Ras history: The saga continues

Abstract

Although the roots of Ras sprouted from the rich history of retrovirus research, it was the discovery of mutationally activated RAS genes in human cancer in 1982 that stimulated an intensive research effort to understand Ras protein structure, biochemistry and biology. While the ultimate goal has been developing anti-Ras drugs for cancer treatment, discoveries from Ras have laid the foundation for three broad areas of science. First, they focused studies on the origins of cancer to the molecular level, with the subsequent discovery of genes mutated in cancer that now number in the thousands. Second, elucidation of the biochemical mechanisms by which Ras facilitates signal transduction established many of our fundamental concepts of how a normal cell orchestrates responses to extracellular cues. Third, Ras proteins are also founding members of a large superfamily of small GTPases that regulate all key cellular processes and established the versatile role of small GTP-binding proteins in biology. We highlight some of the key findings of the last 28 years.

Figure 1

Timeline of representative key discoveries in Ras research. See Suppl. Table 1 for references.

Figure 1

Timeline of representative key discoveries in Ras research. See Suppl. Table 1 for references.

Figure 1

Timeline of representative key discoveries in Ras research. See Suppl. Table 1 for references.

Figure 1

Timeline of representative key discoveries in Ras research. See Suppl. Table 1 for references.

Figure 2

Ras is a GDP/GTP-regulated binary switch. (A) The three RAS genes encode four 188–189 amino acid proteins that share 82–90% overall sequence identity; KRAS encodes two splice variants due to alternative exon 4 utilization, leading to divergent C-terminal sequences. Exons 4A and 4B encode 39 and 38 amino acids, respectively, with 19 identical and 4 conserved substitutions. K-Ras4A is most similar to viral K-ras while K-Ras4B is the predominant isoform expressed in human cells. Residues 1–164 comprise the G domain that contains six conserved sequence motifs shared with other Ras superfamily and GTP-binding proteins. These motifs are involved in binding either phosphate/Mg²⁺ (PM) or the guanine base (G) of GDP and GTP. Residues in Switch I (aa 30–38) and II (aa 60–76) change in conformation during GDP/GTP cycling. The core effector binding domain (E; residues 32–40) and flanking sequences are involved in effector binding specificity. (B) Regulators of the Ras GDP/GTP cycle. RasGEFs stimulate GDP/GTP exchange. With the 10-fold higher cellular concentrations of GTP over GDP, the net result of RasGEF stimulation is formation of active Ras-GTP. Ras-GTP binds preferentially to downstream effectors. RasGAPs accelerate the intrinsic GTP hydrolysis activity of Ras to promote formation of inactive Ras-GDP. Shown are “classic” missense mutants of Ras proteins that have been useful for dissection of Ras function. The Ras(S17N) dominant negative sequesters and blocks RasGEF activity, preventing Ras activation. The G12V and Q61L mutations, found in human cancers, impair GAP-stimulated GTP hydrolysis. The T35S, E37G and Y40C effector domain mutants (EDMs) differentially impair effector binding. The T35S mutant retains efficient binding to Raf but not PI3K or RalGEF, whereas the E37G mutant retains efficient binding to RalGEF but not Raf or PI3K, and the Y40C mutant retains efficient binding to PI3K but not Raf or RalGEF.

Figure 3

Detection of activated and mutated HRAS in human EJ/T24 bladder carcinoma cells. The NIH/3T3 focus formation assay was used to detect activated oncogenes present in human tumor but not normal genomic DNA. High molecular weight DNA was isolated from the EJ/T24 human bladder carcinoma cell line, converted to a calcium phosphate precipitant, and added to the growth medium of a monolayer of NIH/3T3 cells. After 14 days, foci of morphologically and growth transformed cells can be detected in cultures treated with DNA from tumor cells but not in parallel cultures treated with DNA from normal human cells. The active HRAS fragment from EJ/T24 bladder cells lies within a 4.6 kDa XhoI-SphI fragment. Human H-Ras protein is encoded by sequences spanning four exons. Exon 1 encodes amino acids 1–37. Sequence comparison of the bladder carcinoma-derived HRAS DNA identified a single base substitution at codon 12, resulting in a single missense mutation (G12V).

Figure 4

Ras mutations in cancer and developmental disorders. Missense mutations in (A) H-Ras, (B) K-Ras and (C) N-Ras in human cancers were compiled from COSMIC (www.sanger.ac.uk/genetics/CGP/cosmic/). Each specific substitution seen at residue 12, 13 or 61 is indicated separately (pink boxes placed above the Ras protein ribbon), followed in parentheses by the number of cancers identified to have that mutation. The numbers and types of missense mutations in each Ras isoform found in developmental syndromes (RASopathies) were compiled from The Ras/MAPK Syndrome Homepage (www.medgen.med.tohoku.ac.jp/RasMapk%20syndromes.html). Specific substitutions are indicated (green boxes) below the Ras protein ribbon and numbers are given in parentheses after each mutation. (D) Distribution of Ras missense mutations in cancer. The distribution of mutations in H-Ras, K-Ras and N-Ras was calculated from data in COSMIC depicted in (A–C). The percentages of missense mutations at 12, 13, 61 and all other positions were determined for H-Ras (629 total mutations), K-Ras (15,594 total) and N-Ras (2,189 total).

