Failure to farnesylate Rheb protein contributes to the enrichment of G0/G1 phase cells in the Schizosaccharomyces pombe farnesyltransferase mutant.

Protein farnesylation is important for a number of physiological processes, including proliferation and cell morphology. The Schizosaccharomyces pombe mutant, cpp1-, defective in farnesylation, exhibits distinct phenotypes, including morphological changes and sensitivity to the arginine analogue, canavanine. In this work, we report a novel phenotype of this mutant, enrichment of G0/G1 phase cells. This phenotype results mainly from the inability to farnesylate the Rheb G-protein, as normal cell cycle progression can be restored to the mutant by expressing a mutant form of SpRheb (SpRheb-CVIL) that can bypass farnesylation. In contrast, a farnesylation-defective mutant of SpRheb (SpRheb-SVIA) is incapable of restoring the normal cell cycle profile to the cpp1- mutant. Inhibition of SpRheb expression leads to the accumulation of cells at the G0/G1 phase of the cell cycle. This growth arrest phenotype of the sprheb- disruption can be complemented by the introduction of wild-type sprheb+. The complementation is dependent on farnesylation, as the farnesylation-defective SpRheb-SVIA mutant is incapable of complementing the sprheb- disruption. Other mutants of SpRheb, E40K and S20N, are also incapable of complementing the sprheb- disruption. Furthermore, efficient complementation can be obtained by the expression of human Rheb but not Saccharomyces cerevisiae Rheb. Our findings suggest that protein farnesylation is important for cell cycle progression of S. pombe cells and that farnesylated SpRheb is critical in this process.

Protein farnesylation is critical for maintaining normal cell morphology and canavanine resistance in Schizosaccharomyces pombe.

Protein farnesyltransferase (FTase) plays important roles in the growth and differentiation of eukaryotic cells. In this paper, we report the identification of the Schizosaccharomyces pombe gene cpp1(+) encoding the beta-subunit of FTase. The predicted amino acid sequence of the cpp1(+) gene product shares significant similarity with FTase beta-subunits from a variety of organisms. S. pombe FTase purified from E. coli exhibits high enzymatic activity toward the CAAX farnesylation motif substrates (where C represents cysteine, A represents aliphatic amino acid, and X is preferentially methionine, cysteine, serine, alanine, or glutamine) while showing little preference for CAAL geranylgeranylation motif substrates (where L represents leucine or phenylalanine). cpp1(+) is not essential for growth as shown by gene disruption; however, mutant cells exhibit rounded or irregular cell morphology. Expression of a geranylgeranylated mutant form, Ras1-CVIL, which can bypass farnesylation, rescues these morphological defects. We also identify a novel phenotype of cpp1(-) mutants, hypersensitivity to canavanine. This appears to be due to a 3-4-fold increase in the rate of arginine uptake as compared with wild-type cells. Expression of the geranylgeranylated mutant form of a novel farnesylated small GTPase, SpRheb, is able to suppress the elevated arginine uptake rate. These results demonstrate that protein farnesylation is critical for maintaining normal cell morphology through Ras1 and canavanine resistance through SpRheb.

Molecular cloning and characterization of a rice dehydroascorbate reductase.

Plant dehydroascorbate reductase (DHAR), which re-reduces oxidized ascorbate to maintain an appropriate level of ascorbate, is very important, but no gene or cDNA for plant DHAR has been cloned yet. Here, we describe a cDNA for a rice glutathione-dependent DHAR (designated DHAR1). A recombinant Dhar1p produced in Escherichia coli was functional. The expression sequence tag database suggests that Dhar1p homologs exist in various plants. Furthermore, the rice Dhar1p has a low similarity to rat DHAR, although the rice enzyme has a considerably higher specific activity than the mammalian one. The mRNA level of DHAR1, the protein level of Dhar1p and the DHAR activity in rice seedlings were elevated by high temperature, suggesting the protection role of DHAR at high temperature.

