In-frame Deletion Mutation Research Articles

Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3' to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

Nontruncating SCN1A Mutations Associated with Severe Myoclonic Epilepsy of Infancy Impair Cell Surface Expression

Mutations in SCN1A, encoding the voltage-gated sodium channel Na(V)1.1, are the most common cause of severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome. SMEI is most often associated with premature truncations of Na(V)1.1 that cause loss of function, but nontruncating mutations also occur. We hypothesized that some nontruncating mutations might impair trafficking of Na(V)1.1 to the plasma membrane. Here we demonstrated that seven nontruncating missense or in-frame deletion mutations (L986F, delF1289, R1648C, F1661S, G1674R, and G1979E) exhibited reduced cell surface expression relative to wild type (WT) Na(V)1.1 consistent with impaired trafficking. We tested whether two commonly prescribed antiepileptic drugs (phenytoin, lamotrigine), as well as the cystic fibrosis transmembrane conductance regulator (CFTR) trafficking corrector VRT-325, could rescue cell surface and functional expression of two representative Na(V)1.1 mutants (R1648C, G1674R). Treatment of cells with phenytoin increased cell surface expression of WT-Na(V)1.1 and both mutant channels, whereas lamotrigine only increased surface expression of R1648C. VRT-325 did not alter surface expression of WT-Na(V)1.1 or mutant channels. Although phenytoin increased surface expression of G1674R, channel function was not restored, suggesting that this mutation also causes an intrinsic loss of function. Both phenytoin and lamotrigine increased functional expression of R1648C, but lamotrigine also increased persistent sodium current evoked by this mutation. Our findings indicate that certain nontruncating SCN1A mutations associated with SMEI have impaired cell surface expression and that some alleles may be amenable to pharmacological rescue of this defect. However, rescue of dysfunctional Na(V)1.1 channels to the plasma membrane could contribute to exacerbating rather than ameliorating the disease.

Selective Energy Transfer Between Quantum Dots and Gold Nanoparticles for Detection of Multiple Mutations in Epidermal Growth Factor Receptor

Selective energy transfer between quantum dots and gold nanoparticles was used to simultaneously detect mutations in the epidermal growth factor receptor (EGFR) gene. We functionalized the surface of gold nanoparticles and green and red-emitting quantum dots using four different probe DNAs that were designed to be a perfect complementary to an in-frame deletion mutation in exon 19 or L858 R point mutation in exon 21 of EGFR. We found that the presence of the deletion mutation in exon 19 in target oligonucleotides caused fluorescence quenching at 525 nm due to energy transfer from green-emitting quantum dots to gold nanoparticles, whereas point mutation in exon 21 resulted in quenching at 620 nm due to energy transfer from red-emitting quantum dots to gold nanoparticles. This method could successfully be used to simultaneously detect the presence of two types of mutations in EGFR. We also defined a parameter (i.e., the extent of quenching) to quantify fluorescence quenching phenomenon. By varying the fraction of mutant type DNA in target oligonucleotides, we showed that detection sensitivity based on the extent of quenching was about 5%, which is lower than the conventional direct sequencing method.

Use of a Mariner-Based Transposon Mutagenesis System To Isolate Clostridium perfringens Mutants Deficient in Gliding Motility

Clostridium perfringens is an anaerobic Gram-positive pathogen that causes many human and animal diseases, including food poisoning and gas gangrene. C. perfringens lacks flagella but possesses type IV pili (TFP). We have previously shown that C. perfringens can glide across an agar surface in long filaments composed of individual bacteria attached end to end and that two TFP-associated proteins, PilT and PilC, are needed for this. To discover additional gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. More than 10,000 clones were screened for mutants that lacked the ability to move away from the edge of a colony. Twenty-four mutants (0.24%) were identified that fit the criteria. The genes containing insertions that affected gliding motility fell into nine different categories. One gene, CPE0278, which encodes a homolog of the SagA cell wall-dependent endopeptidase, acquired distinct transposon insertions in two independent mutants. sagA mutants were unable to form filaments due to a complete lack of end-to-end connections essential for gliding motility. Complementation of the sagA mutants with a wild-type copy of the gene restored gliding motility. We constructed an in-frame deletion mutation in the sagA gene and found that this mutant had a phenotype similar to those of the transposon mutants. We hypothesize that the sagA mutant strains are unable to form the molecular complexes which are needed to keep the cells in an end-to-end orientation, leading to separation of daughter cells and the inability to carry out gliding motility.

