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Gene Expression Profiling of Human c-Kit Mutant D816V  [PDF]
Shilpa Sharma, Gurudutta Gangenahalli
Journal of Cancer Therapy (JCT) , 2016, DOI: 10.4236/jct.2016.76046
Abstract: The tyrosine kinase receptor III, c-Kit/stem cell factor receptor and its ligand, human stem cell factor (huSCF) are the predominant regulator of mitogenesis in the hematopoietic stem and progenitor cells. However, gain-of-function mutations alter c-Kit auto-regulatory mechanisms to aberrant c-Kit signaling, leading to the onset or progression of cancerous transformations. The most common mutation of c-Kit is the substitution of aspartic acid residue in position 816 to valine (D816V), which is majorly responsible for its ligand-independent constitutive activation, and is implicated in hematopoietic malignancies. Currently, molecular targeted therapy is increasingly becoming a hot spot due to its specificity and low toxicity. As the molecular mechanisms responsible for D816V-c-Kit mediated tumorogenicity are largely unknown, in this study, we aimed to investigate the D816V-c-Kit signaling mediated downstream molecular targets. Specifically, we created c-Kit active mutant form D816V and performed inducible gene expression of mutant D816V-c-Kit in monomyelocytic cell line U937. Mutant D816V-c-Kit expressing cells revealed significantly enhanced cellular mitogenic activity compared to wild-type c-Kit expressing cells independent of huSCF. To examine the molecular targets regulating tumorogenic proliferation, we evaluated the consequences of mutant D816V-c-Kit expression on downstream gene expression profile by high throughput microarray technology. The levels of some of the relevant genes (PIK3CB, eIF4B, PRKCDBP, MOAP1) were validated by quantitative polymerase chain reaction. SLA, STAT5B, MAP3K2 and MAPK14 emerged as important downstream molecular targets of mutant D816V-c-Kit. Further, by dissecting the signaling pathways, we also demonstrated that the D816V-c-Kit mediated hematopoietic cell proliferation is dependent on molecular target p38 MAP kinase.
A c-KIT codon 816 mutation, D816H, in the testicular germ cell tumor: case report of a Japanese patient with bilateral testicular seminomas.
Acta Medica Okayama , 2005,
Abstract: Mutations of the c-KIT gene have been reported not only in gastrointestinal stromal tumors and mast cell tumors, but also in testicular germ cell tumors (TGCTs). In the present study we employed polymerase chain reaction and DNA sequencing analysis to characterize the c-KIT gene in a 29-year-old Japanese patient with bilateral testicular seminomas. Direct sequence analyses revealed a single base substitution in exon 17 in one c-KIT allele, resulting in an amino acid substitution of D816H in this mutated allele. This mutation was found in the left, but not in the right, testicular seminoma. This is the first description of a c-KIT gene mutation in a Japanese patient with bilateral TGCT. The mutational analysis of the c-KIT gene seems to provide crucial information for managing TGCT patients not only in Europe but also in Japan.
Mutation D816V Alters the Internal Structure and Dynamics of c-KIT Receptor Cytoplasmic Region: Implications for Dimerization and Activation Mechanisms  [PDF]
Elodie Laine ,Isaure Chauvot de Beauchêne,David Perahia,Christian Auclair,Luba Tchertanov
PLOS Computational Biology , 2011, DOI: 10.1371/journal.pcbi.1002068
Abstract: The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites.
