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At least 1,531 records · Page 85Linked to original sources
PubMed2004

Regulation of glucose transporter SGLT1 by ubiquitin ligase Nedd4-2 and kinases SGK1, SGK3, and PKB.

OBJECTIVE: Serum- and glucocorticoid-inducible kinase 1 (SGK1) inhibits the ubiquitin ligase neuronal cell expressed developmentally downregulated 4-2 (Nedd4-2), which retards the retrieval of the epithelial Na+ channel ENaC. Accordingly, SGK1 enhances ENaC abundance in the cell membrane. The significance of this effect is shown by an association of an E8CC/CT;I6CC polymorphism in the SGK1 gene with increased blood pressure. However, strong expression of SGK1 in enterocytes not expressing ENaC points to further functions of SGK1. This study was performed to test for regulation of Na+-coupled glucose transporter 1 (SGLT1) by Nedd4-2, SGK1, and/or the related kinases SGK3 and PKB. Additional studies searched for an association of the SGK1 gene with BMI. RESEARCH METHODS AND PROCEDURES: mRNA encoding SGLT1, wild-type Nedd4-2, inactive (C938S)Nedd4-2, wild type SGK1, constitutively active (S422D)SGK1 or inactive (K127N)SGK1, wild-type SGK3, and constitutively active (T308DS473D)PKB or inactive (T308AS473A)PKB were injected into Xenopus oocytes, and glucose transport was quantified from glucose-induced current (I(glc)). BMI was determined in individuals with or without the E8CC/CT;I6CC polymorphism. RESULTS: I(glc) was significantly decreased by coexpression of Nedd4-2 but not of (C938S)Nedd4-2. Coexpression of SGK1, (S422D)SGK1, SGK3, or (T308DS473D)PKB, but not of (K127N)SGK1 or (T308AS473A)PKB, enhanced I(glc) and reversed the effect of Nedd4-2. SGK1 and SGK3 phosphorylated Nedd4-2. Deletion of the SGK/PKB phosphorylation sites in Nedd4-2 blunted the kinase effects. BMI was significantly (p < 0.008) greater in individuals with the E8CC/CT;I6CC polymorphism than in individuals without. DISCUSSION: Overactivity of SGK1 may lead not only to excessive ENaC activity and hypertension but also to enhanced SGLT1 activity and obesity.

Adult↗
PubMed2004

Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV.

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.

Animals↗
PubMed2004

Role of the fission yeast SUMO E3 ligase Pli1p in centromere and telomere maintenance.

Sumoylation represents a conserved mechanism of post-translational protein modification. We report that Pli1p, the unique fission yeast member of the SP-RING family, is a SUMO E3 ligase in vivo and in vitro. pli1Delta cells display no obvious mitotic growth defects, but are sensitive to the microtubule-destabilizing drug TBZ and exhibit enhanced minichromosome loss. The weakened centromeric function of pli1Delta cells may be related to the defective heterochromatin structure at the central core, as shown by the reduced silencing of an ura4 variegation reporter gene inserted at cnt and imr. Interestingly, pli1Delta cells also exhibit enhanced loss of the ura4 reporter at these loci, likely by gene conversion using homologous sequences as information donors. Moreover, pli1Delta cells exhibit consistent telomere length increase, possibly achieved by a similar process. Point mutations within the RING finger of Pli1p totally or partially reproduce the pli1 deletion phenotypes, thus correlating with their sumoylation activity. Altogether, these results strongly suggest that Pli1p, and by extension sumoylation, is involved in mechanisms that regulate recombination in particular heterochromatic repeated sequences.

Amino Acid Sequence↗
PubMed2005

Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis.