Clinical and mutation analyses of a Chinese family with Bethlem myopathy

To explore the clinical features and gene mutation of a Chinese family with Bethlem myopathy in three generations. The clinical data of proband and his family members was collected. Genomic DNA from the patient and his family members was extracted routinely from peripheral blood leukocytes. Polymerase chain reaction and DNA direct sequencing were employed to analyze COL6A1, A2 and A3 genes to determine the mutation. And the relationship between genotype and phenotype was analyzed. Furthermore, the patient's skin fibroblast was cultured and immunofluorescent staining was performed with anti-collagen VI antibody. And the expression pattern of type VI collagen in extracellular matrix between the control and the patient's fibroblast was compared. In this family, 9 patients conformed to the clinical diagnosis of Bethlem myopathy. The features included motor development delay after late infantile period, generalized muscle weakness, walking unstability, distal hyper laxity, proximal joint contractures, skin changes (including hypertrophic scars) and normal intellectual development. Serum creatine kinase (CK) level became mildly elevated and electromyography showed myogenic injury. Disease progressed slowly but the lifespan was not affected. Mutation in exon 2 of COL6A1 gene with c.111-129 deletion was detected in 7 patients in this family. Immunofluorescent staining of type VI collagen in cultured skin fibroblast showed reduced expression of collagen VI in extracellular matrix in the patient compared with the control. Our study has defined the clinical features of Bethlem myopathy. According to molecular genetic analysis, 7 patients in this family have in-frame deletion mutations of COL6A1 and they conform to autosomal dominant inheritance. And genetic counseling and prenatal diagnosis are available. This is the first Chinese report of Bethlem myopathy family.

Genotype–phenotype correlations in 17 Chinese patients with autosomal recessive Alport syndrome

Autosomal recessive Alport syndrome (ARAS) results from mutations in the COL4A3 or COL4A4 gene. We analyzed the genotype and phenotype of 17 unrelated Chinese patients with ARAS. Clinical data were reviewed. All coding exons of COL4A3 and COL4A4 genes were PCR-amplified and sequenced from genomic DNA. We identified pathologic mutations in all patients, giving a mutation detection rate of 100%, with 82% in COL4A3 gene and 18% in COL4A4 gene. Sixteen novel mutations in COL4A3 gene and four novel mutations in COL4A4 gene were identified. Furthermore, a previously reported in-frame deletion mutation (40_63del24) in exon 1 of the COL4A3 gene was found in four patients in our study. A single 40_63del24 mutation in COL4A3 seems to result in mild or no renal manifestations, whereas the homozygous state of 40_63del24 in COL4A3 gene or compound heterozygous mutation of 40_63del24 plus another nonsense or frameshift mutation in COL4A3 gene seems to result severe ARAS with hearing loss. Half of the probands' parents had hematuria with or without mild proteinuria. Therefore, we recommend that ARAS be considered when a patient has a positive family history of hematuria, and screening for COL4A3 mutations firstly may be an efficient strategy for molecular diagnosis of ARAS.

