Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Transient Mutator”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,135 records · Page 63Linked to original sources
PubMed1990

Schindler disease: the molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes an infantile neuroaxonal dystrophy.

Schindler disease is a recently recognized infantile neuroaxonal dystrophy resulting from the deficient activity of the lysosomal hydrolase, alpha-N-acetylgalctosaminidase (alpha-GalNAc). The recent isolation and expression of the full-length cDNA encoding alpha-GalNAc facilitated the identification of the molecular lesions in the affected brothers from family D, the first cases described with this autosomal recessive disease. Southern and Northern hybridization analyses of DNA and RNA from the affected homozygotes revealed a grossly normal alpha-GalNAc gene structure and normal transcript sizes and amounts. Therefore, the alpha-GalNAc transcript from an affected homozygote was reverse-transcribed, amplified by the polymerase chain reaction (PCR), and sequenced. A single G to A transition at nucleotide 973 was detected in multiple subclones containing the PCR products. This point mutation resulted in a glutamic acid to lysine substitution in residue 325 (E325K) of the alpha-GalNAc polypeptide. The base substitution was confirmed by dot blot hybridization analyses of PCR-amplified genomic DNA from family members with allele-specific oligonucleotides. Furthermore, transient expression of an alpha-GalNAc construct containing the E325K mutation resulted in the expression of an immunoreactive polypeptide which had no detectable alpha-GalNAc activity.

Base Sequence↗
PubMed1994

Characterization of thrombomodulin expression in response to retinoic acid and identification of a retinoic acid response element in the human thrombomodulin gene.

Thrombomodulin (TM) is an essential cofactor for the physiologic activation of the anticoagulant protein C by thrombin. We have observed that the expression of TM mRNA in response to retinoic acid was markedly increased in human U937 monoblast-like cells, and human MEG01 megakaryocyte-like cells, but not in human umbilical vein cells, murine hemangioma cells, human K562 erythroblast-like cells, and murine HSD fibroblast-like cells. TM activity in U937 cells and MEG01 cells was not detectable in untreated cells, but developed rapidly after treatment with retinoic acid. In endothelial cells there was minimal change in TM activity in response to retinoic acid treatment. We have isolated clones for the genes for murine and human TM and have identified potential retinoic acid response elements in the 5'-flanking region of the human gene. In U937 cells the increase in mRNA levels was associated with increased transcription, and transient transfection studies with reporter plasmids demonstrate functional retinoic acid response elements present in the 5'-flanking region of the gene. Deletion of, and mutations introduced into, the potential retinoic acid response element confirm the functional response in transient transfections.

Animals↗
PubMed1994

The molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes angiokeratoma corporis diffusum with glycopeptiduria.

Angiokeratoma corporis diffusum with glycopeptiduria is a recently recognized inborn error of glycoprotein catabolism resulting from the deficient activity of human alpha-N-acetylgalactosaminidase (E.C. 3.2.1.49; alpha-GalNAc). The first patient with this autosomal recessive disorder, a 46-yr-old consanguineous Japanese woman, presented with diffuse angiokeratoma, mild intellectual impairment, and peripheral neuroaxonal degeneration. Deficient alpha-GalNAc activity also has been reported in consanguineous brothers with an infantile-onset form of neuroaxonal dystrophy resulting from a missense mutation (designated E325K) in the alpha-GalNAc gene. To identify the mutation causing the phenotypically distinct adult-onset disorder, Southern and Northern hybridization analyses of DNA and RNA from the affected homozygote were performed which revealed a grossly normal alpha-GalNAc gene structure and normal transcript size and abundancy. Reverse transcription, amplification, and sequencing of the alpha-GalNAc transcript identified a single C to T transition at nucleotide (nt) 985 that predicted an arginine to tryptophan substitution in residue 329 (designated R329W) of the alpha-GalNAc polypeptide. This base substitution was confirmed by hybridization of PCR-amplified genomic DNA from family members with allele-specific oligonucleotides. Transient expression of an alpha-GalNAc construct containing the R329W mutation resulted in the expression of an immunoreactive polypeptide which had no detectable alpha-GalNAc activity. Comparison of the biosynthesis and stabilities of the transiently expressed and radiolabeled normal, E325K (infantile-onset) and R329W (adult-onset) alpha-GalNAc polypeptides in COS-1 cells indicated that both the mutant precursors were processed to the mature form; however, the E325K mutant polypeptide was more rapidly degraded than the R329W subunit, thereby providing a basis for the distinctly different infantile- and adult-onset phenotypes.

