The ras superfamily of GTP-binding proteins: guidelines on nomenclature.
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Animals↗
Biomedical subjects
C J Der
Publications and source records attributed to C J Der.
Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.
While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.
3T3 Cells↗
Protein prenylation: key to ras function and cancer intervention?
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Alkyl and Aryl Transferases↗
The ras/cholesterol connection: implications for ras oncogenicity.
The frequent detection of mutated ras genes in a variety of cancers (reviewed in Bos, 1988, 1989; Der, 1988) suggests that ras makes a significant contribution to human malignancies (reviewed in Barbacid, 1987; Lacal and Tronick, 1988; Der, 1989). While the role of ras in malignancy is unclear, it is well-established that the association of ras protein with the inner surface of the plasma membrane is critical for triggering ras oncogenicity. The trafficking of ras proteins to the plasma membrane requires a series of three closely linked posttranslational modifications (farnesylation, proteolysis, and carboxymethylation) that are signaled by the consensus C-terminal CAAX motif present on all ras proteins (reviewed in Rine and Kim, 1990; Gibbs, 1991; Der and Cox, 1991). THe recent discovery that an essential intermediate in cholesterol biosynthesis, the isoprenoid farnesol, is attached covalently to ras proteins has stimulated considerable interest and has identified important new directions for studies of ras function. First, understanding the role of farnesol-linked interactions with the plasma membrane may identify the biochemical basis for the oncogenic actions of ras proteins. Second, the enzymes that catalyze the processing steps that trigger membrane association of ras proteins are promising targets for pharmacologic intervention in ras-associated disease. In this review, we summarize our current knowledge of the role of posttranslational processing in ras-membrane interaction and transforming activity. We also provide an update of recent studies addressing the role of isoprenoid modification in the function of ras and of other isoprenoid-modified proteins (reviewed in James and Olson, 1990; Glomset et al., 1990; Maltese, 1990). While this role is likely to be specific for each protein, ras proteins can provide an excellent prototype for understanding the role of isoprenoid modification in protein function.
Amino Acid Sequence↗
Prenoids and palmitate: lipids that control the biological activity of Ras proteins.
Ras proteins can be modified by two types of lipids--an isoprenoid and the fatty acid palmitate. These lipids help the otherwise cytoplasmic Ras protein to interact with the plasma membrane of a cell. The biological consequences of this association between Ras and membranes are dramatic, and can alter a cell's behavior from normal growth into malignancy. The scope and limits of our knowledge of the steps, structures and enzymes involved in this molecular transformation from soluble inactivity to membrane-bound potency are offered below. The prospects of regulating Ras function by controlling its intracellular location provides a tantalizing opportunity to translate research into a novel therapeutic reality.
Amino Acid Sequence↗
Alternate mechanisms of ras activation are complementary and favor and formation of ras-GTP.
The mechanisms of ras activation by mutations in residue 61 and in the NKXD guanine nucleotide-binding consensus sequence (ras residues 116-119) have been evaluated. Weakly transforming mutations that either reduce intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (61P) or enhance guanine nucleotide exchange rates (116H, 119E) were combined into the same H-ras proteins. The resulting double-mutant proteins exhibited significantly stronger transforming forming activities than are observed with each individual mutation, suggesting that the consequences of these two different mechanisms of activation favor maintenance of ras in the active form, which is GTP bound. In vivo nucleotide association analysis demonstrated a direct relationship between ras-GTP formation and transforming activity. Although both 61P and 61L mutations result in reduced intrinsic GTPase activity and loss of GAP stimulation in vitro, only H-ras(61L) exhibits strong transforming activity. While H-ras(61L) is found predominantly in the GTP-bound form, H-ras(61P) is predominantly complexed with GDP in vivo. Thus, in vitro GAP stimulation of GTPase activity does not directly correlate with transforming potential, suggesting that other ras-specific regulatory components may also be important in regulating the cycling of ras between CDP- and GTP-bound states.
3T3 Cells↗
Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils.