Abstract 9: Imbalanced oncogenic signaling: Analogies between structurally distinct EGFR mutants of relevance to lung and brain cancers

Abstract The epidermal growth factor receptor (EGFR) is expressed at elevated levels in a large fraction of brain, lung, breast, prostate, and head-and-neck cancers. Efforts to treat such cancers with EGFR-targeted antibodies and kinase inhibitors have had limited success, suggesting that the level of EGFR expression in a tumor is not generally a good predictor of response to drugs targeting EGFR. In some cases, however, response to EGFR-targeted agents correlates with the presence of certain EGFR mutations. For example, in non-small cell lung cancer (NSCLC), the small percentage of patients harboring kinase-activating mutations of EGFR (mostly small in-frame deletion and point mutations) respond unusually well to EGFR kinase inhibitors such as gefitinib and erlotinib. In glioblastoma multiforme (GBM), the presence of the EGFRvIII mutation (a distinct deletion mutation which also promotes elevated EGFR activity) is also associated with increased response to EGFR inhibitors, at least in cells expressing PTEN. Settings where the elevated activity of a particular kinase promotes cellular responsiveness to drugs targeting the kinase have been identified as examples of the phenomenon of “oncogene addiction.” One of the prevailing models for EGFR oncogene addiction holds that EGFR mutations simultaneously lead to the increased activities of pro-survival and pro-apoptotic signaling pathways, with the latter persisting longer in response to EGFR inhibition and ultimately prevailing in determining cellular phenotypes. Here, we offer an alternative explanation for at least part of the altered cellular sensitivity to EGFR inhibition observed in the context of EGFR mutation in NSCLC and GBM. Specifically, we demonstrate that downstream of these structurally distinct EGFR mutants an important pathway involving the activation of ERK via the protein tyrosine phosphatase SHP2 is functionally impaired, and that this perturbs cellular responsiveness to gefitinib. Based upon immunoprecipitation, immunofluorescence, activity, and receptor trafficking studies, this mechanism appears to involve the sequestration of biochemically active SHP2 with endocytosis-impaired EGFR mutants at the plasma membrane in a manner which prevents the participation of SHP2 in the complete activation of ERK. Thus, our findings demonstrate that distinct EGFR-activating mutations may result in qualitatively similar perturbations to downstream signaling leading to the surprising impairment of receptor-mediated ERK activation. Given that elevated expression of these EGFR mutants generally promotes cellular response to EGFR inhibitors, this work identifies SHP2 and ERK as potentially critical nodes in the signaling network downstream of EGFR for co-inhibition in settings where EGFR inhibitors are not effective as single agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 9. doi:1538-7445.AM2012-9

Novel in-frame deletion mutation in FLCN gene in a Korean family with recurrent primary spontaneous pneumothorax

Birt–Hogg–Dubé syndrome (BHDS) is an autosomal dominant disease presenting with skin fibrofolliculomas, pulmonary cysts, primary spontaneous pneumothorax (PSP), and renal cancer. It is caused by germline mutations in the FLCN gene, which encodes folliculin. Here we report a novel in-frame deletion mutation p.F143del (c.427_429delTTC) in exon 6 of FLCN gene in the proband and her two sisters. The proband was a 40-year-old Korean woman who presented with right-sided pneumothorax and papular lesions on the face and neck area but without renal cancer. Her father also had a history of PSP and died of renal cancer at the age of 75. Her older sisters have been treated for recurrent PSP but did not have skin lesions suspicious of fibrofolliculoma. The relative expression of FLCN was significantly reduced in the proband and one of the sibling who was confirmed to have FLCN mutation. In-frame deletions in the FLCN gene have rarely been reported but have been shown to impose significant effect on protein stability of FLCN. Identification of a novel genotype in BHDS will provide clues to the phenotype–genotype relations and may aid in explaining the molecular pathogenesis of diseases related to FLCN mutation.

