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PubMed1994

Genetic interactions among genes involved in the STT4-PKC1 pathway of Saccharomyces cerevisiae.

Loss of yeast protein kinase C function results in three distinct phenotypes: staurosporine sensitivity, cell lysis and blockage of cell cycle progression at the G2/M boundary. Genetic analysis of the PKC1/STT1 protein kinase C gene and its interactions with STT4, encoding an upstream phosphatidylinositol 4-kinase, and BCK1, encoding a downstream protein kinase, reveal that they form part of a single pathway. However, the BCK1-20 mutation (a gain-of-function mutation of BCK1) or overexpression of PKC1 cannot suppress all of the phenotypes caused by the loss of STT4 function, strongly suggesting the existence of a branch point between STT4 and PKC1. We also describe the MSS4 gene, a multicopy suppressor of the temperature-sensitive stt4-1 mutation. MSS4 is predicted to encode a hydrophilic protein of 779 amino acid residues and is essential for cell growth. Based on genetic and biochemical data, we suggest that MSS4 acts downstream of STT4, but in a pathway that does not involve PKC1.

1-Phosphatidylinositol 4-Kinase↗
PubMed1996

Role of c-kit receptor tyrosine kinase in the development, survival and neoplastic transformation of mast cells.

The c-kit gene is allelic with the dominant spotting (W) locus on mouse chromosome 5 and encodes a receptor tyrosine kinase. The ligand for c-kit receptor is stem cell factor (SCF), which is the principal growth factor for mast cells. The loss-of-function mutations of c-kit receptor affect the development of mast cells, thereby resulting in a depletion of mast cells. The abundant expression of c-kit receptor is indispensable for the survival of mast cells. In addition, the gain-of-function mutations of c-kit receptor were found in several tumor mast cell lines. When these gain-of-function mutations were introduced to cells of murine interleukin (IL)-3-dependent cell lines, the expression of c-kit receptor with constitutive tyrosine kinase activity not only abrogated the IL-3 requirement of the cells, but also caused them to become tumorigenic in nude athymic mice. The gain-of-function mutations of c-kit receptor appear to result in the malignant transformation of mast cells. Taken together, the signals from the c-kit receptor are essential for the development, survival, and malignant transformation of mast cells.

Animals↗
PubMed2004

Different inhibitory effect of imatinib on phosphorylation of mitogen-activated protein kinase and Akt and on proliferation in cells expressing different types of mutant platelet-derived growth factor receptor-alpha.

Most gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of the c-kit gene. Previously, we found 2 types of gain-of-function mutation of the PDGFRA gene, Val561 to Asp and Asp842 to Val, in about half of GISTs without c-kit gene mutations. Although the inhibitory effect of imatinib on various types of activating mutant KIT has been well examined, that on the activating mutant PDGFRA has not been fully investigated. In the present study, we examined the effect of imatinib on autophosphorylation of mutant PDGFRA, phosphorylation of MAPK and of Akt and in vitro cell proliferation using murine Ba/F3 cells stably transfected with one of the 2 murine-type mutated PDGFRA cDNAs. Imatinib almost completely inhibited autophosphorylation of mutant PDGFRA, phosphorylation of MAPK and Akt as well as in vitro cell proliferation at the concentration of 0.01 microM in cells expressing mutant PDGFRA with Val561 to Asp. However, in cells expressing mutant PDGFRA with Asp842 to Val, imatinib almost completely inhibited autophosphorylation of mutant PDGFRA and phosphorylation of MAPK and Akt at 1.0 microM. The concentration contributing to complete inhibition of in vitro cell proliferation was 10 microM. Ba/F3 cells expressing mutant PDGFRA are a good model to investigate the mechanism of cell proliferation or growth inhibition by imatinib in mutant PDGFRA-driven cells.

Amino Acid Substitution↗
PubMed2003

A point mutation uncouples transducin-alpha from the photoreceptor RGS and effector proteins.