Individual ubiquitin (Ub)-protein ligases (E3s) cooperate with specific Ub-conjugating enzymes (E2s) to modify cognate substrates with polyubiquitin chains. E3s belonging to the Really Interesting New Gene (RING) and Homologous to E6-Associated Protein (E6AP) C-Terminus (HECT) domain families utilize distinct molecular mechanisms. In particular, HECT E3s, but not RING E3s, form a thiol ester with Ub before transferring Ub to the substrate lysine. Here we report that different HECT domain E3s can employ distinct mechanisms of polyubiquitin chain synthesis. We show that E6AP builds up a K48-linked chain on its HECT cysteine residue, while KIAA10 builds up K48- and K29-linked chains as free entities. A small region near the N-terminus of the conserved HECT domain helps to bring about this functional distinction. Thus, a given HECT domain can specify both the linkage of a polyubiquitin chain and the mechanism of its assembly.

Binding Sites↗
PubMed2006

Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells.

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1(-/-) DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSB-binding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1(-/-) cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.

Animals↗
PubMed2003

Negative regulation of Rap1 activation by the Cbl E3 ubiquitin ligase.

Cbl functions as an adaptor protein by interacting with other signalling molecules to form multimolecular complexes. Previous studies have proposed that Cbl is also a positive regulator of CrkL-C3G signalling, which leads to Rap1 activation. However, there is a lack of genetic evidence for a physiological function of Cbl in regulating this pathway. Here, we show that Cbl deficiency results in enhanced activation of Rap1. Cbl was shown to promote the ubiquitylation of CrkL without any apparent effect on its stability. Remarkably, the membrane translocation of C3G, its association with CrkL, and the guanine-nucleotide exchange activity of C3G were all increased in Cbl(-/-) thymocytes. Consistent with a function of Rap1 in integrin activation, enhanced integrin-mediated cell adhesion was also seen in Cbl(-/-) thymocytes. Thus, Cbl negatively regulates Rap1 activation, probably through a proteolysis-independent E3-ubiquitin-ligase activity of Cbl that modulates protein-protein interactions.

Animals↗
PubMed2004

Negative regulation of SCFSkp2 ubiquitin ligase by TGF-beta signaling.

TGF-beta is a multifunctional growth factor whose best-known function is to inhibit cell growth and suppress tumor formation. TGF-beta causes cells to accumulate in mid-to-late G1 phase by blocking the transition from G1 to S. It has been shown that TGF-beta inhibits Cdk2-cyclin E kinase activity by promoting the binding of cell cycle inhibitor p27Kip1 to the kinase complexes. Here, we show that TGF-beta treatment leads to stabilization of p27Kip1 during G1 to S transition. We found that TGF-beta negatively regulates components of the SCF complex, which degrades the p27Kip1 during the G1 to S transition, through two distinct mechanisms. Using a pulse-chase analysis, we demonstrated that the stability of Skp2 decreases in the presence of TGF-beta. Destabilization of Skp2 by ubiquitin-mediated proteolysis was also demonstrated that in an in vitro degradation system, using cell extracts prepared from TGF-beta-treated cultured cells. In addition, TGF-beta treatment decreases the levels of Cks1 mRNA. The deficiency of Cks1 in TGF-beta-treated cells likely contributes to the stabilization of p27Kip1 and destabilization of Skp2, because in the absence of Cks1, SCFSkp2 cannot ubiquitinate p27Kip1; instead, self-ubiquitination of Skp2 occurs. Thus, stabilization of the cell cycle inhibitor p27Kip1 and cell growth inhibition in response to TGF-beta occur in part through limiting the threshold of the SCFSkp2 ubiquitin ligase by transcriptional and post-transcriptional mechanisms.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2006

Mouse major satellite DNA is prone to eccDNA formation via DNA Ligase IV-dependent pathway.

Elevated levels of extrachromosomal circular DNA (eccDNA or spcDNA) are closely associated with genomic instability and aging. Despite extensive studies, the mechanism of its generation in mammalian cells is unknown. We report here that mouse major satellite DNA (MSD) is prone to eccDNA formation and that the resulting molecules are multimeres of the basic repeat. Extrachromosomal circular major satellite (ECMS) DNA constitutes the majority of eccDNA in B16 mouse melanoma cells and is highly abundant in other mouse cells. Production of these molecules is enhanced in proliferating cells, suggesting that processes associated with DNA replication are involved in their appearance. Using siRNA technique we show that DNA Ligase IV is engaged in ECMS synthesis. Based on our findings we propose a novel two-step model for eccDNA formation in mammalian cells.