Genetic Background Modulates the Phenotype of a Mouse Model of DYT1 Dystonia

DYT1 dystonia is a debilitating neurological disease characterized by involuntary twisting movements. The disease is caused by an in-frame deletion (GAG, “ΔE”) mutation in the TOR1A gene that encodes the torsinA protein. Intriguingly, only 30% of mutation carriers exhibit motor symptoms despite the fact that functional brain imaging studies show abnormal brain metabolism in all carriers. Because genetic modifiers may be a determinant of this reduced penetrance, we examined the genetic contribution of three different inbred strains of mice on the DYT1 mutation in animals that are homozygous (Tor1aΔE/ΔE) or heterozygous (Tor1aΔE/+; disease state) for the disease-causing ΔE mutation. We find that the DBA/2J, C57BL/6J, and CD1-ICR contribution of genes significantly alter lifespan in Tor1aΔE/ΔE mice, which die during the first few days of life on the 129S6/SvEvTac (129) background. The C57BL/6J (B6) strain significantly decreases life expectancy of Tor1aΔE/ΔE animals but, like 129S6/SvEvTac Tor1aΔE/+ mice, congenic C57BL/6J Tor1aΔE/+ mice do not exhibit any motor abnormalities. In contrast, the DBA/2J (D2) strain significantly increases life expectancy. This effect was not present in congenic DBA/2J Tor1aΔE/ΔE mice, indicating that the extended lifespan of F2 129/D2 mice was due to a combination of homozygous and heterozygous allelic effects. Our observations suggest that genetic modifiers may alter the penetrance of the ΔE mutation, and that mapping these modifiers may provide fresh insight into the torsinA molecular pathway.

Sialic Acid Catabolism and Transport Gene Clusters Are Lineage Specific in Vibrio vulnificus

Sialic or nonulosonic acids are nine-carbon alpha ketosugars that are present in all vertebrate mucous membranes. Among bacteria, the ability to catabolize sialic acid as a carbon source is present mainly in pathogenic and commensal species of animals. Previously, it was shown that several Vibrio species carry homologues of the genes required for sialic acid transport and catabolism, which are genetically linked. In Vibrio cholerae on chromosome I, these genes are carried on the Vibrio pathogenicity island-2 region, which is confined to pathogenic isolates. We found that among the three sequenced Vibrio vulnificus clinical strains, these genes are present on chromosome II and are not associated with a pathogenicity island. To determine whether the sialic acid transport (SAT) and catabolism (SAC) region is universally present within V. vulnificus, we examined 67 natural isolates whose phylogenetic relationships are known. We found that the region was present predominantly among lineage I of V. vulnificus, which is comprised mainly of clinical isolates. We demonstrate that the isolates that contain this region can catabolize sialic acid as a sole carbon source. Two putative transporters are genetically linked to the region in V. vulnificus, the tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM and a component of an ATP-binding cassette (ABC) transporter. We constructed an in-frame deletion mutation in siaM, a component of the TRAP transporter, and demonstrate that this transporter is essential for sialic acid uptake in this species. Expression analysis of the SAT and SAC genes indicates that sialic acid is an inducer of expression. Overall, our study demonstrates that the ability to catabolize and transport sialic acid is predominately lineage specific in V. vulnificus and that the TRAP transporter is essential for sialic acid uptake.