A novel gain-of-function mutation, R243Q, has been recently identified in the Candida elegans Gqalpha protein EGL-30. The position corresponding to Arg243 in EGL-30 is absolutely conserved among heterotrimeric G proteins. This mutation appears to be the first gain-of-function mutation in the switch III region of Galpha subunits. To investigate consequences of the R-->Q mutation we introduced the corresponding R238Q mutation into transducin-like Gtalpha* subunit. The mutant retained intact interactions with Gtbetagamma and rhodopsin but exhibited a twofold reduction in the kcat value for guanosine 5'-triphosphate (GTP) hydrolysis. The GTPase activity of R238Q was not accelerated by the RGS domain of the visual GTPase-activating protein, RGS9-1. In addition, R238Q displayed a significant impairment in the effector function. Our data and the crystal structures of transducin suggest that the major reason for the reduced intrinsic GTPase activity of R238Q and the lack of RGS9 function is the break of the conserved ionic contact between Arg238 and Glu39, which apparently stabilizes the transitional state for GTP hydrolysis. We hypothesize that the R243Q mutation in EGL-30 severs the ionic interaction of Arg243 with Glu43, leading to a defective inactivation of the mutant by the C. elegans RGS protein EAT-16.

Amino Acid Sequence↗
PubMed2004

KIT exon 8 mutations associated with core-binding factor (CBF)-acute myeloid leukemia (AML) cause hyperactivation of the receptor in response to stem cell factor.

KIT exon 8 mutations are located in the extracellular portion of the receptor and are strongly associated with core-binding factor (CBF)-acute myeloid leukemia (AML). To characterize the functional role of these mutants, we analyzed the proproliferative and antiapoptotic potential of 3 KIT exon 8 mutations in interleukin 3 (IL-3)-dependent Ba/F3 cells. All KIT exon 8 mutants induced receptor hyperactivation in response to stem cell factor (SCF) stimulation in terms of proliferation and resistance toward apoptotic cell death. A representative KIT exon 8 mutant showed spontaneous receptor dimerization, phosphorylation of mitogen-activated protein kinase (MAPK), and conferred IL-3-independent growth to Ba/F3 cells. MAPK and phosphatidylinositol 3-kinase (PI3-kinase) activation was essential for the phenotype of this mutant. Additionally, imatinib inhibited proliferation of KIT exon 8 mutant-expressing Ba/F3 cells. Our data show that KIT exon 8 mutations represent gain-of-function mutations and might represent a new molecular target for treatment of CBF leukemias.

Acute Disease↗
PubMed2003

Mutations in exon 11 of the c-kit gene in a myogenic tumor and a neurogenic tumor as well as in gastrointestinal stromal tumors. Utility of c-kit mutation as a prognostic biomarker for gastrointestinal mesenchymal tumor.

BACKGROUND/AIMS: Gain-of-function mutations in exons 9, 11 and 13 of the c-kit gene in gastrointestinal stromal tumors (GISTs) have been identified, and it has been reported that the prognosis is worse for patients with mutation-positive GISTs than for those with mutation-negative GISTs. We studied c-kit mutations in gastrointestinal mesenchymal tumors. By chance, the c-kit mutation in exon 11 was found in myogenic and neurogenic tumors as well as in GISTs. Furthermore, we studied the clinical prognostic utility of these mutations. METHODS: Ten gastrointestinal mesenchymal tumors were stained with HE and immunohistochemically analyzed with alpha-smooth muscle actin, S-100 protein, CD34 and c-kit. In these tumors, as well as in 11 cases of leiomyomas, PCR-amplified DNA from the juxtamembrane (JM) domain of exon 11, the extracellular domain of exon 9 and the tyrosine kinase domain 1 of exon 13 showed a high frequency of c-kit mutation and was sequenced. RESULTS: Although c-kit mutations have previously been reported only in GISTs, we found c-kit mutations in the JM domain of exon 11 in one myogenic and one neurogenic tumor as well as in two GISTs. No c-kit mutation was seen in the 11 cases of leiomyomas. In addition, all four cases with c-kit mutation in exon 11 suffered a relapse sooner than the other cases without c-kit mutations. CONCLUSION: Clinically, the prognosis was worse for the patients with mutation-positive gastrointestinal mesenchymal tumors than for those with mutation-negative tumors. We therefore conclude that the gain-of-function mutation in exon 11 of the c-kit gene is an important prognostic factor for gastrointestinal mesenchymal tumors, including myogenic and neurogenic tumors as well as GISTs.

