Neurofibromatosis 2 tumour suppressor schwannomin interacts with betaII-spectrin.

NF2 is the most commonly mutated gene in benign tumours of the human nervous system. The NF2 protein, called schwannomin or merlin, is absent in virtually all schwannomas, and many meningiomas and ependymomas. Using the yeast two-hybrid system, we identified betaII-spectrin (also known as fodrin) as a schwannomin-binding protein. Interaction occurred between the carboxy-terminal domain of schwannomin isoform 2 and the ankyrin-binding region of betaII-spectrin. Isoform 1 of schwannomin, in contrast, interacted weakly with betaII-spectrin, presumably because of its strong self-interaction. Thus, alternative splicing of NF2 may regulate betaII-spectrin binding. Schwannomin co-immunoprecipitated with betaII-spectrin at physiological concentrations. The two proteins interacted in vitro and co-localized in several target tissues and in STS26T cells. Three naturally occurring NF2 missense mutations showed reduced, but not absent, betaII-spectrin binding, suggesting an explanation for the milder phenotypes seen in patients with missense mutations. STS26T cells treated with NF2 antisense oligonucleotides showed alterations of the actin cytoskeleton. Schwannomin itself lacks the actin binding sites found in ezrin, radixin and moesin, suggesting that signalling to the actin cytoskeleton occurs via actin-binding sites on betaII-spectrin. Thus, schwannomin is a tumour suppressor directly involved in actin-cytoskeleton organization, which suggests that alterations in the cytoskeleton are an early event in the pathogenesis of some tumour types.

Spatial regulation of the exocyst complex by Rho1 GTPase.

Spatial regulation of membrane traffic is fundamental to many biological processes, including epithelial cell polarization and neuronal synaptogenesis. The multiprotein exocyst complex is localized to sites of polarized exocytosis, and is required for vesicle targeting and docking at specific domains of the plasma membrane. One component of the complex, Sec3, is thought to be a spatial landmark for polarized exocytosis. We have searched for proteins that regulate the polarized localization of the exocyst in the budding yeast Saccharomyces cerevisiae. Here we report that certain rho1 mutant alleles specifically affect the localization of the exocyst proteins. Sec3 interacts directly with Rho1 in its GTP-bound form, and functional Rho1 is needed both to establish and to maintain the polarized localization of Sec3. Sec3 is not the only mediator of the effect of Rho1 on the exocyst, because some members of the complex are correctly targeted independently of the interaction between Rho1 and Sec3. These results reveal the action of parallel pathways for the polarized localization of the exocytic machinery, both of which are under the control of Rho1, a master regulator of cell polarity.

Common modifications of trimeric G proteins and ras protein: involvement of polyisoprenylation.

The heterotrimeric guanine nucleotide-binding regulatory proteins act at the inner surface of the plasma membrane to relay information from cell surface receptors to effectors inside the cell. These G proteins are not integral membrane proteins, yet are membrane associated. The processing and function of the gamma subunit of the yeast G protein involved in mating-pheromone signal transduction was found to be affected by the same mutations that block ras processing. The nature of these mutations implied that the gamma subunit was polyisoprenylated and that this modification was necessary for membrane association and biological activity. A microbial screen was developed for pharmacological agents that inhibit polyisoprenylation and that have potential application in cancer therapy.

Construction of a model cell line for the assay of MDR1 (multi drug resistance gene-1) substrates/inhibitors using HeLa cells.

Cancer cells often become resistant to chemotherapy, and induction of the ABC transporter Multi-drug Resistance gene-1 (MDR1) is a major cause. We established a tool for high-throughput screening of substrates and inhibitors of MDR1, using transformed HeLa cells that over-express MDR1. The cDNA for human MDR1 was subcloned into the eukaryotic expression vector pBK-CMV to produce an MDR1 expression vector, pBK-CMV/MDR1. HeLa cells were transfected with pBK-CMV/MDR1 or the empty vector pBK-CMV. Transfection of the vector sequence for MDR1 and its expression were evaluated by genomic PCR and western blotting, respectively. The efficiency of the MDR1 transporter for pumping a substrate out of the transformed cells was evaluated using rhodamine123 (R-123), a mitochondrial dye that is also an MDR1 substrate. After treatment of the MDR1-expressing HeLa cells with MDR1 substrate vinblastin or inhibitors cyclosporin A and verapamil, the amount of R-123 retained in the cells was increased to 2 to 2.3 times the level in untreated MDR1-expressing HeLa cells. The transfection of empty pBK-CMV had no effect on the R-123 retention in HeLa cells, regardless of drug treatment. In conclusion, we have established a model human carcinoma cell line that expresses functional MDR1 and can be used to screen for substrates and inhibitors of MDR1.