The Ras-related protein, Rap1B, has previously been shown to serve as a PKA substrate in vitro and to be phosphorylated by cAMP elevating agents in human platelets. We have purified a Rap1 protein that serves as a PKA substrate from human neutrophils, and we now identify this protein as Rap1A. A 23-kDa protein that co-migrated with recombinant Rap1A was phosphorylated in electroporated human neutrophils upon stimulation by cAMP in the presence of [gamma-32P]ATP. This protein could be immunoprecipitated by the Rap1A/B-specific antibody, R61. The 23-kDa phosphoprotein was monitored during the purification of Rap1 from neutrophil membrane extracts and was shown to copurify with Rap1 during the DEAE Sephacel, heptylamine Sepharose, and MonoQ chromatography steps utilized. The purified protein was phosphorylated to an extent of 1 mol phosphate/mol GTP gamma S bound. This protein was identified as Rap1A by: 1) amino acid sequence analysis; and 2) immunoblotting with a Rap1A-specific antibody. The amino acid phosphorylated on Rap1A by PKA was a serine residue. The site of phosphorylation was indicated by carboxypeptidase digestion and confirmed using a mutant recombinant Rap1A lacking the relevant serine (serine-180). Rap1A, not Rap1B, appears to be the major 23-kDa PKA substrate in human neutrophils. It is possible that Rap1A plays a role in human neutrophils in mediating the inhibitory effects of cAMP-elevating agents upon chemoattractant-stimulated cell activation.
Electrophoresis, Gel, Two-Dimensional↗
Isoprenoid modification of rab proteins terminating in CC or CXC motifs.
Mevalonate starvation of hamster fibroblasts resulted in a shift of rab1b from the membrane to the cytosolic fraction, suggesting that rab1b depends upon an isoprenoid modification for its membrane localization. rab1b and rab3a proteins expressed in insect cells incorporated a product of [3H]mevalonate, and gas chromatography analysis of material released by Raney nickel cleavage demonstrated that rab1b and rab3a are modified by geranylgeranyl groups. Additionally, in vitro prenylation analysis demonstrated farnesyl modification of H-ras but geranylgeranyl modification of five rab proteins (1a, 1b, 2, 3a, and 6). Together, these results suggest that the carboxyl-terminal CC/CXC motifs (X = any amino acid) specifically signal for addition of geranylgeranyl, but not farnesyl, groups. A rab1b mutant protein lacking the two carboxyl-terminal cysteine residues was not prenylated in vitro. However, since a mutant H-ras protein that terminates with tandem cysteine residues was also not modified, the CC motif may be essential, but not sufficient, to signal prenylation of rab1b. Finally, rab1b and rab3a proteins were not efficient substrates for either farnesyl- or geranylgeranyltransferase activities that modify CAAX-containing proteins (A = any aliphatic amino acid). Therefore, rab proteins may be modified by a prenyltransferase(s) distinct from the prenyltransferases that modify carboxyl-terminal CAAX proteins.
Amino Acid Sequence↗
Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.
We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.
Animals↗
The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.
Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.
Animals↗
Isoprenoid modification and plasma membrane association: critical factors for ras oncogenicity.
Association of ras protein with the plasma membrane is critical for its transforming activity. This association is promoted by a series of post-translational modifications that are signaled by the consensus C-terminal CAAX motif present in all ras proteins. The recent discovery that a 15-carbon isoprenoid (farnesyl) group, derived from an essential intermediate in cholesterol biosynthesis, is attached covalently to ras proteins has stimulated considerable interest and has suggested several important new directions for ras studies. In particular, one promising pharmacologic approach for antagonizing oncogenic ras activity in human malignancies would be to design specific inhibitors of the enzymes that catalyze ras processing and thereby interfere with ras protein association with the plasma membrane.
Amino Acid Sequence↗
Farnesol modification of Kirsten-ras exon 4B protein is essential for transformation.
Oncogenic forms of ras proteins are synthesized in the cytosol and must become membrane associated to cause malignant transformation. Palmitic acid and an isoprenoid (farnesol) intermediate in cholesterol biosynthesis are attached to separate cysteine residues near the C termini of H-ras, N-ras, and Kirsten-ras (K-ras) exon 4A-encoded proteins. These lipid modifications have been suggested to promote or stabilize the association of ras proteins with membranes. Because preventing isoprenylation also prevents palmitoylation, examining the importance of isoprenylation alone has not been possible. However, the oncogenic human [Val12]K-ras 4B protein is not palmitoylated but is isoprenylated, membrane associated, and fully transforming. We therefore constructed mutant [Val12]K-ras 4B proteins that were not isoprenylated to examine the effects of isoprenylation in the absence of palmitoylation. The nonisoprenylated mutant proteins both failed to associate with membranes and did not transform NIH 3T3 cells. In addition, inhibition of isoprenoid and cholesterol synthesis with the drug compactin also decreased [Val12]K-ras 4B protein isoprenylation and membrane association. These results unequivocally demonstrate that isoprenylation, rather than palmitoylation, is essential for ras membrane binding and ras transforming activity. These findings clearly indicate the biological significance of ras protein modification by farnesol and suggest that this modification may be important for facilitating the processing, trafficking, and biological activity of other isoprenylated proteins. Because K-ras is the most frequently activated oncogene in a wide spectrum of human malignancies, study of this pathway could lead to important therapeutic treatments.
