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F-box Protein (f-box + protein)
Selected AbstractsMolecular interactions of fission yeast Skp1 and its role in the DNA damage checkpointGENES TO CELLS, Issue 5 2004Anna Lehmann Skp1 is a central component of the E3 ubiquitin ligase SCF (Skp1-Cullin-1- F -box). It forms an adapter bridge between Cullin-1 and the substrate-determining component, the F-box protein. In order to establish the role of Skp1, a temperature sensitive (ts) screen was carried out using mutagenic PCR (polymerase chain reaction) and 9 independent ts mutants were isolated. Mapping the mutated residues on the 3-D structure of human Skp1 suggested that the mutants would be compromised in binding to F-box proteins but not Cullin-1 (Pcu1). In order to assess the binding properties of ts Skp1, 12 F-box proteins and Pcu1 were epitope-tagged, and co-immunoprecipitation performed. This systematic analysis showed that ts Skp1 retains binding to Pcu1. However, binding to three specific F-box proteins, essential Pof1, Pof3 involved in maintaining genome integrity, and nonessential Pof10, was reduced. skp1ts cells exhibit a G2 cell cycle delay, which is attributable to activation of the DNA damage checkpoint. Intriguingly, contrary to pof3 mutants, in which this checkpoint is required for survival, checkpoint abrogation in skp1ts suppresses a G2 delay and furthermore almost rescues the ts phenotype. The activation mechanism of the DNA damage checkpoint therefore differs between pof3, and skp1ts, implicating a novel role for Skp1 in the checkpoint-signalling cascade. [source] Proteasome- and SCF-dependent degradation of yeast adenine deaminase upon transition from proliferation to quiescence requires a new F-box protein named Saf1pMOLECULAR MICROBIOLOGY, Issue 4 2006Stéphanie Escusa Summary In response to nutrient limitation, Saccharomyces cerevisiae cells enter into a non-proliferating state termed quiescence. This transition is associated with profound changes in gene expression patterns. The adenine deaminase encoding gene AAH1 is among the most precociously and tightly downregulated gene upon entry into quiescence. We show that AAH1 downregulation is not specifically due to glucose exhaustion but is a more general response to nutrient limitation. We also found that Aah1p level is tightly correlated to RAS activity indicating thus an important role for the protein kinase A pathway in this regulation process. We have isolated three deletion mutants, srb10, srb11 and saf1 (ybr280c) affecting AAH1 expression during post-diauxic growth and in early stationary phase. We show that the Srb10p cyclin-dependent kinase and its cyclin, Srb11p, regulate AAH1 expression at the transcriptional level. By contrast, Saf1p, a previously uncharacterized F-box protein, acts at a post-transcriptional level by promoting degradation of Aah1p. This post-transcriptional regulation is abolished by mutations affecting the proteasome or constant subunits of the SCF (Skp1,Cullin,F -box) complex. We propose that Saf1p targets Aah1p for proteasome-dependent degradation upon entry into quiescence. This work provides the first direct evidence for active degradation of proteins in quiescent yeast cells. [source] Remodeling of the SCF complex-mediated ubiquitination system by compositional alteration of incorporated F-box proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2010Mitsunori Kato Abstract Ubiquitination regulates not only the stability but the localization and activity of substrate proteins involved in a plethora of cellular processes. The Skp1,Cullin,F-box protein (SCF) complexes constitute a major family of ubiquitin protein ligases, in each member of which an F-box protein serves as the variable component responsible for substrate recognition, thereby defining the function of each complex. Here we studied whether the composition of F-box proteins in the SCF complexes is remodeled under different conditions. We exploited stable isotope labeling and MS for relative quantification of F-box proteins in the SCF complexes affinity-purified en masse from budding yeast cells at log and post-diauxic phases, and revealed an increment of Saf1, an F-box protein involved in entry into quiescence, during the diauxic shift. Similarly, we found that Met4 overexpression induces a specific increment of Met30, the F-box protein responsible for ubiquitination of Met4. These results illustrate a cellular response to environmental and genetic perturbations