scholarly journals Loss of ATM kinase activity leads to embryonic lethality in mice

2012 ◽  
Vol 198 (3) ◽  
pp. 295-304 ◽  
Author(s):  
Jeremy A. Daniel ◽  
Manuela Pellegrini ◽  
Baeck-Seung Lee ◽  
Zhi Guo ◽  
Darius Filsuf ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Point Mutations ◽  
Adenosine Diphosphate ◽  
Embryonic Lethality ◽  
Dominant Negative ◽  
Cell Cycle Checkpoints ◽  
Missense Mutations ◽  
Atm Kinase ◽  
Adult Mice ◽  
Conditional Deletion

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.

c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 139-146 ◽  
Author(s):  
Matthias Ballmaier ◽  
Manuela Germeshausen ◽  
Harald Schulze ◽  
Klara Cherkaoui ◽  
Sabine Lang ◽  
...  
Keyword(s):  
Clinical Course ◽  
Flow Cytometric Analysis ◽  
Point Mutations ◽  
Adenosine Diphosphate ◽  
Surface Expression ◽  
Missense Mutations ◽  
Hematopoietic Progenitor ◽  
Nonsense Mutations ◽  
Flow Cytometric ◽  
Main Regulator

Abstract Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disease presenting with isolated thrombocytopenia in infancy and developing into a pancytopenia in later childhood. Thrombopoietin (TPO) is the main regulator of thrombocytopoiesis and has also been demonstrated to be an important factor in early hematopoiesis. We analyzed 9 patients with CAMT for defects in TPO production and reactivity. We found high levels of TPO in the sera of all patients. However, platelets and hematopoietic progenitor cells of patients with CAMT did not show any reactivity to TPO, as measured by testing TPO-synergism to adenosine diphosphate in platelet activation or by megakaryocyte colony assays. Flow cytometric analysis revealed absent surface expression of the TPO receptor c-Mpl in 3 of 3 patients. Sequence analysis of the c-mpl gene revealed point mutations in 8 of 8 patients: We found frameshift or nonsense mutations that are predicted to result in a complete loss of c-Mpl function in 5 patients. Heterozygous or homozygous missense mutations predicted to lead to amino acid exchanges in the extracellular domain of the receptor were found in 3 other patients. The type of mutations correlated with the clinical course of the disease. We propose a defective c-Mpl expression due to c-mpl mutations as the cause for thrombocytopenia and progression into pancytopenia seen in patients with CAMT.

scholarly journals Siah-1 N-Terminal RING Domain Is Required for Proteolysis Function, and C-Terminal Sequences Regulate Oligomerization and Binding to Target Proteins

1999 ◽  
Vol 19 (1) ◽  
pp. 724-732 ◽  
Author(s):  
Gang Hu ◽  
Eric R. Fearon
Keyword(s):  
Cell Fate ◽  
Point Mutations ◽  
Dominant Negative ◽  
Ring Domain ◽  
Dominant Negative Mutant ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Target Proteins ◽  
Proteasome Pathway ◽  
Seven In Absentia

ABSTRACT The Drosophila seven in absentia (sina) gene was initially discovered because its inactivation leads to R7 photoreceptor defects. Recent data indicate that Sina binds to the Sevenless pathway protein Phyllopod, and together they mediate degradation of Tramtrack, a transcriptional repressor of R7 cell fate. Independent studies have shown that Sina and its highly related mammalian homologues Siah-1 and Siah-2 bind to the DCC (deleted in colorectal cancer) protein and promote its proteolysis via the ubiquitin-proteasome pathway. To determine the roles of mammalian Siahs in proteolysis and their interactions with target proteins, we sought to define Siah-1 domains critical for regulation of DCC. Mutant Siah-1 proteins, harboring missense mutations in the carboxy (C)-terminal domain analogous to those present in Drosophila sinaloss-of-function alleles, failed to promote DCC degradation. Point mutations and deletion of the amino (N)-terminal RING finger domain of Siah-1 abrogated its ability to promote DCC proteolysis. In the course of defining Siah-1 sequences required for DCC degradation, we found that Siah-1 is itself rapidly degraded via the proteasome pathway, and RING domain mutations stabilized the Siah-1 protein. Siah-1 was found to oligomerize with itself and other Sina and Siah proteins via C-terminal sequences. Finally, evidence that endogenous Siah-1 regulates DCC proteolysis in cells was obtained through studies of an apparent dominant negative mutant of Siah-1, as well as via an antisense approach. The data indicate that the Siah-1 N-terminal RING domain is required for its proteolysis function, while the C-terminal sequences regulate oligomerization and binding to target proteins, such as DCC.

A Novel Mutation Glyl672→Arg in Type 2A and a Homozygous Mutation in Type 2B von Willebrand Disease

1996 ◽  
Vol 76 (02) ◽  
pp. 253-257 ◽  
Author(s):  
Takeshi Hagiwara ◽  
Hiroshi Inaba ◽  
Shinichi Yoshida ◽  
Keiko Nagaizumi ◽  
Morio Arai ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Novel Mutation ◽  
Point Mutations ◽  
Japanese Patients ◽  
Von Willebrand Disease ◽  
Homozygous Mutation ◽  
Missense Mutations ◽  
Reaction Products ◽  
Type 3 ◽  
Von Willebrand

SummaryGenetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.