The Saccharomyces cerevisiae Rheb G-protein is involved in regulating canavanine resistance and arginine uptake.

The new member of the Ras superfamily of G-proteins, Rheb, has been identified in rat and human, but its function has not been defined. We report here the identification of Rheb homologues in the budding yeast Saccharomyces cerevisiae (ScRheb) as well as in Schizosaccharomyces pombe, Drosophila melanogaster, zebrafish, and Ciona intestinalis. These proteins define a new class of G-proteins based on 1) their overall sequence similarity, 2) high conservation of their effector domain sequence, 3) presence of a unique arginine in their G1 box, and 4) presence of a conserved CAAX farnesylation motif. Characterization of an S. cerevisiae strain deficient in ScRheb showed that it is hypersensitive to growth inhibitory effects of canavanine and thialysine, which are analogues of arginine and lysine, respectively. Accordingly, the uptake of arginine and lysine was increased in the ScRheb-deficient strain. This increased arginine uptake requires the arginine-specific permease Can1p. The function of ScRheb is dependent on having an intact effector domain since mutations in the effector domain of ScRheb are incapable of complementing canavanine hypersensitivity of scrheb disruptant cells. Furthermore, the conserved arginine in the G1 box plays a role in the activity of ScRheb, as a mutation of this arginine to glycine significantly reduced the ability of ScRheb to complement canavanine hypersensitivity of ScRheb-deficient yeast. Finally, a mutation in the C-terminal CAAX farnesylation motif resulted in a loss of ScRheb function. This result, in combination with our finding that ScRheb is farnesylated, suggests that farnesylation plays a key role in ScRheb function. Our findings assign the regulation of arginine and lysine uptake as the first physiological function for this new farnesylated Ras superfamily G-protein.

A mutant form of human protein farnesyltransferase exhibits increased resistance to farnesyltransferase inhibitors.

Protein farnesyltransferase (FTase) is a key enzyme responsible for the lipid modification of a large and important number of proteins including Ras. Recent demonstrations that inhibitors of this enzyme block the growth of a variety of human tumors point to the importance of this enzyme in human tumor formation. In this paper, we report that a mutant form of human FTase, Y361L, exhibits increased resistance to farnesyltransferase inhibitors, particularly a tricyclic compound, SCH56582, which is a competitive inhibitor of FTase with respect to the CAAX (where C is cysteine, A is an aliphatic amino acid, and X is the C-terminal residue that is preferentially serine, cysteine, methionine, glutamine or alanine) substrates. The Y361L mutant maintains FTase activity toward substrates ending with CIIS. However, the mutant also exhibits an increased affinity for peptides terminating with CIIL, a motif that is recognized by geranylgeranyltransferase I (GGTase I). The Y361L mutant also demonstrates activity with Ha-Ras and Cdc42Hs proteins, substrates of FTase and GGTase I, respectively. In addition, the Y361L mutant shows a marked sensitivity to a zinc chelator HPH-5 suggesting that the mutant has altered zinc coordination. These results demonstrate that a single amino acid change at a residue at the active site can lead to the generation of a mutant resistant to FTase inhibitors. Such a mutant may be valuable for the study of the effects of FTase inhibitors on tumor cells.

Farnesyltransferase inhibitors induce cytochrome c release and caspase 3 activation preferentially in transformed cells.

