Mutations in RIT1 cause Noonan syndrome - additional functional evidence and expanding the clinical phenotype.

RASopathies are a clinically heterogeneous group of conditions caused by mutations in 1 of 16 proteins in the RAS-mitogen activated protein kinase (RAS-MAPK) pathway. Recently, mutations in RIT1 were identified as a novel cause for Noonan syndrome. Here we provide additional functional evidence for a causal role of RIT1 mutations and expand the associated phenotypic spectrum. We identified two de novo missense variants p.Met90Ile and p.Ala57Gly. Both variants resulted in increased MEK-ERK signaling compared to wild-type, underscoring gain-of-function as the primary functional mechanism. Introduction of p.Met90Ile and p.Ala57Gly into zebrafish embryos reproduced not only aspects of the human phenotype but also revealed abnormalities of eye development, emphasizing the importance of RIT1 for spatial and temporal organization of the growing organism. In addition, we observed severe lymphedema of the lower extremity and genitalia in one patient. We provide additional evidence for a causal relationship between pathogenic mutations in RIT1, increased RAS-MAPK/MEK-ERK signaling and the clinical phenotype. The mutant RIT1 protein may possess reduced GTPase activity or a diminished ability to interact with cellular GTPase activating proteins; however the precise mechanism remains unknown. The phenotypic spectrum is likely to expand and includes lymphedema of the lower extremities in addition to nuchal hygroma.

Rit, a non-lipid-modified Ras-related protein, transforms NIH3T3 cells without activating the ERK, JNK, p38 MAPK or PI3K/Akt pathways.

The biological functions of Rit (Ras-like protein in tissues) and Rin (Ras-like protein in neurons), members of a novel branch of Ras-related GTP-binding proteins that are approximately 50% identical to Ras, have not been characterized. Therefore, we assessed their activity in growth control, transformation and signaling. NIH cells stably expressing a constitutively activated mutant of Rit [Rit(79L)] (analogous to the oncogenic mutant H-Ras(61L)) demonstrated strong growth transformation, proliferating rapidly in low serum and forming colonies in soft agar and tumors in nude mice. Although Rit(79L) alone did not promote morphologically transformed foci, it cooperated with both Raf and Rho A to form Rac/Rho-like foci. Rin [Rin(78L)] cooperated only with Raf. Rit(79L) but not Rin(78L) stimulated transcription from luciferase reporter constructs regulated by SRF, NF-kappaB, Elk-1 and Jun. However, neither activated ERK, JNK or p38, or PI3-K/Akt kinases in immune complex kinase assays. Interestingly, although Rit lacks any known recognition signal for C-terminal lipidation, Rit-transformed cell growth and survival in low serum is dependent on a farnesylated protein, as treatment with farnesyltransferase inhibitors caused apoptosis. Rin cooperated with Raf in focus assays but did not otherwise function in these assays, perhaps due to a lack of appropriate effector pathways in NIH3T3 fibroblasts for this neural-specific Ras family member. In summary, although Rit shares most core effector domain residues with Ras, our results suggest that Rit uses novel effector pathways to regulate proliferation and transformation.

Oncogenic RIT1 mutations in lung adenocarcinoma.

Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF and ERBB2, and translocations involving ALK, RET and ROS1. Identification of these oncogenic events has transformed the treatment of lung adenocarcinoma via application of therapies targeted toward specific genetic lesions in stratified patient populations. However, such mutations have been reported in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy are involved in the remaining cases. Here we report somatic mutations in the small GTPase gene RIT1 in ∼2% of lung adenocarcinoma cases that cluster in a hotspot near the switch II domain of the protein. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces cellular transformation in vitro and in vivo, which can be reversed by combined PI3K and MEK inhibition. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas and suggest PI3K and MEK inhibition as a potential therapeutic strategy in RIT1-mutated tumors.

An expression cloning method to identify monomeric GTP-binding proteins by GTP overlay.