In Caenorhabditis elegans, the RNA-Binding Domains of the Cytoplasmic Polyadenylation Element Binding Protein FOG-1 Are Needed to Regulate Germ Cell Fates

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1617-1630
Author(s):  
Suk-Won Jin ◽  
Nancy Arno ◽  
Adam Cohen ◽  
Amy Shah ◽  
Qijin Xu ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Germ Cell ◽  
Rna Binding ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Biochemical Tests ◽  
Cell Fates ◽  
Cytoplasmic Polyadenylation ◽  
Binding Domains ◽  
Rna Binding Domains

Abstract FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3′-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

scholarly journals Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Seung Won Choi ◽  
Yeri Lee ◽  
Kayoung Shin ◽  
Harim Koo ◽  
Donggeon Kim ◽  
...  
Keyword(s):  
Functional Properties ◽  
Therapeutic Target ◽  
Medical Center ◽  
Dominant Negative ◽  
The Cancer Genome Atlas ◽  
Dominant Negative Effect ◽  
Cell Periphery ◽  
Missense Mutations ◽  
Phosphatase Domain ◽  
Mutant Proteins

AbstractPTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

scholarly journals NF-κB-Inducing Kinase (NIK) Mediates Skeletal Muscle Insulin Resistance: Blockade by Adiponectin

Endocrinology ◽  
2011 ◽  
Vol 152 (10) ◽  
pp. 3622-3627 ◽  
Author(s):  
Sanjeev Choudhary ◽  
Sandeep Sinha ◽  
Yanhua Zhao ◽  
Srijita Banerjee ◽  
Padma Sathyanarayana ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Muscle Insulin Resistance ◽  
Akt Phosphorylation ◽  
Skeletal Muscle Insulin Resistance ◽  
Obese Subjects

Enhanced levels of nuclear factor (NF)-κB-inducing kinase (NIK), an upstream kinase in the NF-κB pathway, have been implicated in the pathogenesis of chronic inflammation in diabetes. We investigated whether increased levels of NIK could induce skeletal muscle insulin resistance. Six obese subjects with metabolic syndrome underwent skeletal muscle biopsies before and six months after gastric bypass surgery to quantitate NIK protein levels. L6 skeletal myotubes, transfected with NIK wild-type or NIK kinase-dead dominant negative plasmids, were treated with insulin alone or with adiponectin and insulin. Effects of NIK overexpression on insulin-stimulated glucose uptake were estimated using tritiated 2-deoxyglucose uptake. NF-κB activation (EMSA), phosphatidylinositol 3 (PI3) kinase activity, and phosphorylation of inhibitor κB kinase β and serine-threonine kinase (Akt) were measured. After weight loss, skeletal muscle NIK protein was significantly reduced in association with increased plasma adiponectin and enhanced AMP kinase phosphorylation and insulin sensitivity in obese subjects. Enhanced NIK expression in cultured L6 myotubes induced a dose-dependent decrease in insulin-stimulated glucose uptake. The decrease in insulin-stimulated glucose uptake was associated with a significant decrease in PI3 kinase activity and protein kinase B/Akt phosphorylation. Overexpression of NIK kinase-dead dominant negative did not affect insulin-stimulated glucose uptake. Adiponectin treatment inhibited NIK-induced NF-κB activation and restored insulin sensitivity by restoring PI3 kinase activation and subsequent Akt phosphorylation. These results indicate that NIK induces insulin resistance and further indicate that adiponectin exerts its insulin-sensitizing effect by suppressing NIK-induced skeletal muscle inflammation. These observations suggest that NIK could be an important therapeutic target for the treatment of insulin resistance associated with inflammation in obesity and type 2 diabetes.

Site-directed mutagenesis of the SH2- and SH3-coding domains of c-src produces varied phenotypes, including oncogenic activation of p60c-src

1990 ◽  
Vol 10 (4) ◽  
pp. 1307-1318
Author(s):  
H Hirai ◽  
H E Varmus
Keyword(s):  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Kinase Domain ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Positive Effects ◽  
Src Gene ◽  
Carboxy Terminal