Adult↗
PubMed2000

Genetic heterogeneity in hereditary thrombophilia.

Venous thromboembolism is a multifactorial disease that depends on variable combinations of acquired and genetic risk factors. The genetic risk factors include loss-of-function mutations in the genes that encode proteins with clot-restraining function, and gain-of-function mutations in procoagulant factors. The loss-of-function mutations are heterogeneous and comprise any mutation that impairs gene function. On the whole, these mutations are rare, with fewer than 1/200--500 individuals affected. This low prevalence in the population is probably caused by the loss of mutant alleles from the gene pool through critically ill homozygous subjects. The gain-of-function mutations in procoagulant proteins differ from the loss-of-function mutations in at least three important respects: these are more homogeneous (factor V Leiden and PT 20210), homozygous individuals are relatively mildly affected, and these are relatively prevalent (3--15%) in Caucasian populations.

3' Untranslated Regions↗
PubMed2004

CTNNB1 mutations and overexpression of Wnt/beta-catenin target genes in WT1-mutant Wilms' tumors.

Gain-of-function mutations in exon 3 of beta-catenin (CTNNB1) are specific for Wilms' tumors that have lost WT1, but 50% of WT1-mutant cases lack such "hot spot" mutations. To ask whether stabilization of beta-catenin might be essential after WT1 loss, and to identify downstream target genes, we compared expression profiles in WT1-mutant versus WT1 wild-type Wilms' tumors. Supervised and nonsupervised hierarchical clustering of the expression data separated these two classes of Wilms' tumor. The WT1-mutant tumors overexpressed genes encoding myogenic and other transcription factors (MOX2, LBX1, SIM2), signaling molecules (TGFB2, FST, BMP2A), extracellular Wnt inhibitors (WIF1, SFRP4), and known beta-catenin/TCF targets (FST, CSPG2, CMYC). Beta-Catenin/TCF target genes were overexpressed in the WT1-mutant tumors even in the absence of CTNNB1 exon 3 mutations, and complete sequencing revealed gain-of-function mutations elsewhere in the CTNNB1 gene in some of these tumors, increasing the overall mutation frequency to 75%. Lastly, we identified and validated a novel direct beta-catenin target gene, GAD1, among the WT1-mutant signature genes. These data highlight two molecular classes of Wilms' tumor, and indicate strong selection for stabilization of beta-catenin in the WT1-mutant class. Beta-Catenin stabilization can initiate tumorigenesis in other systems, and this mechanism is likely critical in tumor formation after loss of WT1.

Cells, Cultured↗
PubMed1998

Genetic evidence that Notch affects independently the development of machrochaeta and wing venation in Drosophila melanogaster.

Temperature sensitive experiments on the interaction of Abruptex mutations of the Notch locus and Enhancer of split indicate that the effect of temperature on the development of macrochaeta and wing venation is different. This suggest that Notch affects the development of macrochaeta and wing venation in different and independent ways being in accordance with the proposition that the signal pathway from the Notch receptor at the cell membrane to Enhancer of split complex in the nucleus is different in the case of the development of macrochaeta and wing venation. The Abruptex mutations as gain-of-function mutations of the Notch locus cannot rescue the Enhancer of split phenotype.

Animals↗
PubMed2002

Searching for Arg201 mutations in the GNAS1 gene in Italian patients with McCune-Albright syndrome.

McCune-Albright syndrome (MAS) is a rare disease caused by somatic postzygotic mutations at Arg201 in the GNAS1 gene that encodes for the Gsalpha protein. Arg201 mutations are gain-of-function mutations in affected tissues (including bone, skin, endocrine glands and other tissues) that result in the activation of cAMP. We used a polymerase chain reaction(PCR)-based technique for the selective enrichment and analysis of the Arg201 mutant allele in 27 different tissues from 24 Italian patients with one or more signs of MAS. Arg201 mutations were identified in 13 different tissues (48.1%) from 11 patients (45.8%). Mutation detection rates differed across the various types of tissue samples, and the mutation was not always found in every tissue sample from the same patient. To overcome problems in the analysis of mutations in somatic mosaicism, as occurs in MAS, a highly sensitive molecular technique should be applied, the most appropriate tissue source selected, and various affected tissues from the same patient analyzed.