Inhibitors of Ras farnesyltransferases.

Farnesyltransferase catalyses the post-translational modification of proteins by a cholesterol precursor, farnesylpyrophosphate. One of the substrates of this enzyme is the product of the ras oncogene. Recently, inhibitors of farnesyltransferase have been identified through two different approaches: microbial screens for natural compounds, and substrate analogues. These inhibitors may be useful in blocking the action of Ras proteins, in further characterizing protein prenyltransferases, and in elucidating the regulation of cholesterol metabolism.

Functional significance of lysine 1423 of neurofibromin and characterization of a second site suppressor which rescues mutations at this residue and suppresses RAS2Val-19-activated phenotypes.

Lysine 1423 of neurofibromin (neurofibromatosis type I gene product [NF1]) plays a crucial role in the function of NF1. Mutations of this lysine were detected in samples from a neurofibromatosis patient as well as from cancer patients. To further understand the significance of this residue, we have mutated it to all possible amino acids. Functional assays using yeast ira complementation have revealed that lysine is the only amino acid that produced functional NF1. Quantitative analyses of different mutant proteins have suggested that their GTPase-activating protein (GAP) activity is drastically reduced as a result of a decrease in their Ras affinity. Such a requirement for a specific residue is not observed in the case of other conserved residues within the GAP-related domain. We also report that another residue, phenylalanine 1434, plays an important role in NF1 function. This was first indicated by the finding that defective NF1s due to an alteration of lysine 1423 to other amino acids can be rescued by a second site intragenic mutation at residue 1434. The mutation partially restored GAP activity in the lysine mutant. When the mutation phenylalanine 1434 to serine was introduced into a wild-type NF1 protein, the resulting protein acquired the ability to suppress activated phenotypes of RAS2Val-19 cells. This suppression, however, does not involve Ras interaction, since the phenylalanine mutant does not stimulate the intrinsic GTPase activity of RAS2Val-19 protein and does not have an increased affinity for Ras proteins.

IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein.

The IRA1 gene is a negative regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. To identify other genes involved in this pathway, we screened yeast genomic DNA libraries for genes that can suppress the heat shock sensitivity of the ira1 mutation on a multicopy vector. We identified IRA2, encoding a protein of 3,079 amino acids, that is 45% identical to the IRA1 protein. The region homologous between the IRA1 protein and ras GTPase-activating protein is also conserved in IRA2. IRA2 maps 11 centimorgans distal to the arg1 locus on the left arm of chromosome XV and was found to be allelic to glc4. Disruption of the IRA2 gene resulted in (i) increased sensitivity to heat shock and nitrogen starvation, (ii) sporulation defects, and (iii) suppression of the lethality of the cdc25 mutant. Analysis of disruption mutants of IRA1 and IRA2 indicated that IRA1 and IRA2 proteins additively regulate the RAS-cyclic AMP pathway in a negative fashion. Expression of the IRA2 domain homologous with GAP is sufficient for complementation of the heat shock sensitivity of ira2, suggesting that IRA down regulates RAS activity by stimulating the GTPase activity of RAS proteins.

Identification of neurofibromin mutants that exhibit allele specificity or increased Ras affinity resulting in suppression of activated ras alleles.