Base Sequence↗
PubMed2004

Structure-function relationship of the extracellular calcium-sensing receptor.

The extracellular calcium-sensing receptor (CaR) originally cloned from bovine parathyroid gland is a G protein-coupled receptor. The physiological relevance of the cloned CaR for sensing and regulating the extracellular calcium concentration has been established by identifying hyper- and hypocalcemic disorders resulting from inactivating and activating mutations, respectively, in the CaR. The cloned CaR has been stably or transiently expressed in human embryonic kidney cells and significant progress has been made in elucidating its regulation and activation process using physiological, biochemical and molecular biological methods. A large collection of naturally occurring CaR mutations offers a valuable resource for studies aimed at understanding the structure-function relationships of the receptor, including functional importance of CaR dimerization. In turn, characterization of these naturally occurring mutations has clarified the pathogenesis of clinical conditions involving abnormalities in the CaR, such as familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.

Allosteric Regulation↗
PubMed1996

YY1 and NF1 both activate the human p53 promoter by alternatively binding to a composite element, and YY1 and E1A cooperate to amplify p53 promoter activity.

A novel transcription factor binding element in the human p53 gene promoter has been characterized. It lies about 100 bp upstream of the major reported start site for human p53 gene transcription. On the basis of DNase I footprinting studies, electromobility shift assay patterns, sequence specificity of binding, the binding pattern of purified transcription factors, effects of specific antibodies, and methylation interference analysis we have identified the site as a composite element which can bind both YY1 and NF1 in an independent and mutually exclusive manner. The site is conserved in the human, rat, and mouse p53 promoters. The occupancy of the site varies in a tissue-specific manner. It binds principally YY1 in nuclear extracts of rat testis and spleen and NF1 in extracts of liver and prostate. This may facilitate tissue-specific control of p53 gene expression. When HeLa cells were transiently transfected with human p53 promoter-chloramphenicol acetyltransferase reporter constructs, a mutation in this composite element which disabled YY1 and NF1 binding caused a mean 64% reduction in basal p53 promoter activity. From mutations which selectively impaired YY1 or NF1 binding and the overexpression of YY1 or NF1 in HeLa cells we concluded that both YY1 and NF1 function as activators when bound to this site. In transient cotransfections E1A could induce the activity of the p53 promoter to a high level; 12S E1A was threefold as efficient as 13S E1A in this activity, and YY1 bound to the composite element was shown to mediate 55% of this induction. Overexpressed YY1 was shown to be able to synergistically activate the p53 promoter with E1A when not specifically bound to DNA. Deletion of an N-terminal domain of E1A, known to be required for direct E1A-YY1 interaction and E1A effects mediated through transcriptional activator p300, blocked the E1A induction of p53 promoter activity.

Animals↗
PubMed1999

Structure and function of the extracellular calcium-sensing receptor (Review).

The extracellular calcium-sensing receptor (CaR) originally cloned from bovine parathyroid gland is a G protein-coupled receptor. The physiological relevance of the cloned CaR for sensing and regulating the extracellular calcium concentration has been established by identifying hyper- and hypocalcemic disorders resulting from inactivating and activating mutations, respectively, in the CaR. The cloned CaR has been stably or transiently expressed in human embryonic kidney cells and significant progress has been made in elucidating its biochemical and functional features using physiological, biochemical and molecular biological methods. A large collection of naturally occurring CaR mutations offers a valuable resource for studies aimed at understanding the structure-function relationships of the receptor, including receptor-receptor interactions. In turn, characterization of these naturally occurring mutations has clarified the pathogenesis of clinical conditions involving abnormalities in the CaR, such as familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, and the physiology of certain cell types, such as keratinocytes.

Humans↗
PubMed2000

[Audiological findings and mitochondrial DNA mutation in a large family with matrilineal sensorineural hearing loss].