Farnesyltransferase inhibitors (FTIs) represent a new class of anticancer drugs that show promise in blocking the growth of tumors. Here, we report that FTIs are capable of inducing apoptosis of transformed but not untransformed cells. Treatment of v-K-ras-transformed normal rat kidney (KNRK) cells with FTIs leads to the induction of apoptotic cell morphology, chromatin condensation and DNA fragmentation. In addition, fluorescence-activated cell sorter analysis of FTI-treated KNRK cells shows a sub-G1 apoptotic peak (chromosome content of <2 N). This FTI-induced apoptosis is evident only when the cells are grown in low serum conditions (0.1% fetal calf serum) and is observed selectively with transformed KNRK cells and not with untransformed NRK cells. Further analysis of the mechanism underlying this apoptosis has shown that FTI treatment of KNRK cells results in the activation of caspase 3 but not caspase 1. Moreover, the addition of Z-DEVD-fmk, an agent that interferes with caspase 3 activity, can inhibit FTI-induced apoptosis in a dose-dependent manner. Introduction of the CASP-3 gene into MCF7 cells, which lack caspase 3 activity, results in a significant increase of FTI-induced apoptosis. Furthermore, FTI induces the release of cytochrome c into the cytosol. This release is an important feature of caspase 3-mediated apoptosis. These results suggest that FTIs induce apoptosis through the release of cytochrome c from the mitochondria resulting in caspase 3 activation.

The Sex-lethal early splicing pattern uses a default mechanism dependent on the alternative 5' splice sites.

The Sex-lethal (Sxl) early transcripts have a unique 5' exon and a splicing pattern that differs from that of the late transcripts. While the late transcripts are regulated sex specifically by control of exon 3 inclusion, the early transcripts are not. While the late transcripts include exon 3 by default, the early transcripts skip exon 3. Splicing patterns of a reporter gene that mimics the early transcript, and its variants, were analyzed in Drosophila transformants and tissue culture cells. The results demonstrate that the early, in contrast to the late, splicing pattern is not regulated by stage-specific or sex-specific trans-acting factors, and so the pattern appears to arise from some type of intrinsic splice site preference or compatibility. Inclusion or exclusion of exon 3 is determined by the identity of the upstream 5' splice site region as late or early. The important region of the early exon lies within 233 nucleotides of the immediately adjacent intron.

Advances in the development of farnesyltransferase inhibitors: substrate recognition by protein farnesyltransferase.

A variety of compounds that show promise in cancer chemotherapy and chemoprevention have been identified as farnesyltransferase inhibitors. These can be classified into mainly two different types of inhibitors, farnesyl diphosphate competitors and CAAX peptidomimetics. The former type acts by competitively inhibiting farnesyltransferase with respect to one of the substrates, farnesyl diphosphate, whereas the latter type acts by mimicking the other substrate, the C-terminal CAAX motif of Ras protein. One example of a farnesyl diphosphate competitor is manumycin, an antibiotic detected in the culture media of a Streptomyces strain. The CAAX peptidomimetics were developed based on the unique property of farnesyltransferase to recognize the CAAX motif at the C-terminus of the protein substrate. Our recent studies have focused on understanding the structural basis of this CAAX recognition. By using in vitro mutagenesis, residues of yeast farnesyltransferase important for the recognition of the CAAX motif have been identified. Two of these residues are closely located at the C-terminal region of the beta-subunit of farnesyltransferase. These and other results on the structural basis of the CAAX recognition may provide information valuable for structure-based design of farnesyltransferase inhibitors.

The role of yan in mediating the choice between cell division and differentiation.

An allele of the yan locus was isolated as an enhancer of the Ellipse mutation of the Drosophila epidermal growth factor receptor (Egfr) gene. This yan allele is an embryonic lethal and also fails to complement the lethality of anterior open (aop) mutations. Phenotypic and complementation analysis revealed that aop is allelic to yan and genetically the lethal alleles act as null mutations for the yan gene. Analysis of the lethal alleles in the embryo and in mitotic clones showed that loss of yan function causes cells to overproliferate in the dorsal neuroectoderm of the embryo and in the developing eye disc. Our studies suggest that the role of yan is defined by the developmental context of the cells in which it functions. An important role of this gene is in allowing a cell to choose between cell division and differentiation. The relationship of the Egfr and Notch pathways to this developmental role of yan is discussed.

GapIII, a new brain-enriched member of the GTPase-activating protein family.