through remodeling of the SCF complex-mediated ubiquitination system. Compositional alteration of incorporated F-box proteins may redirect the activity of this system toward appropriate substrates to be ubiquitinated under individual conditions for the maintenance of cellular homeostasis. [source] S-phase kinase protein 2 is an attractive therapeutic target in a subset of diffuse large B-cell lymphoma,THE JOURNAL OF PATHOLOGY, Issue 4 2008S Uddin Abstract S-phase kinase protein 2 (SKP2), an F-box protein, targets cell-cycle regulators including cycle-dependent kinase inhibitor p27KiP1 via ubiquitin-mediated degradation. SKP2 is frequently overexpressed in a variety of cancer cells and has been implicated in oncogenesis; however, its role in diffuse large B-cell lymphoma (DLBCL) has not been elucidated. Therefore, we investigated the role of SKP2 and its ubiquitin-proteasome pathway in a large series (301) of DLBCL patient samples and a panel of DLBCL cell lines. Using immunohistochemistry, SKP2 was detected in 41.6% of DLBCL tumours and was inversely associated with p27Kip1 protein level. The DLBCL subset with high SKP2 and low p27Kip1 showed a strong correlation with the proliferating index marker Ki-67 (p < 0.0001) and also with the germinal centre phenotype (p = 0.0147). Treatment of DLBCL cell lines with bortezomib or expression of SKP2-specific siRNA causes down-regulation of SKP2 and accumulation of p27Kip1, leading to suppression of growth by inducing apoptosis. Furthermore, treatment of DLBCL cells with bortezomib causes apoptosis via involving the mitochondrial pathway and activation of caspases. Finally, treatment of DLBCL cells with bortezomib down-regulated the expression of XIAP, cIAP1, and survivin. Altogether, these results suggest that SKP2 and the ubiquitin-proteasome pathway may be a potential target for therapeutic intervention in DLBCL. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] An F-box gene, CPR30, functions as a negative regulator of the defense response in ArabidopsisTHE PLANT JOURNAL, Issue 5 2009Mingyue Gou Summary Arabidopsis gain-of-resistance mutants, which show HR-like lesion formation and SAR-like constitutive defense responses, were used well as tools to unravel the plant defense mechanisms. We have identified a novel mutant, designated constitutive expresser of PR genes 30 (cpr30), that exhibited dwarf morphology, constitutive resistance to the bacterial pathogen Pseudomonas syringae and the dramatic induction of defense-response gene expression. The cpr30 -conferred growth defect morphology and defense responses are dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), and NONRACE-SPECIFIC DISEASE RESISTANCE 1 (NDR1). Further studies demonstrated that salicylic acid (SA) could partially account for the cpr30 -conferred constitutive PR1 gene expression, but not for the growth defect, and that the cpr30 -conferred defense responses were NPR1 independent. We observed a widespread expression of CPR30 throughout the plant, and a localization of CPR30-GFP fusion protein in the cytoplasm and nucleus. As an F-box protein, CPR30 could interact with multiple Arabidopsis-SKP1-like (ASK) proteins in vivo. Co-localization of CPR30 and ASK1 or ASK2 was observed in Arabidopsis protoplasts. Based on these results, we conclude that CPR30, a novel negative regulator, regulates both SA-dependent and SA-independent defense signaling, most likely through the ubiquitin-proteasome pathway in Arabidopsis. [source] Auxin-induced, SCFTIR1 -mediated poly-ubiquitination marks AUX/IAA proteins for degradationTHE PLANT JOURNAL, Issue 1 2009Felipe Dos Santos Maraschin Summary The plant hormone auxin (indole-3-acetic acid or IAA) regulates plant development by inducing rapid cellular responses and changes in gene expression. Auxin promotes the degradation of Aux/IAA transcriptional repressors, thereby allowing auxin response factors (ARFs) to activate the transcription of auxin-responsive genes. Auxin enhances the binding of Aux/IAA proteins to the receptor TIR1, which is an F-box protein that is part of the E3 ubiquitin ligase complex SCFTIR1. Binding of Aux/IAA proteins leads to degradation via the 26S proteasome, but evidence for SCFTIR1 -mediated poly-ubiquitination of Aux/IAA proteins is lacking. Here we used an Arabidopsis cell suspension-based protoplast system to find evidence for SCFTIR1 -mediated ubiquitination of the Aux/IAA proteins SHY2/IAA3 and BDL/IAA12. Each of these proteins showed a distinct