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Dominant Negative Effect (dominant + negative_effect)
Selected AbstractsThe first CH domain of affixin activates Cdc42 and Rac1 through ,PIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factorGENES TO CELLS, Issue 3 2004Wataru Mishima Rho GTPases, Cdc42 and Rac1, play pivotal roles in cell migration by efficiently integrating cell-substrate adhesion and actin polymerization. Although it has been suggested that integrins stimulate these Rho GTPases via some of integrin binding proteins such as focal adhesion kinase (FAK) and paxillin, the precise molecular mechanism is largely unknown. In this study, we showed that the over-expression of RP1 corresponding to the first CH domain (CH1) of affixin, an integrin-linked kinase (ILK)-binding protein, induced a significant actin reorganization in MDCK cells by activating Cdc42/Rac1. Affixin full length and RP1 co-immunoprecipitated with ,PIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor (GEF), and they co-localized at the tips of lamellipodia in motile cells. The involvement of ,PIX in the RP1-induced Cdc42 activation was demonstrated by the significant dominant negative effect of a point mutant of ,PIX, ,PIX (L383R, L384S), lacking GEF activity. Our data strongly support that ILK and affixin provide a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation. [source] NKX2-1 mutations leading to surfactant protein promoter dysregulation cause interstitial lung disease in "Brain-Lung-Thyroid Syndrome",HUMAN MUTATION, Issue 2 2010Loďc Guillot Abstract NKX2-1 (NK2 homeobox 1) is a critical regulator of transcription for the surfactant protein (SP)-B and -C genes (SFTPB and SFTPC, respectively). We identified and functionally characterized two new de novo NKX2-1 mutations c.493C>T (p.R165W) and c.786_787del2 (p.L263fs) in infants with closely similar severe interstitial lung disease (ILD), hypotonia, and congenital hypothyroidism. Functional analyses using A549 and HeLa cells revealed that NKX2-1-p.L263fs induced neither SFTPB nor SFTPC promoter activation and had a dominant negative effect on wild-type (WT) NKX2-1. In contrast,NKX2-1-p.R165W activated SFTPC, to a significantly greater extent than did WTNKX2-1,whileSFTPB activation was only significantly reduced in HeLa cells. In accordance with our in vitro data, we found decreased amounts of SP-B and SP-C by western blot in bronchoalveolar lavage fluid (patient with p.L263fs) and features of altered surfactant protein metabolism on lung histology (patient with NKX2-1-p.R165W). In conclusion, ILD in patients with NKX2-1 mutations was associated with altered surfactant protein metabolism, and both gain and loss of function of the mutated NKX2-1 genes on surfactant protein promoters were associated with ILD in "Brain-Lung-Thyroid syndrome". © 2009 Wiley-Liss, Inc. [source] Cancer-associated missplicing of exon 4 influences the subnuclear distribution of the DNA replication factor CIZ1,HUMAN MUTATION, Issue 10 2007Faisal Abdel Rahman Abstract Cip1-interacting zinc finger protein 1 (CIZ1, also known as CDKN1A-interacting zinc finger protein 1) stimulates initiation of mammalian DNA replication and is normally tethered to the nuclear matrix within DNA replication foci. Here, we show that an alternatively spliced human CIZ1 variant, lacking exon 4 (, E4), is misexpressed as a consequence of intronic mutation in Ewing tumor (ET) cell lines. In all ET lines tested, exon 4 is skipped and an upstream mononucleotide repeat element is expanded to contain up to 28 thymidines, compared to 16 in controls. In exon-trap experiments, a 24T variant produced three-fold more exon skipping than a 16T variant, demonstrating a direct effect on splicing. In functional assays, , E4 protein retains replication activity, but fails to form subnuclear foci. Furthermore, coexpression of mouse , E4 with Ciz1 prevents Ciz1 from localizing appropriately, having a dominant negative effect on foci formation. The data show that conditional exclusion of exon 4 influences the spatial distribution of the Ciz1 protein within the nucleus, and raise the possibility that CIZ1 alternative splicing could influence organized patterns of DNA replication. Hum Mutat 28(10), 993,1004, 2007. © 2007 Wiley-Liss, Inc. [source] ARC