Animals↗
Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.
Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.
Animals↗
GTP-binding protein-stimulated phospholipase C and phospholipase D activities in ras-transformed NIH 3T3 fibroblasts.
The stable GTP analog, guanosine 5'-(3-O-thiotriphosphate), GTP gamma S, stimulated both inositol trisphosphate (InsP3) and choline generation by NIH 3T3 cell membranes. Choline generation was stimulated by GTP gamma S over the dose range for activation of GTP-binding proteins. Membranes from control and c-Ha-ras- or c-Ha-ras(61 leu)-transformed cells did not differ in the extent to which GTP gamma S stimulated InsP3 or choline formation despite 5-10 fold over expression of Ras in the transformed cells. Unlike GTP gamma S, GTP did not stimulate phospholipid hydrolysis, even in membranes from cells expressing Ras61leu, a mutant protein having reduced GTPase activity. Thus there is G protein regulation of both phosphatidylcholine-specific phospholipase D and polyphosphoinositide-specific phospholipase C in NIH 3T3 cell membranes. However, the lack of difference in GTP gamma S-stimulated phospholipid metabolism between control and ras-transformed cell membranes suggests that Ras does not function as the G protein(s) that directly regulate either phospholipase.
Adenosine Triphosphatases↗
Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation.
We have located the distal boundary of the tyrosine kinase domain of the EGF receptor and have identified a distinct sequence in the C' terminus required for EGF-dependent receptor internalization, leading to receptor down-regulation and degradation. Within this receptor domain, an 18 amino acid highly negatively charged region of predicted helical structure is required both for endocytosis via a high-affinity, saturable pathway and for ligand-stimulated increases in cytosolic calcium. In contrast to kinase-inactive, internalization-competent receptors, kinase-active, internalization-defective receptors effectively signaled gene transcription, morphological transformation, and growth. These observations support the hypothesis that mitogenic responses to EGF are mediated by activation of the intrinsic protein tyrosine kinase activity of the membrane-bound receptor, with ligand-induced internalization serving to terminate the signal.
Amino Acid Sequence↗
Activation of the cellular proto-oncogene product p21Ras by addition of a myristylation signal.
The 21-kD proteins encoded by ras oncogenes (p21Ras) are modified covalently by a palmitate attached to a cysteine residue near the carboxyl terminus. Changing cysteine at position 186 to serine in oncogenic forms produces a nonpalmitylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. Nonpalmitylated p21Ras derivatives were constructed that contained myristic acid at their amino termini to determine if a different form of lipid modification could restore either membrane association or transforming activity. An activated p21Ras, altered in this way, exhibited both efficient membrane association and full transforming activity. Surprisingly, myristylated forms of normal cellular Ras were also transforming. This demonstrates that Ras must bind to membranes in order to transmit a signal for transformation, but that either myristate or palmitate can perform this role. However, the normal function of cellular Ras is diverted to transformation by myristate and therefore must be regulated ordinarily by some unique property of palmitate that myristate does not mimic. Myristylation thus represents a novel mechanism by which Ras can become transforming.
Animals↗
Activation of ras oncogenes in chemically transformed BALB/MK-2 mouse keratinocytes.
BALB/MK-2 cells are an epidermal growth factor (EGF)-dependent cell line derived from BALB/c mouse epidermis that can undergo terminal differentiation under appropriate conditions. Previous studies have shown that transformation of these cells by retroviral oncogenes relieves the EGF requirement while blocking the terminal differentiation program. In this report we show that BALB/MK-2 cells are sensitive to transformation by the chemical carcinogens dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (MCA), and N-methyl-N'-nitro-N'-nitroso-guanidine (MNNG). BALB/MK-2 cells transformed by these carcinogens proliferate in the absence of EGF and do not undergo terminal differentiation in response to calcium. However, the cells retain their anchorage growth dependence and are nontumorigenic in nude mice. NIH 3T3 transfection analysis showed that the endogenous Ha-ras gene had been activated in both DMBA- and MNNG-transformed cells and the Ki-ras gene had been activated in the MCA-transformed cells. Additionally, non-ras transforming activity was detected in some MNNG-transformed BALB/MK-2 cells. Thus, the BALB/MK-2 cell line provides a reproducible in vitro assay system for chemical transformation of epithelial cells and for identification of oncogene activations associated with changes in growth control and differentiation.
9,10-Dimethyl-1,2-benzanthracene↗
The ras family of oncogenes.
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Amino Acid Sequence↗