OBJECTIVE: To explore audiological features of matrilineal non-syndromic deafness and its molecular mechanism. METHODS: A large family with 41 members having inherited deafness was studied. Complete history and the data of general and otolaryngological examinations were collected. All subjects were screened for mitochondrial DNA A1555G mutation by molecular analysis. Audiological evaluation included puretone audiometry, auditory brainstem responses and transiently evoked otoacoustic emissions. RESULTS: All subjects were in good health generally. Molecular analysis showed that all maternal relatives with or without hearing loss harbored the A1555G mitochondrial mutation. No mutation was found among spouses and paternal relatives. Audiological results showed notable symmetric bilateral sensorineural hearing loss in 17 of 20 maternal relatives, in which 5 cases had a progressive hearing loss in the recent 11 years. The age of appearance of hearing loss ranged from 1 to 50 years. CONCLUSION: All hearing-impaired subjects of this family had late-onset sensorineural hearing loss. Most of which were progressive. The A1555G mitochondrial mutation in the 12S rRNA gene is responsible for the disorder. Other factors, such as nuclear genes or environmental determinants, may influence the clinical expression of mutant mtDNA.

Acoustic Impedance Tests↗
PubMed1993

Phosphorylation sites at the C-terminus of the platelet-derived growth factor receptor bind phospholipase C gamma 1.

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1.

Animals↗
PubMed2005

The mutational spectrum of PTPN11 in juvenile myelomonocytic leukemia and Noonan syndrome/myeloproliferative disease.

Germ line PTPN11 mutations cause 50% of cases of Noonan syndrome (NS). Somatic mutations in PTPN11 occur in 35% of patients with de novo, nonsyndromic juvenile myelomonocytic leukemia (JMML). Myeloproliferative disorders (MPDs), either transient or more fulminant forms, can also occur in infants with NS (NS/MPD). We identified PTPN11 mutations in blood or bone marrow specimens from 77 newly reported patients with JMML (n = 69) or NS/MPD (n = 8). Together with previous reports, we compared the spectrum of PTPN11 mutations in 3 groups: (1) patients with JMML (n = 107); (2) patients with NS/MPD (n = 19); and (3) patients with NS (n = 243). Glu76 was the most commonly affected residue in JMML (n = 45), with the Glu76Lys alteration (n = 29) being most frequent. Eight of 19 patients with NS/MPD carried the Thr73Ile substitution. These data suggest that there is a genotype/phenotype correlation in the spectrum of PTPN11 mutations found in patients with JMML, NS/MPD, and NS. This supports the need to characterize the spectrum of hematologic abnormalities in individuals with NS and to better define the impact of the PTPN11 lesion on the disease course in patients with NS/MPD and JMML.

Child↗
PubMed2000

Channelopathies: potassium-related periodic paralyses and similar disorders.

Channelopathy is a term used to describe clinical problems caused by disorders of membrane ion channels. Included in this disease category are certain types of periodic paralyses, ataxia, myotonia, migraine headache, epilepsy, nephrolithiasis, and long QT syndrome. This article briefly summarizes membrane ion channel structure and function and details several relatively common channelopathies. In hyperkalemic periodic paralysis, mutant skeletal muscle sodium channels fail to close completely after an action potential. This evokes two apparently opposite symptoms: myotonia (caused by a small depolarization and repetitive excitation) or paralysis (caused by larger depolarization and inexcitability). In hypokalemic periodic paralysis, mutation affects the closing of skeletal muscle calcium channels, causing transient paresis or paralysis. The task of the advanced practice nurse is to recognize these disorders, institute appropriate prophylactic measures and treatments, monitor symptom progression, and avoid complications. Understanding of channelopathies is advancing rapidly. On the horizon are therapies tailored to counter specific membrane ion channel defects.

Humans↗
PubMed2002

Trafficking of PLP/DM20 and cAMP signaling in immortalized jimpy oligodendrocytes.