Adolescent↗
PubMed2002

Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome.

The extracellular Ca(2+)-sensing receptor (CaSR) plays an essential role in extracellular Ca(2+) homeostasis by regulating the rate of parathyroid hormone (PTH) secretion and the rate of calcium reabsorption by the kidney. Activation of the renal CaSR is thought to inhibit paracellular divalent cation reabsorption in the cortical ascending limb (cTAL) both directly and indirectly via a decrease in NaCl transport. However, in patients with autosomal dominant hypocalcemia (ADH), caused by CaSR gain-of-function mutations, a defect in tubular NaCl reabsorption with renal loss of NaCl has not been described so far. This article describes a patient with ADH due to a gain-of-function mutation in the CaSR, L125P, associated with a Bartter-like syndrome that is characterized by a decrease in distal tubular fractional chloride reabsorption rate and negative NaCl balance with secondary hyperaldosteronism and hypokalemia. The kinetics of activation of the L125P mutant receptor expressed in HEK-293 cells, assessed by measuring CaSR-stimulated changes in intracellular Ca(2+) and ERK activity, showed a dramatic reduction in the EC(50) for extracellular Ca(2+) compared with the wild-type and a loss-of-function mutant CaSR (I40F). This study describes the first case of ADH associated with a Bartter-like syndrome. It is herein proposed that the L125P mutation of the CaSR, which represents the most potent gain-of-function mutation reported so far, may reduce NaCl reabsorption in the cTAL sufficiently to result in renal loss of NaCl with secondary hyperaldosteronism and hypokalemia.

Bartter Syndrome↗
PubMed2003

Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome.

We took advantage of overlapping interstitial deletions at chromosome 8p11-p12 in two individuals with contiguous gene syndromes and defined an interval of roughly 540 kb associated with a dominant form of Kallmann syndrome, KAL2. We establish here that loss-of-function mutations in FGFR1 underlie KAL2 whereas a gain-of-function mutation in FGFR1 has been shown to cause a form of craniosynostosis. Moreover, we suggest that the KAL1 gene product, the extracellular matrix protein anosmin-1, is involved in FGF signaling and propose that the gender difference in anosmin-1 dosage (because KAL1 partially escapes X inactivation) explains the higher prevalence of the disease in males.

Cell Adhesion Molecules↗
PubMed2002

RET receptor tyrosine kinase isoforms in kidney function and disease.

The RET proto-oncogene encodes two major isoforms, RET9 and RET51, which differ at the carboxyl-terminal. Loss-of-function mutations in RET result in gut aganglionosis while gain of function mutations result in cancer syndromes. From studies on transgenic mice, RET9 is important for early development of the kidney and the enteric nervous system. Little is known about the function of RET isoforms in later life. Here we report the expression of RET isoforms and its signalling complex, GDNF and GFRalpha1, in foetal and adult human kidneys. We found their expression in both the developing and the adult renal collecting system. We further show that only RET51 but not RET9 could promote the survival and tubulogenesis of mIMCD3 (mouse inner medullary collecting duct) cells in collagen gel. Our results agree with the hypothesis that RET51 signalling is related to differentiation events in later kidney organogenesis. In addition, it may also have a function in the adult kidney. We further extend our study by showing increased RET and GDNF expression in collecting duct cysts of polycystic kidney patients. This suggests that GDNF/RET signalling may contribute to proliferation of the collecting duct epithelium in an autocrine/paracrine manner.

Aged↗
PubMed1997

Identification and characterization of genes that interact with lin-12 in Caenorhabditis elegans.

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.

Animals↗
PubMed2006

FLT3 K663Q is a novel AML-associated oncogenic kinase: Determination of biochemical properties and sensitivity to Sunitinib (SU11248).