Animals↗
PubMed2005

The human checkpoint sensor and alternative DNA clamp Rad9-Rad1-Hus1 modulates the activity of DNA ligase I, a component of the long-patch base excision repair machinery.

The human checkpoint sensor and alternative clamp Rad9-Rad1-Hus1 can interact with and specifically stimulate DNA ligase I. The very recently described interactions of Rad9-Rad1-Hus1 with MutY DNA glycosylase, DNA polymerase beta and Flap endonuclease 1 now complete our view that the long-patch base excision machinery is an important target of the Rad9-Rad1-Hus1 complex, thus enhancing the quality control of DNA.

Cell Cycle Proteins↗
PubMed1990

Effects of DNA-binding drugs on T4 DNA ligase.

A number of DNA intercalating and externally binding drugs have been found to inhibit nick sealing, cohesive and blunt end ligation, AMP-dependent DNA topoisomerization and EDTA-induced DNA nicking mediated by bacteriophage T4 DNA ligase. The inhibition seems to arise from drug-substrate interaction so that formation of active DNA-Mg2(+)-AMP-enzyme complex is impaired while assembled and active complexes are not disturbed by drug binding to the substrate.

Adenosine Monophosphate↗
PubMed1995

Characterization of human DNA ligase I expressed in a baculovirus-insect cell system.

The baculovirus expression system was used to overexpress human DNA ligase I (hLig I). Approx. 2 mg of recombinant hLig I was produced per 10(8) Spodoptera frugiperda Sf9 insect cells infected with the recombinant baculovirus. The optimum time point for the production of biologically active recombinant hLig I was 48 h post-infection. Lig I activity was demonstrated by auto-adenylating, polynucleotide joining and DNA relaxation assays. The baculovirus system has the advantage over previously described methods for producing hLig I of generating large amounts of a full-length active protein.

Animals↗
PubMed1998

Human ubiquitin-protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin-conjugating enzymes.

BACKGROUND: Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. RESULTS: Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, approximately 120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed the both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. CONCLUSION: Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

Cell Differentiation↗
PubMed2005

Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1.

DNA nonhomologous end-joining in vivo requires the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4 (LX) complexes. Here, we have examined the impact of histone octamers and linker histone H1 on DNA end-joining in vitro. Packing of the DNA substrate into dinucleosomes does not significantly inhibit ligation by LX. However, LX ligation activity is substantially reduced by the incorporation of linker histones. This inhibition is independent of the presence of core histone octamers and cannot be restored by addition of Ku alone but can be partially rescued by DNA-PK. The kinase activity of DNA-PK is essential for the recovery of end-joining. DNA-PK efficiently phosphorylates histone H1. Phosphorylated histone H1 has a reduced affinity for DNA and a decreased capacity to inhibit end-joining. Our findings raise the possibility that DNA-PK may act as a linker histone kinase by phosphorylating linker histones in the vicinity of a DNA break and coupling localized histone H1 release from DNA ends, with the recruitment of LX to carry out double-stranded ligation. Thus, by using histone H1-bound DNA as a template, we have reconstituted the end-joining step of DNA nonhomologous end-joining in vitro with a requirement for DNA-PK.

Animals↗
PubMed2002

FRS2 alpha attenuates FGF receptor signaling by Grb2-mediated recruitment of the ubiquitin ligase Cbl.

Attenuation of growth factor signaling is essential for the regulation of developmental processes and tissue homeostasis in most organisms. The product of Cbl protooncogene is one such regulator, which functions as an ubiquitin ligase that ubiquitinates and promotes the degradation of a variety of cell signaling proteins. Here, we demonstrate that Grb2 bound to tyrosine-phosphorylated FRS2 alpha forms a ternary complex with Cbl by means of its Src homology 3 domains resulting in the ubiquitination of fibroblast growth factor (FGF) receptor and FRS2 alpha in response to FGF stimulation. These observations highlight the importance of FRS2 alpha in the assembly of both positive (i.e., Sos, phosphatidylinositol 3-kinase) and negative (i.e., Cbl) signaling proteins to mediate a balanced FGF signal transduction. However, the partial inhibition of FGF receptor down-regulation in FRS2 alpha-/- cells indicates that the attenuation of signaling by FGF receptor is regulated by redundant or multiple mechanisms.

