Impact of different K-ras mutations on tumor progression and drug sensitivity in non-small-cell lung cancer

Ras proteins are signal switch molecules that regulate cell fates by coupling receptor activation to downstream effector pathways that control different cellular responses, like proliferation, differentiation and survival. The 3 members of the ras gene family are the N-rass gene, located at chromosome 1, the H-rass gene at chromosome 11, and the K-rass gene at chromosome 12. These 3 genes encode four highly homologous 21 kD proteins: H-rass, N-rass, K-rass4A and K-rass4B.

The Ras proteins are 188 amino acids in lenght for H-rass, N-rass and K-rass4A, while K-rass4B contains an additional amino acid. Amino acids 1-165 are highly conserved between isoforms, while carboxy terminal 25 amino acids show some variations. In the conserved domain, there are several motifs important for protein function, including GTP binding domain, effector binding domain and Switch I and Swtich II loops that are responsible for Guanine nucleotide Exchange Factor (GEF) and GTPase Activating Protein (GAP) interactions. The variable region within the carboxy.terminus, instead, contains sequences important for post-translational modification, including the CAAX box responsible for targeting lipid modification (K-rass4A and K-rass4B differ in this region, because of differential post-translational modification). Indeed, membrane localization of Ras proteins is fundamental for its function, so these molecules must undergo a four stage post-translational lipid modification, that is necessary because they are normally small and hydrophilic.

First of all, proteins undergo prenylation, the addition of a farnesyl group to the carboxy terminus catalyzed by the enzyme farnesyltransferase. In the presence of a farnesyltransferase inhibitor, a geranylgeranyl group may be added, except for H-rass, to the carboxy terminus by geranylgeranyl transferase type I (GGT-1); these enzymes, however, recognize the same CAAX motif. In the second step of post-translational modification, prenylated Ras proteins undergo proteolytic cleavage of the terminal AAX motif by CAAX proteases Rce1 or Acf1. Third, carboxymethylation occurs at the terminus via s-adenosyl methionine and a specific transferase. Finally, all Ras proteins, except K-ras4B, undergo palmitoylation of SH group of cysteine residues close to the carboxy terminus. K.ras4B, instead, is able to associate with the membrane due to electrostatic interactions through a cluster of positively charged lysine residues, so it is not necessary to undergo the final step for it.