Figure 4

Ras mutations in cancer and developmental disorders. Missense mutations in (A) H-Ras, (B) K-Ras and (C) N-Ras in human cancers were compiled from COSMIC (www.sanger.ac.uk/genetics/CGP/cosmic/). Each specific substitution seen at residue 12, 13 or 61 is indicated separately (pink boxes placed above the Ras protein ribbon), followed in parentheses by the number of cancers identified to have that mutation. The numbers and types of missense mutations in each Ras isoform found in developmental syndromes (RASopathies) were compiled from The Ras/MAPK Syndrome Homepage (www.medgen.med.tohoku.ac.jp/RasMapk%20syndromes.html). Specific substitutions are indicated (green boxes) below the Ras protein ribbon and numbers are given in parentheses after each mutation. (D) Distribution of Ras missense mutations in cancer. The distribution of mutations in H-Ras, K-Ras and N-Ras was calculated from data in COSMIC depicted in (A–C). The percentages of missense mutations at 12, 13, 61 and all other positions were determined for H-Ras (629 total mutations), K-Ras (15,594 total) and N-Ras (2,189 total).

Figure 5

Colorectal and pancreatic cancer progression. (A) Colorectal cancer progression and gene mutations. Colonic epithelial cells undergo a histologic transition from normal to malignant state that is driven by specific genetic events including inactivation of tumor suppressors (APC, SMAD4 and TP53) and activation of the KRAS oncogene. The three stages of adenomas represent tumors of increasing size, dysplasia, and villous content. (B) Pancreatic cancer progression and gene mutations. Multiple tumor types arise from the exocrine pancreas, of which greater than 95% are pancreatic ductal adenocarcinoma (PDAC). Normal ductal epithelium progression to infiltrating cancer (left to right) is illustrated through a series of histologically defined precursor lesions (PanINs) that show increasing degrees of disruption of cellular morphology, nuclear atypia and dysplastic growth. High grade PanIN-3 progresses to invasive PDAC. Activating point mutations in the KRAS gene occur early, inactivation of the p16/INK4A gene occurs at an intermediate stage, and inactivation of the TP53, SMAD4/DPC4 and BRCA2 genes occurs relatively late. Oncogenes are indicated in green text and tumor suppressors in red text.

Figure 6

Conservation of Ras proteins in evolution. (A) Domain architecture of Ras proteins and the boundaries of the constant G domains were determined using SMART (http://smart.embl-heidelberg.de/). (B) Clustal/W was then used to align the G domain sequences, and the percent of amino acid identity was determined and used to generate the dendrogram.

Figure 7

Human and invertebrate Ras superfamily proteins. (A) The numbers of members of each species and of each Ras superfamily branch were obtained from the references cited in Suppl. Table 2, and then used to generate the graphs. Numbers indicate total members per family. The graph of human family proteins was generated using the numbers in reference . (B) Human Ras family proteins. Dendrogram generated by Clustal/W and adapted from reference . (C) Human Rho family proteins. Adapted from reference .

Figure 7

Human and invertebrate Ras superfamily proteins. (A) The numbers of members of each species and of each Ras superfamily branch were obtained from the references cited in Suppl. Table 2, and then used to generate the graphs. Numbers indicate total members per family. The graph of human family proteins was generated using the numbers in reference . (B) Human Ras family proteins. Dendrogram generated by Clustal/W and adapted from reference . (C) Human Rho family proteins. Adapted from reference .

Figure 8

Ras interactome. Proteins that regulate Ras GDP/GTP cycling, catalyze posttranslational modification, or serve as immediate downstream effectors are indicated. Compiled in part from Table 1 in reference .

Figure 9

Ras signaling. (A) Conservation of Raf-MEK-ERK cascade downstream of Ras. (B) Ras effector pathways implicated in Ras-mediated oncogenesis. Compiled in part from literature cited in reference .

Figure 9

Ras signaling. (A) Conservation of Raf-MEK-ERK cascade downstream of Ras. (B) Ras effector pathways implicated in Ras-mediated oncogenesis. Compiled in part from literature cited in reference .