The synthesis, transport, and insertion of jimpy proteolipid protein and DM20 were studied in normal (158N) and jimpy (158JP) immortalized oligodendrocyte lines. Four different expression vectors encoding fusion proteins composed of native PLP and DM20 or jimpy PLP or DM20 were linked to enhanced green fluorescent protein (EGFP). All four transfected fusion proteins had similar distributions in the cell bodies and processes of the two cell types. Both normal and jimpy PLP-EGFP and DM20-EGFP were detected in both cell lines as far as 200 microM from the cell body, indicating synthesis and transport of mutated PLP and DM20 toward the plasma membrane. Immunocytochemistry of fixed normal and jimpy cells with the O10 antibody, which recognizes a conformationally sensitive PLP/DM20 epitope, confirmed that normal and jimpy PLP and DM20 were transported to the plasma membrane. Live staining of normal and jimpy cells transiently transfected with the native PLP showed positive staining, indicating PLP was correctly inserted into the membrane of both normal and jimpy oligodendrocytes. However, live staining of normal and jimpy cells transiently transfected with jimpy PLP showed no positive staining, indicating the mutated protein is abnormally inserted into the plasma membrane. Electrophysiological recordings of the resting membrane potential measured in the whole cell mode of the patch-clamp technique showed the absence of a developmentally regulated negative shift in the membrane potential in jimpy cells compared to normal native or immortalized oligodendrocytes. Treatment of 158N cells and native oligodendrocytes with dibutyryl cAMP (dbcAMP) caused morphological and biochemical differentiation, but failed to do so in 158JP cells, suggesting an abnormal signaling pathway in jimpy. The defect in cAMP signaling in jimpy oligodendrocytes was associated with the suppression of increase in mRNA level of the inducible cAMP early repressor (ICER). When the jimpy oligodendrocyte line was transfected with normal PLP or DM20 and exposed to dbcAMP, the cells failed to differentiate. This finding suggests that improper insertion of jimpy protein into the plasma membrane alters the membrane in such a way that certain signaling pathways are permanently altered. The abnormal insertion of jimpy PLP/DM20 into the plasma membrane may be the basis for the lack of cell signaling and abnormal resting potential in jimpy oligodendrocytes.

Animals↗
PubMed2006

Replicative fidelity of lentiviral vectors produced by transient transfection.

Previous investigations have estimated the human immunodeficiency virus type 1 (HIV) base pair substitution rate to be approximately 10(-4) to 10(-5) per round of viral replication, and HIV has been hypothesized to be more error-prone than other retroviruses. Using a single cycle reversion assay, we unexpectedly found that the reversion rates of HIV, avian leukosis virus and Moloney murine leukemia virus were the same, within statistical error. Because both the viral enzyme reverse transcriptase (RT) and cellular RNA polymerase II (RNAP) are required for viral replication, we hypothesized that the similar reversion rates actually reflect the intrinsic error rate of RNAP, which is the enzyme common to all three retroviruses in the reversion assay. To address this possibility, HIV vectors with the U3 region replaced by a reporter reversion cassette were constructed and vector supernatant produced by transient transfection. All single integrant revertant cell lines showed the identical mutations at both long terminal repeats. This indicates that either RNAP or another cellular enzyme is responsible for these reversions, or that HIV RT only makes errors during first strand synthesis. Additionally, when HIV particles were rescued from an integrated vector as opposed to being produced by transient transfection, the reversion rate was significantly lower, suggesting that one or more factors in the virus-producing cells plays a role in the fidelity of retroviral replication. These results have implications regarding the fidelity of the transgene after transient transfection production of lentiviral vector supernatants.

Animals↗
PubMed2004

A new mutation in the KIT ATP pocket causes acquired resistance to imatinib in a gastrointestinal stromal tumor patient.

BACKGROUND & AIMS: Imatinib, a tyrosine kinase inhibitor of BCR-ABL, KIT, and platelet-derived growth factor receptor, is used in patients with chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors (GIST). Primary and acquired resistance to the drug can occur in both diseases. Molecular mechanisms have been reported in CML and GIST for primary resistance, whereas extensive studies on the mechanisms responsible for secondary resistance have been almost exclusively reported for CML. METHODS: In a patient with advanced GIST undergoing imatinib therapy, an isolated progressing peritoneal mass was excised, along with 2 still-responding lesions. Complementary DNA and genomic DNA were analyzed by sequencing for c-Kit gene mutations. KIT receptor expression and phosphorylation status were assessed by immunoprecipitation and Western blot. Transient-transfection experiments were performed with mutagenized KIT constructs, and their activation status was assessed. RESULTS: In addition to an exon 11 mutation, shared among all of the analyzed lesions, a novel point mutation in c-Kit exon 14 resulting in T670I substitution was found only in the progressing lesion, which harbored a phosphorylated receptor, as opposed to the finding of an inactive receptor in responding lesions. Functional analyses showed that KIT/T670I is insensitive to imatinib and that T670I mutation, introduced in a receptor responding to imatinib, subverted its sensitivity to the drug. CONCLUSIONS: This new mutation was confined to the progressing lesion; the resulting amino acidic substitution, T670I, affecting the ATP/imatinib pocket of KIT, makes it insensitive to the drug. Interestingly, this substitution is a homologue to the T315I mutation already reported in CML, where it is responsible for acquired resistance to imatinib.