Autoimmune Hepatitis Type 2 in a Child With IPEX Syndrome

Autoimmune Hepatitis Type 2 in a Child With IPEX Syndrome

A meckelin–filamin A interaction mediates ciliogenesis

MKS3, encoding the transmembrane receptor meckelin, is mutated in Meckel-Gruber syndrome (MKS), an autosomal-recessive ciliopathy. Meckelin localizes to the primary cilium, basal body and elsewhere within the cell. Here, we found that the cytoplasmic domain of meckelin directly interacts with the actin-binding protein filamin A, potentially at the apical cell surface associated with the basal body. Mutations in FLNA, the gene for filamin A, cause periventricular heterotopias. We identified a single consanguineous patient with an MKS-like ciliopathy that presented with both MKS and cerebellar heterotopia, caused by an unusual in-frame deletion mutation in the meckelin C-terminus at the region of interaction with filamin A. We modelled this mutation and found it to abrogate the meckelin-filamin A interaction. Furthermore, we found that loss of filamin A by siRNA knockdown, in patient cells, and in tissues from Flna(Dilp2) null mouse embryos results in cellular phenotypes identical to those caused by meckelin loss, namely basal body positioning and ciliogenesis defects. In addition, morpholino knockdown of flna in zebrafish embryos significantly increases the frequency of dysmorphology and severity of ciliopathy developmental defects caused by mks3 knockdown. Our results suggest that meckelin forms a functional complex with filamin A that is disrupted in MKS and causes defects in neuronal migration and Wnt signalling. Furthermore, filamin A has a crucial role in the normal processes of ciliogenesis and basal body positioning. Concurrent with these processes, the meckelin-filamin A signalling axis may be a key regulator in maintaining correct, normal levels of Wnt signalling.

Diffuse and focal palmoplantar keratoderma can be caused by a keratin 6c mutation

The palmoplantar keratodermas (PPKs) are a large group of genodermatoses comprising nearly 60 genetically distinct diseases. They are characterized by hyperkeratosis on the palms and soles with or without extrapalmoplantar hyperkeratotic lesions. Focal PPK is one of the hallmarks of pachyonychia congenita, a rare autosomal dominant disorder resulting from mutations in the keratin genes KRT6A, KRT6B, KRT16 or KRT17. Recently, in-frame deletion mutations of KRT6C have been identified in three families with focal PPK with slight or no nail changes. We report here a novel KRT6C mutation identified in a Japanese family with PPK with phenotypic heterogeneity, presenting with not only focal but also diffuse hyperkeratosis. The proband had diffuse hyperkeratosis on the soles and small focal hyperkeratoses on the palms, while the two other affected individuals showed focal hyperkeratoses on the soles. All three patients were heterozygotes for c.1414G>A in KRT6C, predicted to result in p.Glu472Lys. These findings strongly suggest that screening of patients with nonepidermolytic diffuse PPK, in whom the pathogenic mutations are yet to be determined, might identify mutations in KRT6C.

Kindler syndrome: Extension of FERMT1 mutational spectrum and natural history

Mutations in the FERMT1 gene (also known as KIND1), encoding the focal adhesion protein kindlin-1, underlie the Kindler syndrome (KS), an autosomal recessive skin disorder with an intriguing progressive phenotype comprising skin blistering, photosensitivity, progressive poikiloderma with extensive skin atrophy, and propensity to skin cancer. Herein we review the clinical and genetic data of 62 patients, and delineate the natural history of the disorder, for example, age at onset of symptoms, or risk of malignancy. Although most mutations are predicted to lead to premature termination of translation, and to loss of kindlin-1 function, significant clinical variability is observed among patients. There is an association of FERMT1 missense and in-frame deletion mutations with milder disease phenotypes, and later onset of complications. Nevertheless, the clinical variability is not fully explained by genotype-phenotype correlations. Environmental factors and yet unidentified modifiers may play a role. Better understanding of the molecular pathogenesis of KS should enable the development of prevention strategies for disease complications.

Oculodentodigital Dysplasia

To describe new ocular findings associated with oculodentodigital dysplasia (ODDD) and a novel mutation in the connexin 43 transmembrane domain. Oculodentodigital dysplasia is a rare autosomal dominant disease characterized by multiple systemic abnormalities, most commonly of the ocular, nasal, dental, and limb structures. Herein, we studied 2 patients with ODDD. We describe their clinical findings and 2 ocular abnormalities not previously emphasized or reported. Optic nerve and retinal dysplasia was observed in both patients, and ciliary body cysts were observed in 1 patient. Both patients carried isolated in-frame deletion and missense mutations of the GJA1 gene on chromosome 6. Optic nerve and retinal dysplasia had not been emphasized as ocular manifestations of ODDD. Ciliary body cysts have not previously been reported in association with ODDD. Our findings support the potential significance of connexin 43 in the retina, optic nerve, and ciliary body. Retinal and optic nerve dysplasia may be more common than previously appreciated and may be associated with reduced vision. In addition, the ciliary body cysts observed in 1 patient may be secondary to weakened cellular adhesions between ciliary body pigmented and nonpigmented epithelium associated with the in-frame deletion identified in the affected patient. The presence of these cysts may exacerbate glaucoma or complicate its management.