abundance and repressor activity when expressed in this cell system. Moreover, the amount of endogenous TIR1 protein appeared to be rate-limiting for a proper auxin response measured by the co-transfected DR5::GUS reporter construct. Co-transfection with 35S::TIR1 led to auxin-dependent degradation, and excess of 35S::TIR1 even led to degradation of Aux/IAAs in the absence of auxin treatment. Expression of the mutant tir1-1 protein or the related F-box protein COI1, which is involved in jasmonate signaling, had no effect on Aux/IAA degradation. Our results show that SHY2/IAA3 and BDL/IAA12 are poly-ubiquitinated and degraded in response to increased auxin or TIR1 levels. In conclusion, our data provide experimental support for the model that SCFTIR1 -dependent poly-ubiquitination of Aux/IAA proteins marks these proteins for degradation by the 26S proteasome, leading to activation of auxin-responsive gene expression. [source] AXL and AXR1 have redundant functions in RUB conjugation and growth and development in ArabidopsisTHE PLANT JOURNAL, Issue 1 2007Nihal Dharmasiri Summary Cullin-RING ubiquitin-protein ligases such as the Skp1, cullin, F-box protein (SCF) have been implicated in many growth and developmental processes in plants. Normal SCF function requires that the CUL1 subunit be post-translationally modified by related to ubiquitin (RUB), a protein related to ubiquitin. This process is mediated by two enzymes: the RUB-activating and RUB-conjugating enzymes. In Arabidopsis, the RUB-activating enzyme is a heterodimer consisting of AXR1 and ECR1. Mutations in the AXR1 gene result in a pleiotropic phenotype that includes resistance to the plant hormone auxin. Here we report that the AXL (AXR1-like) gene also functions in the RUB conjugation pathway. Overexpression of AXL in the axr1-3 background complements the axr1-3 phenotype. Biochemical analysis indicates that AXL overexpression restores CUL1 modification to the wild-type level, indicating that AXR1 and AXL have the same biochemical activity. Although the axl mutant resembles wild-type plants, the majority of axr1 axl-1 double mutants are embryo or seedling lethal. Furthermore, the axl-1 mutation reveals novel RUB-dependent processes in embryo development. We conclude that AXR1 and AXL function redundantly in the RUB conjugating pathway. [source] Proliferating Floral Organs (Pfo), a Lotus japonicus gene required for specifying floral meristem determinacy and organ identity, encodes an F-box proteinTHE PLANT JOURNAL, Issue 4 2003Shulu Zhang Summary To study flower development in the model legume Lotus japonicus, a population of transgenic plants containing a maize transposable element (Ac) in their genome was screened for floral mutants. One mutation named proliferating floral organs (pfo) causes plants to produce a large number of sepal-like organs instead of normal flowers. It segregates as a single recessive Mendelian locus, and causes sterility. Scanning electron microscopy revealed that pfo affects the identity, number and arrangement of floral organs. Sepal-like organs form in the first whorl, and secondary floral meristems are produced in the next whorl. These in turn produce sepal-like organs in the first whorl and floral meristems in the second whorl, and the process is reiterated. Petals and stamens are absent while carpels are either absent or reduced. The pfo phenotype was correlated with the presence of an Ac insertion yielding a 1.6-kb HindIII restriction fragment on Southern blots. Both the mutant phenotype and this Ac element are unstable. Using the transposon as a tag, the Pfo gene was isolated. Conceptual translation of Pfo predicts a protein containing an F-box, with high overall similarity to the Antirrhinum FIMBRIATA, Arabidopsis UNUSUAL FLORAL ORGANS and Pisum sativum Stamina pistilloida proteins. This suggests that Pfo may regulate floral organ identity and meristem determinacy by targeting proteins for ubiquitination. [source] Crystallization and preliminary X-ray characterization of the Skp1,Fbg3 complexACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Taichi Kumanomidou F-box proteins are the substrate-recognition components of Skp1,Cullin1,F-box protein,Rbx1 (SCF) ubiquitin ligase complexes. Fbs1, an F-box protein, binds specifically to proteins modified with high-mannose oligosaccharides. Fbg3, another F-box protein, has 51% sequence identity to Fbs1. Although the residues that are necessary for binding to oligosaccharides are conserved between