The products of the viral and cellular src genes, p60v-src and p60c-src, appear to be composed of multiple functional domains. Highly conserved regions called src homology 2 and 3 (SH2 and SH3), comprising amino acid residues 88 to 250, are believed to modulate the protein-tyrosine kinase activity present in the carboxy-terminal halves of the src proteins. To explore the functions of these regions more fully, we have made 34 site-directed mutations in a transformation-competent c-src gene encoding phenylalanine in place of tyrosine 527 (Y527F c-src). Twenty of the new mutations change only one or two amino acids, and the remainder delete small or large portions of the SH2-SH3 region. These mutant alleles have been incorporated into a replication-competent Rous sarcoma virus vector to examine the biochemical and biological properties of the mutant proteins after infection of chicken embryo fibroblasts. Four classes of mutant proteins were observed: class 1, mutants with only slight differences from the parental gene products; class 2, mutant proteins with diminished transforming and specific kinase activities; class 3, mutant proteins with normal or enhanced specific kinase activity but impaired biological activity, often as a consequence of instability; and class 4, mutant proteins with augmented biological and catalytic activities. In general, there was a strong correlation between total kinase activity (or amounts of intracellular phosphotyrosine-containing proteins) and transforming activity. Deletion mutations and some point mutations affecting residues 109 to 156 inhibited kinase and transforming functions, whereas deletions affecting residues 187 to 226 generally had positive effects on one or both of those functions, confirming that SH2-SH3 has complex regulatory properties. Five mutations that augmented the transforming and kinase activities of Y527F c-src [F172P, R175L, delta(198-205), delta(206-226), and delta(176-226)] conferred transformation competence on an otherwise normal c-src gene, indicating that mutations in SH2 (like previously described lesions in SH3, the kinase domain, and a carboxy-terminal inhibitory domain) can activate c-src.

scholarly journals PCNT point mutations and familial intracranial aneurysms

Neurology ◽  
2018 ◽  
Vol 91 (23) ◽  
pp. e2170-e2181 ◽  
Author(s):  
Oswaldo Lorenzo-Betancor ◽  
Patrick R. Blackburn ◽  
Emily Edwards ◽  
Rocío Vázquez-do-Campo ◽  
Eric W. Klee ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Intracranial Aneurysms ◽  
Point Mutations ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Interaction Domain ◽  
Knockout Mouse Model ◽  
Whole Exome ◽  
Primordial Dwarfism

ObjectiveTo identify novel genes involved in the etiology of intracranial aneurysms (IAs) or subarachnoid hemorrhages (SAHs) using whole-exome sequencing.MethodsWe performed whole-exome sequencing in 13 individuals from 3 families with an autosomal dominant IA/SAH inheritance pattern to look for candidate genes for disease. In addition, we sequenced PCNT exon 38 in a further 161 idiopathic patients with IA/SAH to find additional carriers of potential pathogenic variants.ResultsWe identified 2 different variants in exon 38 from the PCNT gene shared between affected members from 2 different families with either IA or SAH (p.R2728C and p.V2811L). One hundred sixty-four samples with either SAH or IA were Sanger sequenced for the PCNT exon 38. Five additional missense mutations were identified. We also found a second p.V2811L carrier in a family with a history of neurovascular diseases.ConclusionThe PCNT gene encodes a protein that is involved in the process of microtubule nucleation and organization in interphase and mitosis. Biallelic loss-of-function mutations in PCNT cause a form of primordial dwarfism (microcephalic osteodysplastic primordial dwarfism type II), and ≈50% of these patients will develop neurovascular abnormalities, including IAs and SAHs. In addition, a complete Pcnt knockout mouse model (Pcnt−/−) published previously showed general vascular abnormalities, including intracranial hemorrhage. The variants in our families lie in the highly conserved PCNT protein-protein interaction domain, making PCNT a highly plausible candidate gene in cerebrovascular disease.

scholarly journals In Planta Mutagenesis Determines the Functional Regions of the Wheat Puroindoline Proteins

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 853-860 ◽  
Author(s):  
Leila Feiz ◽  
Brian S. Beecher ◽  
John M. Martin ◽  
Michael J. Giroux
Keyword(s):  
Functional Analysis ◽  
Allelic Variation ◽  
Screening Method ◽  
Point Mutations ◽  
Missense Mutations ◽  
Grain Hardness ◽  
In Planta ◽  
Rich Region ◽  
Critical Function ◽  
The Impact

In planta analysis of protein function in a crop plant could lead to improvements in understanding protein structure/function relationships as well as selective agronomic or end product quality improvements. The requirements for successful in planta analysis are a high mutation rate, an efficient screening method, and a trait with high heritability. Two ideal targets for functional analysis are the Puroindoline a and Puroindoline b (Pina and Pinb, respectively) genes, which together compose the wheat (Triticum aestivum L.) Ha locus that controls grain texture and many wheat end-use properties. Puroindolines (PINs) together impart soft texture, and mutations in either PIN result in hard seed texture. Studies of the PINs' mode of action are limited by low allelic variation. To create new Pin alleles and identify critical function-determining regions, Pin point mutations were created in planta via EMS treatment of a soft wheat. Grain hardness of 46 unique PIN missense alleles was then measured using segregating F2:F3 populations. The impact of individual missense alleles upon PIN function, as measured by grain hardness, ranged from neutral (74%) to intermediate to function abolishing. The percentage of function-abolishing mutations among mutations occurring in both PINA and PINB was higher for PINB, indicating that PINB is more critical to overall Ha function. This is contrary to expectations in that PINB is not as well conserved as PINA. All function-abolishing mutations resulted from structure-disrupting mutations or from missense mutations occurring near the Tryptophan-rich region. This study demonstrates the feasibility of in planta functional analysis of wheat proteins and that the Tryptophan-rich region is the most important region of both PINA and PINB.

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