We have developed a method for identifying monomeric GTP-binding proteins that is based on probing plasmid expression libraries with [alpha-32P]GTP. The method involves the production of nitrocellulose replica filter lifts from a plasmid cDNA expression library and treatment of the filters with chloroform vapor to lyse the Escherichia coli and to denature and inactivate endogenous E. coli GTP-binding proteins, thus allowing the direct identification of cDNA clones which encode Ras-like small GTP-binding proteins by ligand blotting. Using this procedure we have cloned a series of small Ras-like GTP-binding proteins from human retina. The method relies on a functional test, ligand specificity of the expressed proteins, to identify candidate molecules. This results in the isolation of predominantly full-length cDNA clones without relying on DNA sequence similarity. Thus, this method may be particularly useful for the cloning of novel Ras-related GTP-binding proteins which share limited sequence similarity with previously identified members of the Ras superfamily.

Rem is a new member of the Rad- and Gem/Kir Ras-related GTP-binding protein family repressed by lipopolysaccharide stimulation.

We report the cDNA cloning and characterization of a novel GTP-binding protein, termed Rem (for Rad and Gem-related), that was identified as a product of polymerase chain reaction amplification using oligonucleotide primers derived from conserved regions of the Rad, Gem, and Kir Ras subfamily. Alignment of the full-length open reading frame of mouse Rem revealed the encoded protein to be 47% identical to the Rad, Gem, and Kir proteins. The distinct structural features of the Rad, Gem, and Kir subfamily are maintained including a series of nonconservative amino acid substitutions at positions important for GTPase activity and a unique sequence motif thought to direct membrane association. Recombinant Rem binds GTP in a specific and saturable manner. Ribonuclease protection analysis found Rem to be expressed at comparatively high levels in cardiac muscle and at moderate levels in lung, skeletal muscle, and kidney. The administration of lipopolysaccharide to mice, a potent activator of the inflammatory and immune systems, results in the general repression of Rem mRNA levels in a dose- and time-dependent manner. Thus, Rem is the first Ras-related gene whose mRNA levels have been shown to be regulated by repression.

A novel RalGEF-like protein, RGL3, as a candidate effector for rit and Ras.

The small GTPase Rit is a close relative of Ras, and constitutively active Rit can induce oncogenic transformation. Although the effector loops of Rit and Ras are highly related, Rit fails to interact with the majority of the known Ras candidate effector proteins, suggesting that novel cellular targets may be responsible for Rit transforming activity. To gain insight into the cellular function of Rit, we searched for Rit-binding proteins by yeast two-hybrid screening. We identified the C-terminal Rit/Ras interaction domain of a protein we have designated RGL3 (Ral GEF-like 3) that shares 35% sequence identity with the known Ral guanine nucleotide exchange factors (RalGEFs). RGL3, through a C-terminal 99-amino acid domain, interacted in a GTP- and effector loop-dependent manner with Rit and Ras. Importantly, RGL3 exhibited guanine nucleotide exchange activity toward the small GTPase Ral that was stimulated in vivo by the expression of either activated Rit or Ras. These data suggest that RGL3 functions as an exchange factor for Ral and may serve as a downstream effector for both Rit and Ras.

Phosphorylation-dependent association of the Ras-related GTP-binding protein Rem with 14-3-3 proteins.

Rem belongs to a subfamily of Ras-related GTPases that includes Rad, Gem, and Kir. These proteins are unique among the Ras superfamily since their expression is under transcriptional regulation and they contain distinct amino and carboxyl termini. To gain insight into the cellular function of Rem, we have undertaken an expression screen using a mouse embryo cDNA library to identify Rem-interacting proteins and find that Rem interacts with a series of 14-3-3 isoforms (epsilon, eta, theta, and zeta). Immunoprecipitation studies demonstrate an interaction that is independent of the nucleotide state of Rem. Rem is phosphorylated in vivo, and binding of Rem to 14-3-3zeta is abolished by pretreating Rem with protein phosphatase 1. Thus, the association of Rem and 14-3-3zeta is phosphorylation-dependent. Examination of the interaction between 14-3-3zeta and various Rem deletion mutants mapped a critical binding site to the C-terminus of Rem. Finally, we demonstrate the interaction of Rad but not the newly identified Rem2 protein with 14-3-3 proteins. These results suggest that 14-3-3 may allow the recruitment of distinct proteins that participate in Rem-mediated signal transduction pathways.

Farnesol is utilized for protein isoprenylation and the biosynthesis of cholesterol in mammalian cells.