Homozygous lethality and heterozygous spotting due to a novel missense mutation in the mouse Kit gene  [cached]
Baojin WU, Lijing YIN, Xiaoshu YIN, Weiwei YANG, Bing CHEN, Zhengfeng XUE, Peilin WU
Current Zoology , 2009,
Abstract: N-ethyl-N-nitrosourea (ENU) mutagenesis in mice can be used to study gene function in vivo and to establish genetic mouse models of human disease. In this study, a white spotted mouse (named KitW-1Bao) was obtained by ENU-induced mutagenesis. Inheritance testing showed a single-gene dominant mutation and lethality in the KitW-1Bao homozygous mice. The mutation was mapped to Chromosome 5 between markers D5Mit356 and D5Mit308. The region contains the Kit gene, whose mutations are known to lead to pigmentation defects in mice. Sequence analysis of the Kit cDNA from KitW-1Bao heterozygotes revealed an A to T missense mutation resulting in an amino acid substitution of Asp (D) by Val (V) at amino acid position 849 within a highly conserved tyrosine kinase domain. The combined phenotype displayed by the KitW-1Bao heterozygous and homozygous mutant mice demonstrates the critical function of the highly conserved aspartic acid residue at position 849 in the Kit gene product [Current Zoology 55 (6): 430–434, 2009].
Differential Effects of CSF-1R D802V and KIT D816V Homologous Mutations on Receptor Tertiary Structure and Allosteric Communication  [PDF]
Priscila Da Silva Figueiredo Celestino Gomes, Nicolas Panel, Elodie Laine, Pedro Geraldo Pascutti, Eric Solary, Luba Tchertanov
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0097519
Abstract: The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.
A study of KIT activating mutations in acute myeloid leukemia M0 subtype in north India
SR Hussain, H Naqvi, P Singh, SG Babu, F Mahdi
Egyptian Journal of Medical Human Genetics , 2012,
Abstract: Acute Myeloid Leukemia (AML)-M0 is a cancer of blood-forming cells in the bone marrow. KIT gene is a receptor tyrosine kinase class III that is expressed on by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. Mutations of KIT receptor tyrosine kinase are involved in the constitutive activation and development of human hematologic malignancies. We have designed this study aiming to identify and determine the frequency and prevalence of mutations in North Indian patients suffering from AML-M0. To perceive the KIT gene mutations, we have carried out PCR–SSCP followed by direct DNA sequencing in 50 AML-M0 cases. We have found eight cases (24.2%) with t(8;21) having 12 point mutations whereas three cases (17.6%) with inv(16) having four point mutations. The point mutation detected at exon 9 in five cases is Asp496Val. Eight different point mutations were identified at exon 11 in seven AML-M0 cases that include Lys550Asn, Tyr568Ser, Ile571Leu, Tyr578Pro, Trp582Ser and Arg588Met. Point mutations at codons Ile571Leu and Trp582Ser was found in two independent cases. Three point mutations were found in exon 17 (Leu813Pro, Lys818Arg, Val825Ala) in three AML-M0 cases. The results underline that the KIT gene appears to be most frequently mutated target in AML-M0 cases. These observations suggest that mutations in exon 11 of the KIT gene might be useful molecular genetic markers in AML-M0 and these mutations might be related to progression and clinical pathogenesis.
Characterization of a Novel BCHE “Silent” Allele: Point Mutation (p.Val204Asp) Causes Loss of Activity and Prolonged Apnea with Suxamethonium  [PDF]
Herve Delacour, Sofya Lushchekina, Isabelle Mabboux, Aurore Bousquet, Franck Ceppa, Lawrence M. Schopfer, Oksana Lockridge, Patrick Masson
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0101552
Abstract: Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a “silent” phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 μM for BTC, two times higher than that of the atypical enzyme, and a low Vmax are consistent with the absence of activity against suxamethonium. Molecular dynamic (MD) simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric enzyme in the plasma and non-detectable fast moving-bands on electrophoresis gels.