Somatic mutations of FLT3 resulting in constitutive kinase activation are the most common acquired genomic abnormality found in acute myeloid leukemia (AML). The majority of these mutations are internal tandem duplications (ITD) of the juxtamembrane region (JM). In addition, a minority of cases of AML are associated with mutation of the FLT3 activation loop (AL), typically involving codons D835 and/or I836. We hypothesized that other novel mutations of FLT3 could also contribute to leukemogenesis. We genotyped 109 cases of AML and identified two novel gain-of-function mutations. The first mutation, N841 H, is similar to previously described mutations involving amino-acid substitutions of codon 841. The other novel mutation, FLT3 K663Q, is the first AML-associated gain-of-function mutation located outside the JM and AL domains. Of note, this mutation was potently inhibited by Sunitinib (SU11248), a previously described FLT3 kinase inhibitor. Sunitinib reduced the proliferation and induced apoptosis of transformed Ba/F3 cells expressing FLT3 K663Q. The potency of Sunitinib against FLT3 K663Q was similar to its potency against FLT3 ITD mutations. We conclude that FLT3 mutations in AML can involve novel regions of the TK1. Future studies are needed to define the incidence and prognostic significance of FLT3 mutations outside the well-established JM and AL regions.

Acute Disease↗
PubMed1993

C-terminal truncation of the transcriptional activator encoded by areA in Aspergillus nidulans results in both loss-of-function and gain-of-function phenotypes.

Mutations truncating as many as 143 C-terminal residues from the transcriptional activator encoded by the areA gene, mediating nitrogen metabolite repression in Aspergillus nidulans, do not significantly reduce the ability of the areA product to activate expression of most genes under areA control. Such mutations can even have a gain-of-function, derepressed phenotype, consistent with a critical role for this region in modulating the activity of the areA protein. However, expression of a few genes under areA control is substantially impaired by such C-terminal truncations, indicating that regions of an activator protein can play differing roles in the control of different structural genes. This underlines the advantages of being able to monitor effects of areA mutations on expression of large numbers of structural genes. Additionally, it is shown that truncation of as many as 153 C-terminal residues, virtually all amino acids C-terminal to the DNA-binding region, is compatible with retention of some areA function.

Amino Acid Sequence↗
PubMed1996

Cell death mechanisms in ALS.

Mutations in copper-zinc superoxide dismutase (CuZnSOD) that are associated with familial ALS (FALS) are dominant, gain-of-function mutations, but the nature of the function gained has not been identified. In addition to catalyzing the dismutation of superoxide, copper-zinc superoxide dismutase also displays peroxidase activity. Whereas mutants A4V and G93A retained superoxide dismutase activity, they demonstrated a markedly enhanced copper-dependent peroxidase activity in comparison with that of the wild type enzyme as detected by the spin trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) in electron paramagnetic resonance measurements. Two copper chelators, diethyldithiocarbamate and penicillamine, inhibited the mutants' peroxidase activity, but not that of the wild type enzyme, at stoichiometric concentrations; furthermore, these copper chelators enhanced neural survival in a cell-culture model of ALS but did not alter survival of cells expressing only wild type copper-zinc superoxide dismutase. These observations suggest that oxidative reactions catalyzed by mutant copper-zinc superoxide dismutases may initiate the neuropathologic changes of FALS.

Amyotrophic Lateral Sclerosis↗
PubMed2002

Arabidopsis SHY2/IAA3 inhibits auxin-regulated gene expression.

In Arabidopsis, SHY2 encodes IAA3, a member of the auxin-induced Aux/IAA family. Gain-of-function mutations in SHY2/IAA3 cause enlarged cotyledons, short hypocotyls, and altered auxin-regulated root development. Here we show that the gain-of-function mutation shy2-2 decreases both the induction and repression of auxin-regulated genes, suggesting that SHY2/IAA3 acts as a negative regulator in auxin signaling. shy2-2 affects auxin induction of many previously characterized primary response genes, implying that it might repress primary auxin responses. In addition, shy2-2 also affects expression of multiple auxin-nonresponsive genes. Light regulates expression of SHY2/IAA3, suggesting a possible link between light and auxin response pathways.

Arabidopsis↗