Evidence has been obtained indicating that free farnesol (F-OH) can be utilized for isoprenoid biosynthesis in mammalian cells. When rat C6 glial cells and an African green monkey kidney cell line (CV-1) were incubated with [3H]F-OH, radioactivity was incorporated into cholesterol, ubiquinone (CoQ) and isoprenylated proteins. The incorporation of label from [3H]F-OH into cholesterol in C6 and CV-1 cells was blocked by squalestatin 1 (SQ) which specifically inhibits the conversion of farnesyl pyrophosphate (F-P-P) to squalene. This result strongly suggests that cholesterol, and probably CoQ and protein, is metabolically labeled via F-P-P. SDS-PAGE analysis of the delipidated protein fractions from C6 and CV-1 cells revealed several labeled polypeptides. Consistent with these proteins being modified by isoprenylation of cysteine residues. Pronase E digestion released a major labeled product with the chromatographic mobility of [3H]farnesyl-cysteine (F-Cys). A different set of polypeptides was labeled when C6 and CV-1 cells were incubated with [3H]geranylgeraniol (GG-OH). Both sets of proteins appear to be metabolically labeled by [3H]mevalonolactone, and [3H]-labeled F-Cys and geranylgeranyl-cysteine (GG-Cys) were liberated from these proteins by Pronase E treatment. These cellular and biochemical studies indicate that F-OH can be used for isoprenoid biosynthesis and protein isoprenylation in mammalian cells after being converted to F-P-P by phosphorylation reactions that remain to be elucidated.

Geranylgeraniol promotes entry of UT-2 cells into the cell cycle in the absence of mevalonate.

Although UT-2 cells, a mutant clone of Chinese hamster ovary cells, have been shown to require mevalonate for growth due to a deficiency in 3-hydroxy-3-methylglutaryl-CoA reductase, the precise mevalonate-derived product(s) essential for proliferation has not been identified. These studies show that UT-2 cells proliferate in the presence of free geranylgeraniol (GG-OH), as well as mevalonate. Cell growth was optimal when the culture medium was supplemented with 5-10 microM GG-OH. Under these growth conditions [3H]GG-OH is actively incorporated into UT-2 proteins. Prominent [3H]geranylgeranylated polypeptides in the size range (19-27 kDa) of the small GTP-binding proteins are observed by SDS-PAGE. Analysis of the butanol-soluble products released from the metabolically labeled proteins by digestion with Pronase E reveals that the proteins contain [3H]geranylgeranylated cysteine residues. Even though [3H]farnesol is also incorporated into cysteinyl residues of a different set of UT-2 proteins, farnesol added at 10 microM did not satisfy the mevalonate requirement for cell growth. These results show that UT-2 cells divide in the presence of exogenously supplied GG-OH, providing evidence that one or more geranylgeranylated proteins are essential for entry of UT-2 cells, and probably other mammalian cells, into the cell cycle.

Utilization of geranylgeraniol for protein isoprenylation in C6 glial cells.

When rat C6 glial cells were incubated with [3H]geranylgeraniol (GGol), radioactivity was incorporated into a delipidated protein fraction. SDS-PAGE analysis of the protein fraction labeled by [3H]GGol revealed a 46 kDa polypeptide and a group of labeled polypeptides (19-27 kDa) in the same size range as the small GTP-binding proteins. A similar pattern of labeled polypeptides was seen when C6 cells were metabolically labeled with [14C]mevalonolactone. An isotopically labeled product, chromatographically identical to geranylgeranylcysteine (GG-Cys), was released by Pronase E digestion of the [3H]GGol-labeled protein. When the protein fraction from cells metabolically labeled with [3H]mevalonolactone was digested with Pronase E, two radiolabeled products were released with the chromatographic mobilities of farnesyl-cysteine (F-Cys) and GG-Cys. These studies suggest that C6 glial cells are capable of converting GGol to geranylgeranyl pyrophosphate (GG-P-P), or perhaps a novel "activated" form of the allylic isoprenol, that can be utilized for protein isoprenylation reactions.

Variants of the carboxyl-terminal KDEL sequence direct intracellular retention.