Abdominal Neoplasms↗
PubMed1994

Molecular analysis of hprt gene mutations in skin fibroblasts of rats exposed in vivo to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

The granuloma pouch assay in the rat is a model system in which relative frequencies of genetic and (pre-) neoplastic changes induced in vivo by carcinogenic agents can be determined within the same target tissue. The target is granuloma pouch tissue and consists of a population of (transient) proliferating fibroblasts which can be cultured in vitro. hprt gene mutations were studied in granuloma pouch tissue of rats treated with single doses of direct acting alkylating agents N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). Both agents showed an exposure-dependent increase in the hprt mutant frequency. Thirty-seven MNU (60 mg/kg)- and 43 ENU (100 mg/kg)-induced hprt mutant cell clones were analyzed at the molecular level. Twenty-two MNU-induced and 36 ENU-induced mutants carried a single base pair change in exon sequences of the hprt gene. The predominant base pair alterations induced by MNU were GC to AT transitions (18 of 22), which are probably caused by O6-methylguanine lesions. For most of the GC to AT transitions (16 of 18), the G was located in the nontranscribed strand, suggesting a strand bias in the repair of O6-methylguanine lesions. ENU-induced mutations occurred predominantly at AT base pairs (28 of 36), being mostly AT to TA and AT to CG transversions, and are probably caused by O2-ethylthymidine. Also here, DNA repair processes seem to act with different rates/efficiencies on DNA adducts in the 2 strands of the hprt gene, since all the 24 transversions observed at AT base pairs had the thymidine residue in the nontranscribed strand. GC to AT transitions were only present at a low frequency among ENU-induced mutations, suggesting that O6-ethylguanine lesions were repaired efficiently before mutations were fixed during replication. The mutational spectra of MNU- and ENU-induced hprt mutant clones were different from spontaneously occurring hprt mutant clones. These results indicate that MNU and ENU induce different mutational spectra in vivo and that DNA repair systems remove O6-methylguanine, O2, and/or O4-ethylthymidine much faster from the transcribed strand than the nontranscribed strand of the hprt gene in these rat fibroblasts.

Amino Acid Sequence↗
PubMed2003

Uncoupling of leading- and lagging-strand DNA replication during lesion bypass in vivo.

Numerous agents attack DNA, forming lesions that impair normal replication. Specialized DNA polymerases transiently replace the replicative polymerase and copy past lesions, thus generating mutations, the major initiating cause of cancer. We monitored, in Escherichia coli, the kinetics of replication of both strands of DNA molecules containing a single replication block in either the leading or lagging strand. Despite a block in the leading strand, lagging-strand synthesis proceeded further, implying transient uncoupling of concurrent strand synthesis. Replication through the lesion requires specialized DNA polymerases and is achieved with similar kinetics and efficiencies in both strands.

2-Acetylaminofluorene↗
PubMed1998

Heat stress-induced life span extension in yeast.

The yeast Saccharomyces cerevisiae has a limited life span that can be measured by the number of times individual cells divide. Several genetic manipulations have been shown to prolong the yeast life span. However, environmental effects that extend longevity have been largely ignored. We have found that mild, nonlethal heat stress extended yeast life span when it was administered transiently early in life. The increased longevity was due to a reduction in the mortality rate that persisted over many cell divisions (generations) but was not permanent. The genes RAS1 and RAS2 were necessary to observe this effect of heat stress. The RAS2 gene is consistently required for maintenance of life span when heat stress is chronic or in its extension when heat stress is transient or absent altogether. RAS1, on the other hand, appears to have a role in signaling life extension induced by transient, mild heat stress, which is distinct from its life-span-curtailing effect in the absence of stress and its lack of involvement in the response to chronic heat stress. This distinction between the RAS genes may be partially related to their different effects on growth-promoting genes and stress-responsive genes. The ras2 mutation clearly hindered resumption of growth and recovery from stress, while the ras1 mutation did not. The HSP104 gene, which is largely responsible for induced thermotolerance in yeast, was necessary for life extension induced by transient heat stress. An interaction between mitochondrial petite mutations and heat stress was found, suggesting that mitochondria may be necessary for life extension by transient heat stress. The results raise the possibility that the RAS genes and mitochondria may play a role in the epigenetic inheritance of reduced mortality rate afforded by transient, mild heat stress.

