrolyzes GTP to GDP and becomes inactive.

Oncogenes

Ras oncogenes in human tumors or chemically-induced animal tumors contain a single missense mutation in codons 12, 13, 59 or 61. Point mutations of codon 12 resulting in missense from Gly to any other amino acid (except Pro) results in a ras oncogene. v-Ha-ras contains two point mutations: Gly 12 changed to Arg 12 and Ala 59 changed to Thr 59.

All of the Ras oncogenic mutations affect its GTPase activity such that Ras canot hydrolyze GTP and is sensitive to GAP. Thus oncogenic Ras is always bound to GTP and active, and its GTP/GDP ratio is no longer responsive to growth factor levels.

Ras can become activate in a variety of ways in cancer cells, not just due to an oncogenic ras gene:

• By an activated or overexpressed receptor tyrosine kinase (e.g. Neu, Src or Bcr/Abl oncogenes)
• Overexpressed exchange factor (e.g. GEF, SOS)
• Loss of GAP proteins (e.g. NF-1)

Ras can also be inactivated by mutations, especially those that affect its farnesilation of its effector domains.

Farnesylation is important for Ras association to the plasma membrane, and also important for its oncogenic action. Experimentally, Ras mutants that can not be farnesylated fail to associate with the plasma membrane and have no oncogenic activity (no growth in soft agar).

The Ras effector domain (Loop 2) interacts with the effector protein. Deletion of or point mutations in the effector domain abolish transformation by the ras oncogene. Effector domain mutations do not impair GTP or GDP binding. Effector domain mutations also do not impair Ras farnesylation, methylation, palmitylation, or membrane localization.

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