Soluble proteins which reside in the lumen of the endoplasmic reticulum share a common carboxyl-terminal tetrapeptide Lys-Asp-Glu-Leu (KDEL). Addition of the tetrapeptide to a normally secreted protein is both necessary and sufficient to cause retention in the endoplasmic reticulum. In order to characterize the critical residues in the KDEL signal, cDNAs encoding proneuropeptide Y (pro-NPY) with the 4-amino acid carboxyl-terminal extension KDEL or a series of KDEL variants were expressed in the AtT-20 cell line. AtT-20 cells, a mouse anterior pituitary corticotrope cell line, synthesize, process, and secrete the pro-ACTH/endorphin precursor. Since post-translational processing in AtT-20 cells has been extensively characterized, it provides a model system in which the processing of a foreign peptide precursor (pro-NPY) and the endogenous precursor (pro-ACTH/endorphin) can be compared. Altered cDNAs encoding pro-NPY with KDEL, DKEL, RDEL, KNEL, KDQL, or KDEA at the COOH terminus were used to generate stable AtT-20 cell lines. The processing of pro-NPY to neuropeptide Y and the carboxyl-terminal peptide was studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tryptic peptide mapping, and radiosequencing. Addition of the tetrapeptides KDEL, DKEL, RDEL, or KNEL to the COOH terminus of the neuropeptide Y precursor, a peptide hormone normally processed and secreted from neuronal cells, caused complete intracellular retention of the unprocessed prohormone in AtT-20 cells. However, KDQL and KDEA-extended pro-NPY molecules were processed and secreted like wild-type pro-NPY when expressed in AtT-20 cells. The secretion of proNPY-derived peptides in these cell lines paralleled secretion of endogenous pro-ACTH/endorphin-derived products under both basal and stimulated conditions. These mutagenesis studies demonstrate that variants of the KDEL retention signal can direct intracellular retention.

Geranylgeraniol restores cell proliferation to lovastatin treated C6 glial cells.

Since we have previously shown that farnesol (F-OH) and geranylgeraniol (GG-OH) can be utilized for protein isoprenylation, the ability of the free allylic isoprenols to overcome the lovastatin-induced block on rat C6 glial cell proliferation has been investigated. Lovastatin an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in mevalonate biosynthesis, inhibited the rate of cell growth in a concentration dependent manner. The addition of mevalonate and GG-OH to lovastatin treated cells was shown to overcome the drug induced inhibition of cell proliferation. This result indicates that geranylgeraniol is capable of providing the mevalonic acid-derived products necessary to support cell growth. These results suggest that isoprenols are converted to an "activated' form, possibly the corresponding allylic pyrophosphate intermediate by reactions that remain to be characterized, prior to being utilized for sterol biosynthesis and protein isoprenylation.

Protein farnesyltransferase isoprenoid substrate discrimination is dependent on isoprene double bonds and branched methyl groups.

Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized the benzyloxyisoprenyl pyrophosphate (BnPP) series of transferable farnesyl pyrophosphate (FPP) analogues (1a-e) to test the length dependence of the isoprenoid substrate on the FTase-catalyzed transfer of lipid to protein substrate. Kinetic analyses show that pyrophosphates 1a-e and geranyl pyrophosphate (GPP) transfer with a lower efficiency than FPP whereas geranylgeranyl pyrophosphate (GGPP) does not transfer at all. While a correlation was found between K(m) and analogue hydrophobicity and length, there was no correlation between k(cat) and these properties. Potential binding geometries of FPP, GPP, GGPP, and analogues 1a-e were examined by modeling the molecules into the active site of the FTase crystal structure. We found that analogue 1d displaces approximately the same volume of the active site as does FPP, whereas GPP and analogues 1a-c occupy lesser volumes and 1e occupies a slightly larger volume. Modeling also indicated that GGPP adopts a different conformation than the farnesyl chain of FPP, partially occluding the space occupied by the Ca(1)a(2)X peptide in the ternary X-ray crystal structure. Within the confines of the FTase pocket, the double bonds and branched methyl groups of the geranylgeranyl chain significantly restrict the number of possible conformations relative to the more flexible lipid chain of analogues 1a-e. The modeling results also provide a molecular explanation for the observation that an aromatic ring is a good isostere for the terminal isoprene of FPP.

Cloning and expression of a cDNA encoding the alpha subunit of rat p21ras protein farnesyltransferase.