protects rat cardiomyocytes against oxidative stress through inhibition of caspase-2 mediated mitochondrial pathwayJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Yi-Qiang Zhang Abstract Apoptosis repressor with a CARD domain (ARC) has been demonstrated to protect heart cells against ischemia/reperfusion (I/R) injury. In this study, we investigated the mechanism by which ARC protects heart cells against oxidative stress. We monitored the extent of apoptosis and activity of multiple components of the intrinsic apoptotic pathway in rat cardiac myoblast cell line H9c2 with either reduced or increased expression of ARC during oxidative stress. Overexpression of ARC-inhibited oxidative stress-induced caspase-2/3 activation, cytochrome c release, and translocation of Bax to mitochondria. Furthermore, phosphorylation of ARC at threonine 149 was found to be critical to its function. ARC containing a T149A mutation failed to translocate to mitochondria, did not inhibit caspase-2 activation, and had a dominant negative effect against the protective effect of endogenous ARC during oxidative stress. In addition, wild-type ARC but not the T149A mutant inhibited cell death induced by overexpression of caspase-2. Using a yeast two-hybrid (YTH) screening approach and co-immunoprecipitation (Co-IP), we found that protein phosphatase 2C (PP2C) interacted with ARC and that PP2C mediated-dephosphorylation of ARC inhibited its anti-apoptotic activity. Eliminating either the N-terminal CARD domain or the C-terminal P/E domain also abolished the anti-apoptotic function of ARC, suggesting that full-length ARC is required for its apoptotic inhibition. These results indicate that ARC plays an important role in protection of H9c2 cells against oxidative stress-induced apoptosis by phosphorylation-dependent suppression of the mitochondria-mediated intrinsic pathway, partially initiated through the activation of caspase-2. J. Cell. Biochem. 99: 575,588, 2006. © 2006 Wiley-Liss, Inc. [source] Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in ArabidopsisTHE PLANT JOURNAL, Issue 2 2003Yafan Huang Summary CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1,8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1,8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway. [source] A non-sense mutation in the corneodesmosin gene in a Mexican family with hypotrichosis simplex of the scalpBRITISH JOURNAL OF DERMATOLOGY, Issue 6 2005N.O. Dávalos Summary Background, Hypotrichosis simplex of the scalp (HSS; MIM 146520) is a rare autosomal dominant form of non-syndromic alopecia that affects men and women equally. Up to now, only a small number of families with HSS have been reported. The affected individuals experience a diffuse progressing hair loss from childhood to adulthood that is confined to the scalp. Recently, HSS has been mapped to the short arm of chromosome 6 (6p21.3), allowing mutations in the corneodesmosin gene (CDSN) to be identified as the cause of the disorder. To date, two stop mutations have been found in three unrelated families with HSS of different ethnic origin. Objectives, To describe the first HSS-family with Latin American (Mexican) background comprising 6 generations and to identify a mutation in the CDSN gene. Patients/Methods, The patients were examined by a clinician and blood samples were taken. After DNA extraction, sequencing analysis of the CDSN gene and restriction enzyme analysis with PsuI were performed. Results, By direct sequencing of the two exons of the CDSN gene, a nonsense mutation was identified in the index patient in exon 2, resulting in a premature stop codon (Y239X). The mutation cosegregates perfectly in the family with the disease and was not found in 300 control chromosomes using a restriction enzyme analysis with PsuI. Conclusions, A nonsense mutation was identified in the first family with HSS of Latin American ethnical background. Our data provide molecular genetic evidence for a 3rd stop mutation in exon 2 of the CDSN gene being responsible for HSS. All to date known nonsense mutations responsible 3 for HSS are clustered in a region of 40 amino acids which is in accordance with a dominant negative effect conferred by aggregates of truncated CDSN proteins. 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