Blotting, Northern↗
PubMed2003

[K(+)](o)-dependent change in conformation of the HERG1 long QT mutation N629D channel results in partial reversal of the in vitro disease phenotype.

OBJECTIVES: We hypothesized that exposure of N629D/wildtype channels to transient increases in [K(+)](o) could alter the conformation of the outer vestibule and thus reverse the disease phenotype. N629D is a recently described mutation of the HERG1 gene that causes familial long QT syndrome. This mutation alters the pore signature sequence resulting in loss of K(+) selectivity. Previous studies have reported that enforced occupancy of [K(+)](o) at sites near the selectivity filter alters the conformation/folding of the outer vestibule of the Kv2.1 channel. METHODS: Since the long QT syndrome is manifest in individuals who are heterozygous for this HERG trait, we co-expressed N629D and the wildtype at equimolar concentrations. RESULTS: Co-expression of N629D/wildtype in Xenopus oocytes and mammalian cells resulted in a channel with a positive shift in reversal potential and a loss in the outward tail current, relative to the wildtype. Exposure of the N629D/wildtype to transient increases in [K(+)](o) from 5 to 40 mM/l changed the tail current from inward to outward during repolarization and restored the reversal potential to values similar to the wildtype. These findings in Xenopus oocytes were also seen when N620D/wildtype channels were expressed in mammalian cells. These [K(+)](o)-dependent changes persisted for hours after the [K(+)](o) was returned to 2.5 mM. This potential therapeutic effect began with increases in [K(+)](o) from 2.5 to 5 mM. CONCLUSIONS: This study reports a novel therapeutic strategy and mechanism to partially restore physiologic function in this HERG LQTS mutation.

Animals↗
PubMed2003

A novel nuclear export signal in Smad1 is essential for its signaling activity.

To investigate the subcellular distributions of Smad proteins, the intracellular mediators of transforming growth factor-beta family cytokines, we examined their sequences for nuclear export signals (NES). We found a leucine-rich NES-like motif (termed NES2) in the central linker region of the receptor-regulated Smads that is absent from the other two classes of Smads (Co-Smads and I-Smads). In microinjection assays, NES2 peptide caused nuclear export of a fused glutathione S-transferase protein. Mutations in NES2 converted Smad1 from an even distribution throughout the cells into an exclusive nuclear localization in both transiently and stably expressing cell lines, and this nuclear enrichment was more pronounced than that induced by mutations in NES1. Furthermore, overexpression of CRM1, the cellular export receptor, transforms Smad1 into a mostly cytoplasmic profile by enhancing its nuclear export. The Smad1 NES2 mutant but not the Smad1 NES1 mutant is mostly resistant to this cytoplasmic targeting, indicating that NES2, not NES1, is the major target for CRM1 in Smad1. We further confirmed the functionality of NES2 by a heterokaryon assay. The Smad1 NES1 mutant displays good ligand responsiveness and moderately lowered transcriptional activity compared with wild type Smad1. In contrast, the Smad1 NES2 mutant shows a severe disruption in reporter gene activation, minimal response to bone morphogenetic protein stimulation, and significantly lowered bone morphogenetic protein-induced phosphorylation, which may be the reason for its deficient transcription activity. Thus, we have defined a major NES in Smad1 that is essential for its ligand-induced coupling with cell surface receptors and hence, transcriptional activity. Our study, along with recent studies of the nucleocytoplasmic shuttling of Smad2 and Smad3 proteins, demonstrate that continued nucleocytoplasmic shuttling is a common requisite for the active signaling of R-Smads. Although conserved in other R-Smads such as Smad3, NES2 is not functional in these R-Smads because CRM1 overexpression fails to target them to cytoplasm. Possible reasons for this discrepancy are discussed.

3T3 Cells↗