Neurofibromin plays a critical role in the downregulation of Ras proteins in neurons and Schwann cells. Thus, the ability of neurofibromin to interact with Ras is crucial for its function, as mutations in NF1 that abolish this interaction fail to maintain function. To investigate the neurofibromin-Ras interaction in a systematic manner, we have carried out a yeast two-hybrid screen using a mutant of H-ras, H-rasD92K, defective for interaction with the GTPase-activated protein-related domain (GRD) of NF1. Two screens of a randomly mutagenized NF1-GRD library led to the identification of seven novel NF1 mutants. Characterization of the NF1-GRD mutants revealed that one class of mutants are allele specific for H-raSD92K. These mutants exhibit increased affinity for H-raSD92K and significantly reduced affinity for wild-type H-ras protein. Furthermore, they do not interact with another H-ras mutant defective for interaction with GTPase-activating proteins. Another class of mutants are high-affinity mutants which exhibit dramatically increased affinity for both wild-type and mutant forms of Ras. They also exhibit a striking ability to suppress the heat shock sensitive traits of activated RAS2G19v in yeast cells. Five mutations cluster within a region encompassing residues 1391 to 1436 (region II). Three NF1 patient mutations have previously been identified in this region. Two mutations that we identified occur in a region encompassing residues 1262 to 1276 (region I). Combining high-affinity mutations from both regions results in even greater affinity for Ras. These results demonstrate that two distinct regions of NF1-GRD are involved in the Ras interaction and that single amino acid changes can affect NF1's affinity for Ras.

Mutation of a termination codon affects src initiation.

The four Rous sarcoma virus messages gag, gag-pol, env, and src all derive from a full-length RNA precursor. All four messages contain the same 5' leader segment. Three of the messages, gag, gag-pol, and env, use an AUG present in this leader to initiate translation. The src AUG initiation codon lies 3' of the leader segment, 90 bases downstream of the gag initiation codon in the spliced src message. However, in the spliced src message a UGA termination codon lies between the gag AUG and the src AUG. All three codons are in the same reading frame. By using oligonucleotide-directed mutagenesis, the UGA termination codon has been converted to CGA. Cells infected with the mutant (called 1057 CGA) were spindle shaped, distinct from the rounded shape of cells infected with the parental Rous sarcoma virus. The mutant virus initiates src translation at the gag AUG, producing a 63,000-dalton src protein. We suggest that the wild-type src message produces two polypeptides, a very small (nine-amino acid) peptide that is initiated at the gag AUG and the 60,000-dalton src protein that is initiated at the src AUG.

A dominant activating mutation in the effector region of RAS abolishes IRA2 sensitivity.

Previously described mutations in RAS genes that cause a dominant activated phenotype affect the intrinsic biochemical properties of RAS proteins, either decreasing the intrinsic GTPase or reducing the affinity for guanine nucleotides. In this report, we describe a novel activating mutation in the RAS2 gene of Saccharomyces cerevisiae that does not alter intrinsic biochemical properties of the mutant RAS2 protein. Rather, this mutation, RAS2-P41S (proline 41 to serine), which lies in the effector region of RAS, is shown to abolish the ability of the IRA2 protein to stimulate the GTPase activity of the mutant RAS protein. This mutation also modestly reduced the ability of the mutant protein to stimulate the target adenylate cyclase in an in vitro assay, although in vivo the phenotypes it induced suggest that it retains potency in stimulation of adenylate cyclase. Our results demonstrate that although the effector region of RAS appears to be important for interaction with both target effector and negative regulators of RAS, it is possible to eliminate negative regulator responsiveness and retain potency in effector stimulation.

Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70.

The Rho3 protein plays a critical role in the budding yeast Saccharomyces cerevisiae by directing proper cell growth. Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton. To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S. cerevisiae cDNA library to identify proteins interacting with Rho3. Two proteins, Exo70 and Myo2, were identified in this screen. Interactions with these two proteins are greatly reduced or abolished when mutations are introduced into the Rho3 effector domain. In addition, a type of mutation known to produce dominant negative mutants of Rho proteins abolished the interaction with both of these proteins. In contrast, Rho3 did not interact with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1, nor did Rho1 interact with Exo70 or Myo2. Rho3 did interact with Bni1, another effector of Rho1, but less efficiently than with Rho1. The interaction between Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified proteins. The interaction between Exo70 and Rho3 in vitro was dependent on the presence of GTP, since Rho3 complexed with guanosine 5'-O-(3-thiotriphosphate) interacted more efficiently with Exo70 than Rho3 complexed with guanosine 5'-O-(3-thiodiphosphate). Overlapping subcellular localization of the Rho3 and Exo70 proteins was demonstrated by indirect immunofluorescence. In addition, patterns of localization of both Exo70 and Rho3 were altered when a dominant active allele of RHO3, RHO3(E129,A131), which causes a morphological abnormality, was expressed. These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton.