Figure 10

Ras posttranslational processing and membrane association. (A) The C-terminal 24–25 residues comprise the membrane targeting sequence which is comprised of the C-terminal CAAX box, required for posttranslational lipid modification, and the hypervariable (HV) domain that includes a second membrane targeting sequence element (palmitoylated cysteine(s) or polybasic stretches). (B) CAAX motif-signaled posttranslational processing. The cysteine residue of the CAAX box (red) is modified posttranslationally by cytosolic farnesyltransferase (FTase)-catalyzed covalent addition of a C15 farnesyl isoprenoid. Processing is completed on the cytosolic leaflet of the endoplasmic reticulum (ER) by Rce1 (Ras and a-factor converting enzyme-1)-catalyzed proteolytic removal of the AAX residues and then by Icmt (isoprenylcysteine carboxyl methyltransferase)-catalyzed carboxylmethylation of the now terminal farnesylated cysteine residue. FTase inhibitor (FTI) treatment blocks farnesylation and all subsequent CAAX modifications, rendering Ras cytosolic and inactive. Similarly, a serine substitution of the cysteine residue of the CAAX motif (“SAAX”) also prevents farnesylation and all CAAX-signaled modifications. The X residue of the CAAX motif dictates prenyltransferase specificity. Substitution of the X residue with leucine generates mutants that undergo modification by GGTase-I and addition of a C20 geranylgeranyl lipid instead of a C15 farnesyl isoprenoid. (C) Ras plasma membrane association is dependent on a second targeting element. The CAAX-signaled modifications alone are not sufficient to promote plasma membrane association. Additional sequence elements in the HV provide a second membrane targeting signal. In H-Ras, N-Ras and K-Ras4A, cysteine residues in the HV region are modified posttranslationally by ER-associated protein acyltransferases (PATs) that promote covalent addition of a C16 palmitate fatty acid. In K-Ras4B, lysine-rich polybasic amino acids comprise the second signal, which facilitate association with the negatively charged head groups of phosphatidylserine and phosphatidylinositol in the cytosolic face of the plasma membrane. Whereas H-Ras and N-Ras (and presumably K-Ras4A) traffic through the classical secretory pathway through the Golgi to the plasma membrane, K-Ras4B bypasses the Golgi and transits to the plasma membrane by a poorly characterized mechanism. Additionally, the differing second signals of K-Ras4B and H-Ras dictate localization to distinct plasma membrane subdomains, that may lead to distinct effector signaling.

Figure 10

Ras posttranslational processing and membrane association. (A) The C-terminal 24–25 residues comprise the membrane targeting sequence which is comprised of the C-terminal CAAX box, required for posttranslational lipid modification, and the hypervariable (HV) domain that includes a second membrane targeting sequence element (palmitoylated cysteine(s) or polybasic stretches). (B) CAAX motif-signaled posttranslational processing. The cysteine residue of the CAAX box (red) is modified posttranslationally by cytosolic farnesyltransferase (FTase)-catalyzed covalent addition of a C15 farnesyl isoprenoid. Processing is completed on the cytosolic leaflet of the endoplasmic reticulum (ER) by Rce1 (Ras and a-factor converting enzyme-1)-catalyzed proteolytic removal of the AAX residues and then by Icmt (isoprenylcysteine carboxyl methyltransferase)-catalyzed carboxylmethylation of the now terminal farnesylated cysteine residue. FTase inhibitor (FTI) treatment blocks farnesylation and all subsequent CAAX modifications, rendering Ras cytosolic and inactive. Similarly, a serine substitution of the cysteine residue of the CAAX motif (“SAAX”) also prevents farnesylation and all CAAX-signaled modifications. The X residue of the CAAX motif dictates prenyltransferase specificity. Substitution of the X residue with leucine generates mutants that undergo modification by GGTase-I and addition of a C20 geranylgeranyl lipid instead of a C15 farnesyl isoprenoid. (C) Ras plasma membrane association is dependent on a second targeting element. The CAAX-signaled modifications alone are not sufficient to promote plasma membrane association. Additional sequence elements in the HV provide a second membrane targeting signal. In H-Ras, N-Ras and K-Ras4A, cysteine residues in the HV region are modified posttranslationally by ER-associated protein acyltransferases (PATs) that promote covalent addition of a C16 palmitate fatty acid. In K-Ras4B, lysine-rich polybasic amino acids comprise the second signal, which facilitate association with the negatively charged head groups of phosphatidylserine and phosphatidylinositol in the cytosolic face of the plasma membrane. Whereas H-Ras and N-Ras (and presumably K-Ras4A) traffic through the classical secretory pathway through the Golgi to the plasma membrane, K-Ras4B bypasses the Golgi and transits to the plasma membrane by a poorly characterized mechanism. Additionally, the differing second signals of K-Ras4B and H-Ras dictate localization to distinct plasma membrane subdomains, that may lead to distinct effector signaling.

Figure 11

Ras signaling components mutated in RASopathies. Generated based on information summarized in Suppl. Table 3 and from references cited in reference .