The complete amino acid sequence of the alpha subunit of heterodimeric p21ras protein farnesyltransferase from rat has been deduced from the sequence of a cloned cDNA. The cDNA encodes a 377-amino acid protein that migrates on NaDodSO4/polyacrylamide gels identically to the alpha subunit purified from rat brain. When introduced into mammalian cells by transfection, the cDNA for the alpha subunit produced no immunodetectable protein or farnesyltransferase activity unless the cells were simultaneously transfected with a cDNA encoding beta subunit. In light of previous evidence that alpha subunit forms a heterodimer with at least two different beta subunits, current data suggest a mechanism for coordinating amounts of alpha and beta subunits. If an alpha subunit were stable only as a complex with a beta subunit, the number of alpha subunits would be automatically maintained at a level just sufficient to balance all beta subunits, thereby avoiding the potentially toxic overaccumulation of free alpha subunits.

Characterization of the carboxyl-terminal sequences responsible for protein retention in the endoplasmic reticulum.

The COOH-terminal sequence KDEL has been shown to be essential for the retention of several proteins in the lumen of the endoplasmic reticulum (Munro S., and Pelham, H. R. B. (1987) Cell 48, 899-907; Pelham, H. R. B. (1988) EMBO J. 7, 913-918; Mazzarella; R. A., Srinivasan, M., Haugejorden, S. M., and Green, M. (1990) J. Biol. Chem. 265, 1092-1101). We have previously demonstrated that variants to the KDEL retention signal, particularly at the initial two positions of the tetrapeptide, can be made without affecting its ability to direct intracellular retention when appended to the neuropeptide Y precursor (pro-NPY) (Andres, D. A., Dickerson, I. M., and Dixon, J. E. (1990) J. Biol. Chem. 265, 5952-5955). To further investigate the nature of the KDEL retention signal, oligonucleotide-directed mutagenesis and transfection was used to generate stable mouse anterior pituitary AtT-20 cell lines expressing pro-NPY mutants with variants of the KDEL sequence added to their direct carboxyl terminus. Analyses of dibasic processing and indirect immunofluorescent microscopy of AtT-20 subclones were consistent with the retention of the pro-NPY mutants bearing the COOH-terminal extensions QDEL, KEDL, or KDEI within the endoplasmic reticulum. A change in the final amino acid of the tetrapeptide from Leu to Val abolished retention completely, and the peptide hormone was processed and secreted. These results indicate that only a limited number of conservative changes can be made to the final two positions of the tetrapeptide without abolishing activity and suggest a highly specific interaction of the retention signal and the KDEL receptor.

Selective inhibition of cholesterol biosynthesis in brain cells by squalestatin 1.

The effect of squalestatin 1 (SQ) on squalene synthase and other enzymes utilizing farnesyl pyrophosphate (F-P-P) as substrate was evaluated by in vitro enzymological and in vivo metabolic labeling experiments to determine if the drug selectively inhibited cholesterol biosynthesis in brain cells. Direct in vitro enzyme studies with membrane fractions from primary cultures of embryonic rat brain (IC50 = 37 nM), pig brain (IC50 = 21 nM), and C6 glioma cells (IC50 = 35 nM) demonstrated that SQ potently inhibited squalene synthase activity but had no effect on the long-chain cis-isoprenyltransferase catalyzing the conversion of F-P-P to polyprenyl pyrophosphate (Poly-P-P), the precursor of dolichyl phosphate (Dol-P). SQ also had no effect on F-P-P synthase; the conversion of [3H]F-P-P to geranylgeranyl pyrophosphate (GG-P-P) catalyzed by partially purified GG-P-P synthase from bovine brain; the enzymatic farnesylation of recombinant H-p21ras by rat brain farnesyltransferase; or the enzymatic geranylgeranylation of recombinant Rab 1A, catalyzed by rat brain geranylgeranyltransferase. Consistent with SQ selectively blocking the synthesis of squalene, when C6 glial cells were metabolically labeled with [3H]mevalonolactone, the drug inhibited the incorporation of the labeled precursor into squalene and cholesterol (IC50 = 3-5 microM) but either had no effect or slightly stimulated the labeling of Dol-P, ubiquinone (CoQ), and isoprenylated proteins. These results indicate that SQ blocks cholesterol biosynthesis in brain cells by selectively inhibiting squalene synthase.(ABSTRACT TRUNCATED AT 250 WORDS)