3T3 Cells↗
PubMed2003

A binding motif for Siah ubiquitin ligase.

The Drosophila SINA (seven in absentia) protein and its mammalian orthologs (Siah, seven in absentia homolog) are RING domain proteins that function in E3 ubiquitin ligase complexes and facilitate ubiquitination and degradation of a wide range of cellular proteins, including beta-catenin. Despite these diverse targets, the means by which SINASiah recognize substrates or binding proteins has remained unknown. Here we identify a peptide motif (RPVAxVxPxxR) that mediates the interaction of Siah protein with a range of protein partners. Sequence alignment and mutagenesis scanning revealed residues that are important to this interaction. This consensus sequence correctly predicted a high-affinity interaction with a peptide from the cytoskeletal protein plectin-1 (residues 95-117). The unusually high-affinity binding obtained with a 23-residue peptide (K(Dapp) = 29 nM with SINA) suggests that it may serve as a useful dominant negative reagent for SINASiah proteins.

Amino Acid Motifs↗
PubMed2003

Promotion of G alpha i3 subunit down-regulation by GIPN, a putative E3 ubiquitin ligase that interacts with RGS-GAIP.

We have isolated an RGS-GAIP interacting protein that links RGS proteins to protein degradation. GIPN (GAIP interacting protein N terminus) is a 38-kDa protein with an N-terminal leucine-rich region, a central RING finger-like domain, and a putative C-terminal transmembrane domain. GIPN binds exclusively to RGS proteins of subfamily A, RGS-GAIP, RGSZ1, and RGSZ2. The N-terminal leucine-rich region of GIPN interacts with the cysteine-rich motif of RGS-GAIP. GIPN mRNA is ubiquitously expressed, and GIPN is found on the plasma membrane of transfected HEK293 cells. Endogenous GIPN is concentrated along the basolateral plasma membrane of proximal and distal tubules in rat kidney, where many G protein-coupled receptors and some G proteins are also located. Two immunoreactive species are found in rat kidney, a 38-kDa cytosolic form and an approximately 94-kDa membrane form. GIPN shows Zn2+- and E1/E2-dependent autoubiquitination in vitro, suggesting that it has E3 ubiquitin ligase activity. Overexpression of GIPN stimulates proteasome-dependent reduction of endogenous G alpha i3 in HEK293 cells and reduces the half-life of overexpressed G alpha i3-YFP. Thus, our findings suggest that GIPN is involved in the degradation of G alpha i3 subunits via the proteasome pathway. RGS-GAIP functions as a bifunctional adaptor that binds to G alpha subunits through its RGS domain and to GIPN through its cysteine string motif.

Amino Acid Motifs↗
PubMed2002

Ubiquitin ligase-associated protein SGT1 is required for host and nonhost disease resistance in plants.

Homologues of the yeast ubiquitin ligase-associated protein SGT1 are required for disease resistance in plants mediated by nucleotide-binding site/leucine-rich repeat (NBS-LRR) proteins. Here, by silencing SGT1 in Nicotiana benthamiana, we extend these findings and demonstrate that SGT1 has an unexpectedly general role in disease resistance. It is required for resistance responses mediated by NBS-LRR and other R proteins in which pathogen-derived elicitors are recognized either inside or outside the host plant cell. A requirement also exists for SGT1 in nonhost resistance in which all known members of a host species are resistant against every characterized isolate of a pathogen. Our findings show that silencing SGT1 affects diverse types of disease resistance in plants and support the idea that R protein-mediated and nonhost resistance may involve similar mechanisms.

Amino Acid Sequence↗
PubMed2002

A self-replicating ligase ribozyme.

A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 min(-1), whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(-11) M.min(-1). Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.

Base Sequence↗