Ras GTPase-activating proteins (GAPs) are negative regulators of ras, which controls proliferation and differentiation in many cells. Ras GAPs have been found in a variety of species from yeast to mammals. We describe here a newly identified mammalian GAP, GapIII, which was obtained by differential screening of a rat oligodendrocyte cDNA library. GapIII putatively encodes a 834 amino acid protein with a predicted molecular weight of 96 kDa, which contains a consensus GAP-related domain (GRD). The protein encoded by this cDNA has high homology with Gap1m, which was recently identified as a putative mammalian homolog of Drosophila Gap1. These proteins contain three structural domains, an N-terminal calcium-dependent phospholipid binding domain, GRD, and a C-terminal PH/Btk domain. Because of the sequence homology and the structural similarities of this protein with Gap1m, we hypothesize that GapIII and Gap1m may be members of a mammalian GAP gene family, separate from p120GAP, neurofibromin (NF1), and IQGAP. To confirm the GapIII protein activity, constructs containing different GapIII-GRD domains were transformed into iral mutant yeast to determine their relative ability to replace IRA1 functionally. Constructs that contained essentially the full-length protein (all three domains), the GRD alone, or the GRD plus PH/Btk domain suppressed heat shock sensitivity of ira1, whereas constructs that contained the GRD with part of the PH/Btk domain had only a weak ability to suppress heat shock sensitivity. These results suggest that the GapIII GRD itself is sufficient to down-regulate ras proteins in yeast. Expression of GapIII mRNA (4.2 kb) was examined by Northern analysis and in situ hybridization. This mRNA was expressed at highest levels in the brain, where its expression increased with development. Lower levels of the mRNA were expressed in the spleen and lung. Among neural cells, GapIII mRNA was expressed in neurons and oligodendrocytes, but not in astrocytes. Interestingly, the expression pattern in brain is reminiscent of type 1 NF1 expression reported by Gutmann et al. (Cell Growth Differ in press, 1995). We propose that in addition to p120GAP and neurofibromin, the GapIII/Gap1m family may be important for modulating ras activity in neurons and oligodendrocytes during normal brain development and in particular in the adult brain.

[Evaluation on facial pattern of early childhood patients with T.M.J. dysfunction occurred after anterior crossbite correction].

The purpose of this study was to investigate the facial pattern of early childhood patient with temporomandibular joint (TMJ) dysfunction (clicking) occurred after anterior cross-bite correction. Chincap appliance was engaged in all cases, with or without minor intraoral mechano-therapy. Materials were consisted of lateral and postero-anterior cephalograms of 50 japanese patients (6-9 years old), all showing anterior cross-bite with normal function of TMJ at pre-treatment stage. They were consisted of two groups, one was the group of 22 patients with TMJ dysfunction occurred after cross-bite correction and the other was the control group of 28 patients with no TMJ problem. Morphological measurements were done and compared between two groups. The results were as follows: 1) There was no significant difference between the TMJ group and the control group from the lateral facial pattern. 2) The upper and middle facial skeleton were symmetry in both groups, but the maxillary alveolar legion (CMo, U 1) of TMJ group was significantly from the antero-posterior view. 3) On the lower face (mandible) of the TMJ group, it was pointed out that the position of Gonion was significantly asymmetry and L 1 point and Menton were disclosed to have severe lateral displacement. It was cleared that a high incidence of temporomandibular joint dysfunction was found in mandibular asymmetry cases. It was concluded that a careful case-management is required for mandibular asymmetry patients during chicap-orthodontic therapy.

Unusual case of progressive systemic sclerosis with onset in early childhood and following infectious mononucleosis.

A rare case of infantile progressive systemic sclerosis is reported. A Japanese girl suffered from infectious mononucleosis at the age of 1 year 3 months, and 5 months later she developed edema and sclerosis of the skin. She has been followed up for 4 years and now has grotesque features, with contractures of hands and feet due to advanced systemic sclerosis. The relationship between infectious mononucleosis and progressive systemic sclerosis is discussed.