Identification of EGFR and KRAS mutations in Chinese patients with esophageal squamous cell carcinoma

The prognosis of esophageal squamous cell carcinoma (ESCC) is poor. It is urgent to improve this situation. Epidermal growth factor receptor (EGFR)-targeted therapy possesses a promising clinical efficacy. Mutations of EGFR and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) have been identified in esophageal carcinoma, but corresponding Chinese data are limited. So we investigated the mutation status of EGFR and KRAS in Chinese patients with ESCC, and explored their correlations with clinicopathological features. Formalin-fixed paraffin-embedded surgically resected tumor samples were obtained from 50 randomly selected Chinese patients with ESCC. EGFR mutations in exons 18-21 were detected by Scorpions amplification refractory mutation system technology. KRAS mutations in codons 12, 13 were detected by direct sequencing of polymerase chain reaction products. The correlations between clinicopathological features and the mutation status of EGFR and KRAS were analyzed using the Statistical Package for the Social Sciences. In the present study, EGFR mutations were found in 7 (14%) out of 50 patients, including G719X missense mutation (n= 1), in-frame deletion (n= 2), and L858R missense mutation (n= 5). Six (12%) out of 50 patients had KRAS mutations in codon 12. Concurrent EGFR and KRAS mutations were detected in one sample. The presences of EGFR and KRAS mutations were not associated with gender, age, smoking history, cell differentiation, or cancer stage. In conclusion, the incidence of EGFR mutations in Chinese patients with ESCC was higher than that of previous reports, and the incidence of KRAS mutations was not low. EGFR and KRAS mutations were mainly located in exons 19 and 21 and codon 12, respectively. Unlike in NSCLC, concurrent EGFR and KRAS mutations existed.

EGFRL858R Mutation and Polymorphisms of Genes Related to Estrogen Biosynthesis and Metabolism in Never-Smoking Female Lung Adenocarcinoma Patients

To assess whether polymorphisms of genes related to estrogen biosynthesis and metabolism are associated with EGFR mutations. We studied 617 patients with lung adenocarcinoma, including 302 never-smoking women. On the basis of multiple candidate genes approach, the effects of polymorphisms of CYP17, CYP19A1, ERα, and COMT in association with the occurrence of EGFR mutations were evaluated using logistic regression analysis. In female never-smokers, significant associations with EGFR L858R mutation were found for the tetranucleotide (TTTA)(n) repeats in CYP19A1 (odds ratio, 2.6; 95%CI, 1.2-5.7 for 1 or 2 alleles with (TTTA)(n) repeats >7 compared with both alleles with (TTTA)(n) repeats ≤ 7), and the rs2234693 in ERα (OR, 2.1; 95% CI, 1.1-4.0 for C/T and C/C genotypes compared with T/T genotype). The C/C genotype (vs. T/T genotype) of ERα was significantly associated with EGFR L858R mutation (OR, 3.0; 95% CI, 1.1-8.1), in-frame deletion (OR, 2.9; 95% CI, 1.1-7.6) and other mutations (OR, 4.3; 95% CI, 1.3-14.0). The genotype of COMT rs4680 was significantly associated with EGFR L858R mutation in female and male never-smokers showing OR's (95% CI) of 1.8 (1.0-3.2) and 3.6 (1.1-11.3), respectively, for genotypes G/A and G/G compared with genotype A/A. The number of risk alleles of CYP17, CYP19A1, ERα, and COMT was associated with an increasing OR of EGFR L858R mutation in female never-smokers (P = 0.0002 for trend). The L858R mutation of EGFR is associated with polymorphisms of genes related to estrogen biosynthesis and metabolism in never-smoking female lung adenocarcinoma patients.