Frequency and significance of the novel single nucleotide missense polymorphism Val109Asp in the human gene encoding omentin in Caucasian patients with type 2 diabetes mellitus or chronic inflammatory bowel diseases
Andreas Sch?ffler, Martina Zeitoun, Hella Wobser, Christa Buechler, Charalampos Aslanidis, Hans Herfarth
Cardiovascular Diabetology , 2007, DOI: 10.1186/1475-2840-6-3
Abstract: The Val109Asp single nucleotide miss-sense polymorphism and the His86His polymorphism in exon-4 of the omentin gene were newly identified by random sequencing. Only the miss-sense polymorphism was investigated further. Genotyping was performed by restriction fragment length polymorphism (RFLP) analysis of amplified DNA fragments. Three different cohorts of well-characterized individuals were included in the study. 114 patients suffering from T2D, 190 patients suffering from IBD (128 with CD and 62 with UC) and 276 non-diabetic healthy controls without any history for IBD were analyzed.The following allelic frequencies were determined: controls: Val-allele: 0.26, Asp-allele: 0.74; T2D: Val-allele: 0.3, Asp-allele: 0.7; IBD: Val-allel: 0.31, Asp-allele: 0.69. UC and CD patients did not differ in regard to the allelic frequency. Similarly, controls, T2D patients and IBD patients did not show significant differences in genotype distribution among each other. Disease manifestation and pattern of infestation were not related to genotype subgroups, neither in CD nor in UC. Furthermore, there was no significant association between genotype subgroups and anthropometric or laboratory parameters in T2D patients.Based on sequence comparisons and homology searches, the amino acid position 109 is conserved in the omentin gene of humans, mice and chimpanzee but is not completely conserved between other omentin homologous genes. Moreover, position 109 lies outside the fibrinogen domain. Due to these structural features and based on the present data, the Val109Asp sequence variation is more a single nucleotide polymorphism than a real disease-causing mutation.Adipokines [1] such as leptin, resistin and adiponectin are currently investigated as potential future drug targets in type 2 diabetes mellitus (T2D), lipid metabolism, endothelial dysfunction and inflammatory diseases in general. Therefore, the discovery of yet unknown adipokines represents a main target in the field of metabo
Allosteric Communication across the Native and Mutated KIT Receptor Tyrosine Kinase  [PDF]
Elodie Laine,Christian Auclair,Luba Tchertanov
PLOS Computational Biology , 2012, DOI: 10.1371/journal.pcbi.1002661
Abstract: A fundamental goal in cellular signaling is to understand allosteric communication, the process by which signals originated at one site in a protein propagate dependably to affect remote functional sites. Here, we describe the allosteric regulation of the receptor tyrosine kinase KIT. Our analysis evidenced that communication routes established between the activation loop (A-loop) and the distant juxtamembrane region (JMR) in the native protein were disrupted by the oncogenic mutation D816V positioned in the A-loop. In silico mutagenesis provided a plausible way of restoring the protein communication detected in the native KIT by introducing a counter-balancing second mutation D792E. The communication patterns observed in the native and mutated KIT correlate perfectly with the structural and dynamical features of these proteins. Particularly, a long-distance effect of the D816V mutation manifested as an important structural re-organization of the JMR in the oncogenic mutant was completely vanished in the double mutant D816V/D792E. This detailed characterization of the allosteric communication in the different forms of KIT, native and mutants, was performed by using a modular network representation composed of communication pathways and independent dynamic segments. Such representation permits to enrich a purely mechanistic interaction-based model of protein communication by the introduction of concerted local atomic fluctuations. This method, validated on KIT receptor, may guide a rational modulation of the physiopathological activities of other receptor tyrosine kinases.
Hotspot Mutations in KIT Receptor Differentially Modulate Its Allosterically Coupled Conformational Dynamics: Impact on Activation and Drug Sensitivity  [PDF]
Isaure Chauvot de Beauchêne,Ariane Allain,Nicolas Panel,Elodie Laine,Alain Trouvé,Patrice Dubreuil,Luba Tchertanov
PLOS Computational Biology , 2014, DOI: doi/10.1371/journal.pcbi.1003749
Abstract: Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D) localized in crucial regulatory segments, the juxtamembrane region (JMR) and the activation (A-) loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts.
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