Fbs1 and Fbg3, Fbg3 does not bind glycoproteins. Skp1 and Fbg3 were co-expressed in Escherichia coli and their complex was purified to homogeneity and crystallized. Microseeding combined with the sandwiched hanging-drop technique improved the quality of the resulting crystals. The plate-shaped crystals belonged to space group P212121, with unit-cell parameters a = 34.1, b = 76.6, c = 193.9,Å and one molecule per asymmetric unit. [source] Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpointGENES TO CELLS, Issue 5 2004Anna Lehmann Skp1 is a central component of the E3 ubiquitin ligase SCF (Skp1-Cullin-1- F -box). It forms an adapter bridge between Cullin-1 and the substrate-determining component, the F-box protein. In order to establish the role of Skp1, a temperature sensitive (ts) screen was carried out using mutagenic PCR (polymerase chain reaction) and 9 independent ts mutants were isolated. Mapping the mutated residues on the 3-D structure of human Skp1 suggested that the mutants would be compromised in binding to F-box proteins but not Cullin-1 (Pcu1). In order to assess the binding properties of ts Skp1, 12 F-box proteins and Pcu1 were epitope-tagged, and co-immunoprecipitation performed. This systematic analysis showed that ts Skp1 retains binding to Pcu1. However, binding to three specific F-box proteins, essential Pof1, Pof3 involved in maintaining genome integrity, and nonessential Pof10, was reduced. skp1ts cells exhibit a G2 cell cycle delay, which is attributable to activation of the DNA damage checkpoint. Intriguingly, contrary to pof3 mutants, in which this checkpoint is required for survival, checkpoint abrogation in skp1ts suppresses a G2 delay and furthermore almost rescues the ts phenotype. The activation mechanism of the DNA damage checkpoint therefore differs between pof3, and skp1ts, implicating a novel role for Skp1 in the checkpoint-signalling cascade. [source] Remodeling of the SCF complex-mediated ubiquitination system by compositional alteration of incorporated F-box proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2010Mitsunori Kato Abstract Ubiquitination regulates not only the stability but the localization and activity of substrate proteins involved in a plethora of cellular processes. The Skp1,Cullin,F-box protein (SCF) complexes constitute a major family of ubiquitin protein ligases, in each member of which an F-box protein serves as the variable component responsible for substrate recognition, thereby defining the function of each complex. Here we studied whether the composition of F-box proteins in the SCF complexes is remodeled under different conditions. We exploited stable isotope labeling and MS for relative quantification of F-box proteins in the SCF complexes affinity-purified en masse from budding yeast cells at log and post-diauxic phases, and revealed an increment of Saf1, an F-box protein involved in entry into quiescence, during the diauxic shift. Similarly, we found that Met4 overexpression induces a specific increment of Met30, the F-box protein responsible for ubiquitination of Met4. These results illustrate a cellular response to environmental and genetic perturbations through remodeling of the SCF complex-mediated ubiquitination system. Compositional alteration of incorporated F-box proteins may redirect the activity of this system toward appropriate substrates to be ubiquitinated under individual conditions for the maintenance of cellular homeostasis. [source] Crystallization and preliminary X-ray characterization of the Skp1,Fbg3 complexACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Taichi Kumanomidou F-box proteins are the substrate-recognition components of Skp1,Cullin1,F-box protein,Rbx1 (SCF) ubiquitin ligase complexes. Fbs1, an F-box protein, binds specifically to proteins modified with high-mannose oligosaccharides. Fbg3, another F-box protein, has 51% sequence identity to Fbs1. Although the residues that are necessary for binding to oligosaccharides are conserved between Fbs1 and Fbg3, Fbg3 does not bind glycoproteins. Skp1 and Fbg3 were co-expressed in Escherichia coli and their complex was purified to homogeneity and crystallized. Microseeding combined with the sandwiched hanging-drop technique improved the quality of the resulting crystals. The plate-shaped crystals belonged to space group P212121, with unit-cell parameters a = 34.1, b = 76.6, c = 193.9,Å and one molecule per asymmetric unit. 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