Potentiation of nitric oxide-induced apoptosis of MDA-MB-468 cells by farnesyltransferase inhibitor: implications in breast cancer.

High amounts of nitric oxide (NO) produced by activated macrophages or NO donors are required to induce cytotoxicity and apoptosis in pathogens and tumor cells. High concentrations of NO may lead to nonspecific toxicity thereby limiting the use of NO donors in the treatment of cancer. In this study, we tested the possibility of potentiating the apoptotic action of NO in a human breast cancer cell line, MDA-MB-468, by combining it with a farnesyltransferase inhibitor (FTI), which has been shown to induce apoptosis in some other cancer cell lines with minimal toxicity to normal cells. DETA-NONOate, a long acting NO donor which has a half-life of 20 h at 37 degrees C, was used in this study. DETA-NONOate (1 mM), which releases NO in the range produced by activated macrophages, induced apoptosis after 36 h in MDA-MB-468 cells via cytochrome c release and caspase-9 and -3 activation. FTI (25 microM) potentiated the action of lower concentrations of DETA-NONOate (25-100 microM) by inducing apoptosis in these cells within 24 h by increasing cytochrome c release and caspase-9 and -3 activation. This effect was observed preferentially in the cancer cell lines studied with no apoptosis induction in normal breast epithelial cells. This novel combination of FTI and NO may emerge as a promising approach for the treatment of breast cancer.

New activated RAS2 mutations identified in Saccharomyces cerevisiae.

Activating mutations in RAS proto-oncogenes encode proteins with greater GTP binding. Such mutant proteins are responsible for many human cancers. Six new amino acids were discovered that can yield an activated Saccharomyces cerevisiae RAS2 protein when they are altered. These new RAS2 alleles were found among a collection of 35 random mutations that exhibit a dominant reduction of glycogen accumulation. The RAS2-P41S and RAS2-E99K alleles encode proteins that have lost responsiveness to GTPase activating proteins. They affect amino acids in loop 2 and helix 3 respectively and illustrate that GTPase activating proteins recognize a larger portion of the RAS structure than previously realized. RAS2 mutations E130K, S153F, A154T, and A157S alter amino acids proximal to the guanine binding site and probably influence nucleotide binding either directly or indirectly.

Cdk inhibitors, roscovitine and olomoucine, synergize with farnesyltransferase inhibitor (FTI) to induce efficient apoptosis of human cancer cell lines.

Farnesyltransferase inhibitor (FTI) induces apoptosis of transformed cells. This involves changes in mitochondria, including decrease of mitochondrial membrane potential and the release of cytochrome c. The released cytochrome c then induces events leading to the activation of caspase-3. In this study, we report that purine derivative cyclin-dependent kinase (Cdk) inhibitors, roscovitine and olomoucine, dramatically enhance this FTI-induced apoptosis of human cancer cell lines. We noticed the synergy between Cdk inhibitors and FTI through our screen to identify compounds that enhance FTI-induced apoptosis of promyelocytic leukemic cell line HL-60. The Cdk inhibitors by themselves do not induce apoptosis at the concentrations used. Roscovitine synergizes with FTI to release cytochrome c from mitochondria. In addition, we detected synergistic effects of FTI and roscovitine to inhibit hyperphosphorylation of retinoblastoma protein. Enhancement of FTI-induced apoptosis by roscovitine is not unique to HL-60 cells, since similar synergy was observed with a leukemic cell line CEM and a prostate cancer cell line LNCaP. In LNCaP cells, in addition to roscovitine and olomoucine, phophatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, was effective in enhancing FTI-induced apoptosis. However, the effects of roscovitine appear to be distinct from those of LY294002, since roscovitine did not affect Akt activity while LY294002 significantly decreased the activity of Akt. Our finding of the synergy between FTI and Cdk inhibitor is significant for understanding the mechanism of action of FTI as well as for clinical use of FTI.