Nuclear localization of the meiosis-specific transcription factor Ndt80 is regulated by the pachytene checkpoint

In budding yeast, the Ndt80 protein is a meiosis-specific transcription factor that is essential for the exit of pachytene and progression into nuclear divisions and spore formation. The pachytene checkpoint responds to defects in meiotic recombination and chromosome synapsis and negatively regulates the activity of Ndt80. The activity of Ndt80 was suggested to be regulated at both transcriptional and posttranslational levels; however, the mechanism for posttranslational regulation of Ndt80 was unclear. From a study of ndt80 in-frame deletion mutations, we have identified a dominant mutation NDT80-bc, which is able to completely bypass the pachytene checkpoint. The NDT80-bc mutation relieves the checkpoint-mediated arrest of the zip1, dmc1, and hop2 mutants, producing spores with low viability. The NDT80-bc mutant provides direct evidence for the posttranslational control of Ndt80 activity. Furthermore, the data presented show that Ndt80 is retained in cytoplasm in the zip1 mutant, whereas Ndt80-bc is found in the nucleus. We propose that the nuclear localization of Ndt80 is regulated by the pachytene checkpoint through a cytoplasmic anchor mechanism.

Analysis of Phosphotyrosine Signaling in Glioblastoma Identifies STAT5 as a Novel Downstream Target of ΔEGFR

An in-frame deletion mutation in Epidermal Growth Receptor (EGFR), ΔEGFR is a common and potent oncogene in glioblastoma (GBM), promoting growth and survival of cancer cells. This mutated receptor is ligand independent and constitutively active. Its activity is low in intensity and thought to be qualitatively different from acutely ligand stimulated wild-type receptor implying that the preferred downstream targets of ΔEGFR play a significant role in malignancy. To understand the ΔEGFR signal, we compared it to that of a kinase-inactivated mutant of ΔEGFR and wild-type EGFR with shotgun phosphoproteomics using an electron-transfer dissociation (ETD) enabled ion trap mass spectrometer. We identified and quantified 354 phosphopeptides corresponding to 249 proteins. Among the ΔEGFR-associated phosphorylations were the previously described Gab1, c-Met and Mig-6, and also novel phosphorylations including that of STAT5 on Y694/9. We have confirmed the most prominent phosphorylation events in cultured cells and in murine xenograft models of glioblastoma. Pathway analysis of these proteins suggests a preference for an alternative signal transduction pathway by ΔEGFR compared to wild-type EGFR. This understanding will potentially benefit the search for new therapeutic targets for ΔEGFR expressing tumors.

Slk19p of Saccharomyces cerevisiae Regulates Anaphase Spindle Dynamics Through Two Independent Mechanisms

Slk19p is a member of the Cdc-14 early anaphase release (FEAR) pathway, a signaling network that is responsible for activation of the cell-cycle regulator Cdc14p in Saccharomyces cerevisiae. Disruption of the FEAR pathway results in defects in anaphase, including alterations in the assembly and behavior of the anaphase spindle. Many phenotypes of slk19Δ mutants are consistent with a loss of FEAR signaling, but other phenotypes suggest that Slk19p may have FEAR-independent roles in modulating the behavior of microtubules in anaphase. Here, a series of SLK19 in-frame deletion mutations were used to test whether Slk19p has distinct roles in anaphase that can be ascribed to specific regions of the protein. Separation-of-function alleles were identified that are defective for either FEAR signaling or aspects of anaphase spindle function. The data suggest that in early anaphase one region of Slk19p is essential for FEAR signaling, while later in anaphase another region is critical for maintaining the coordination between spindle elongation and the growth of interpolar microtubules.