Kir, a novel Ras-family G-protein, induces invasive pseudohyphal growth in Saccharomyces cerevisiae.

Kir belongs to a novel class of Ras-family G-proteins which includes Gem and Rad. These proteins are unique among Ras super-family G-proteins since their expression is under transcriptional regulation in mammalian cells. To gain insight into the function of Kir, we took advantage of the well-defined signal transduction pathways of yeast. When kir is expressed in Saccharomyces cerevisiae, the transformants form pseudohyphae and exhibit invasive properties characteristics of yeast cells undergoing a developmental transition induced by nitrogen starvation. Analysis of pseudohyphal signaling pathway mutants suggests that the Kir-induced pseudohyphae formation requires a MAP kinase cascade involving ste20, ste11, ste7 but not ste5 gene products. Furthermore, our results are consistent with the idea that Kir functions upstream of the STE20 kinase which plays a critical role in two distinct MAP kinase cascades.

The inhibition of protein prenyltransferases by oxygenated metabolites of limonene and perillyl alcohol.

The monoterpenes limonene and perillyl alcohol are effective therapeutic agents against advanced rat mammary cancer. Limonene is currently undergoing clinical testing in cancer patients. These monoterpenes and their oxygenated metabolites have been previously shown to inhibit protein prenylation in cultured cells. Since farnesylation of ras protein is critical for its ability to cause oncogenic transformation, inhibition of protein prenylation may be the basis of the anti-tumor effects of limonene and perillyl alcohol. In this study we test the ability of limonene and its oxygenated analogs to inhibit protein prenylation enzymes in vitro. Limonene and perillyl alcohol and their major in vivo metabolite, perillic acid, are weak inhibitors of both mammalian and yeast protein farnesyl transferase (PFT) and protein geranylgeranyl transferase (PGGT). In contrast, a minor metabolite of both limonene and perillyl alcohol, perillic acid methyl ester, is a potent inhibitor of both enzymes. Perillic acid methyl ester is a competitive inhibitor of yeast PFT with respect to farnesyl pyrophosphate. These studies suggest that if the inhibition of protein prenylation is a mechanism for limonene's and perillyl alcohol's anti-cancer activities, these monoterpenes may be prodrugs that are converted into pharmacologically-active substances by metabolic modification.

Synthesis of various phosphodiesters and phosphomonoesters with ribonuclease N.

1. 3'-Guanylyl-ethanol, 3'-guanylyl-propanol, and 3'-guanylyl-alpha-glycerol were synthesized by ribonuclease N1 [EC 3.1.4.8] using guanosine 2',3'-cyclic phosphate as a phosphate donor and various alcohols as phosphate acceptors. The yields of these phosphodiesters were 15%, 13.5%, 38.2%, respectively, with respect to phosphate donor under the optimum conditions. No phosphodiester was synthesized when 2-propanol was used as a phosphate acceptor. Thus, primary alcoholic hydroxyl groups may be regarded as the preferred phosphate acceptor. 2. 3'-Guanylyl-glucose and 3'-guanylyl-ribose were synthesized using glucose and ribose as phosphate acceptors. Under the optimum conditions, the yields of guanylyl-glucose amounted to 52.0%, while that of guanylyl-ribose was much lower. The guanylyl-glucose can be regarded as 3'-guanylyl-6-glucopyranose, based on the results of periodate oxidation. 3. Neither hydroxyamino acids (serine and threonine) nor N-acetylserinamide could be phosphorylated under the conditions used for the above phosphorylations. 4. 3'-Guanylyl-glycerol obtained as above was hydrolyzed by snake venon phosphodiesterase to produce glycerol 3-phosphate. The latter consisted of L-glycerol 3-phosphate (ca 17%) and the D-isomer (ca. 83%). Ribonuclease N1 thus catalyzes an asymmetric synthesis.