Fbxw4 interacts with components of an E3 ubiquitin ligase complex and the COP9 signalosome.

<p>A. Table representing the number of unique peptides identified from one representative mass spectrometry experiment following FLAG immunoprecipitation from lysates of control cells expressing FLAG only “contr.”, or cells expressing FLAG-Fbxw4 or FLAG-Fbxo46. Column on left indicates the size of the interacting protein, in kilo-daltons (kDa). Gene names of proteins that contain the identified peptides are shown in the right column. Components of an E3 ubiquitin ligase complex are shaded in light gray; components of the COP9 signalosome are shaded dark gray. B. Validation of data mass spectrometry data by immunoprecipitation followed by western blot. 293 T cells were transfected with plasmids containing FLAG- Fbxw4, FLAG-Fbxo46 or an empty vector (v). 48 hours post-transfection cell lysates were prepared and immunoprecipitations were performed with mono-clonal anti-FLAG antibodies (M2) (to immunoprecipitate Fbxw4- or Fbxo46-interacting complexes). Western blots were performed to detect Fbxw4 or Fbxo46 (FLAG rb; polyclonal FLAG antibody; top panel), SKP1, COPS5, or COPS2. C. Expression of Fbxw4 alters the migration of endogenous SKP1 by gel filtration chromatography. 293 T cells were transfected with an empty vector (left panels) or a cplasmid containing FLAG-Fbxw4 (right panels). 48 hours post-transfection cell lysates were prepared and separated on a superpose6 gel filtration column. Western blots were performed on every other fraction to detect Fbxw4 (top panels) or SKP1 (bottom panels). In the absence of Fbxw4 SKP1 elutes with a peak at fraction 23, whereas when Fbxw4 is expressed there is co-elution of Fbxw4 with peaks at fraction 15 and in the void volume. Size standards that elute from given fractions are shown.</p

Fbxw4 interacts with E3 ubiquitin ligase components and with the COP9 signalosome in an F-box dependent manner.

<p>A. Schematic of the Fbxw4^−fbox protein. Numbers represent the amino acids of the protein. Domains contained within Fbxw4^−fbox are indicated. B. Table representing the number of unique peptides identified from one representative mass spectrometry experiment following FLAG immunoprecipitation from lysates of control cells expressing FLAG only “contr.”, or cells expressing FLAG- Fbxw4 or FLAG- Fbxw4^−fbox. Column on left indicates the size of the interacting protein, in kilo-daltons (kDa). Gene names of proteins that contain the identified peptides are shown in the right column. Components of an E3 ubiquitin ligase complex are shaded in light gray; components of the COP9 signalosome are shaded dark gray. C. Validation of data mass spectrometry data by immunoprecipitation followed by western blot. 293 T cells were transfected with plasmids containing FLAG-Fbxw4, FLAG-Fbxw4^−fbox, FLAG-Fbxo46 or an empty vector (v). 48 hours post-transfection cell lysates were prepared and immunoprecipitations were performed with mono-clonal anti-FLAG antibodies (M2) (to immunoprecipitate Fbxw4-, Fbxw4^−fbox-, or Fbxo46-interacting complexes). Western blots were performed to detect Fbxw4, Fbxw4^−fbox or Fbxo46 (FLAG rb; polyclonal FLAG antibody; top panel) or SKP1.</p

FBXW4 associates with ubiquitinated cellular proteins.

<p>A. Fbxw4 can be immunoprecipitated by ubiquitinated proteins and Fbxw4 can immunoprecipitate ubiquitinated proteins. 293 T cells were transfected with empty vector (v), HA-Ubiquitin, or HA-Ubiquitin and FLAG-Fbxw4. 36 hours post-transfection cells were treated with MG132 (+) or left untreated (−) for six hours. Cell lysates were prepared and immunoprecipitations were performed with either anti-HA antibodies (top two panels) or mono-clonal anti-FLAG antibodies (M2) (bottom two panels). Western blots were performed on both sets of immunoprecipitations with anti-FLAG and anti-HA antibodies. B. FBXW4 associates with cellular proteins that are endogenously ubiquitinated. 293 T cells were transfected with plasmids containing FLAG-Fbxw4, FLAG- Fbxw4^−fbox, FLAG-Fbxo46 or an empty vector (v). 36 hours post-transfection cells were treated with MG132 (+) or left untreated (−) for six hours. Cell lysates were prepared and immunoprecipitations were performed with mono-clonal anti-FLAG antibodies (M2) (to immunoprecipitate Fbxw4-, Fbxw4^−fbox-, or FBXO46-interacting complexes). Western blots were performed to detect Fbxw4, Fbxw4^−fbox or Fbxo46 (FLAG rb; polyclonal FLAG antibody; top panel) or Ubiquitin.</p

The Fbxw4 locus is a common site of proviral insertion and is highly expressed in the murine mammary gland during involution.

<p>A. Examination of the UCSC genome browser (genome.ucsc.edu) and the Retroviral Tagged Cancer Gene Database (<a href="http://variation.osu.edu/rtcgd/index.html" target="_blank">http://variation.osu.edu/rtcgd/index.html</a>) shows that multiple retroviral insertions have been cloned from within the transcribed Fbxw4 locus. Arrows indicate the directionality of the inserted provirus. The exons are indicated at the bottom and the position and scale of murine chromosome 19 is shown on top. Names given to the cloned proviral insertions are indicated on the left. Insertion sites beginning with ‘mmt’ were cloned from mouse mammary tumor virus induced mammary carcinomas. Insertion sites beginning with ‘Dkm’, ‘248’ and ‘B5’ were cloned from leukemias from murine leukemia virus accelerated hematopoietic cancers. B. FBXW4 is variably expressed in normal murine tissues. Oligos specific for murine Fbxw4 were used to perform quantitative rt-PCR on the ‘mouse normal cDNA TissueScan array’. Values were normalized to quantitative rt-PCR for GAPDH. C. Schematic of the Fbxw4 protein. Numbers represent the amino acids of the protein. Domains contained within Fbxw4 are indicated.</p

Delangin and MAU-2 Regulate Similar Processes in Xenopus tropicalis Embryonic Development

<p>Antisense morpholino oligonucleotides (MO) were used to target specific mRNAs to inhibit production of X. tropicalis delangin or MAU-2 (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>). Embryos injected with control MO exhibit normal development (A). Embryos targeted to knock down delangin or MAU-2 both exhibit a delay in development from gastrula stages relative to control MO-injected embryos, however they look normal at this stage. By late tailbud stage (stage 28), delangin morphants (B) are severely truncated along the A-P axis and ventralized, exhibiting retarded dorsal tissue development, particularly in the neural tube and somites. Head, eye, and tail development are also defective. MAU-2 morphants (C) exhibit a very similar but less severe phenotype than is evident in delangin morphants, including shortening of the A-P axis, ventralization and defects in neural, somite, head, eye, and tail development relative to the control MO-injected embryos. </p

Human MAU-2 and Delangin Are Nuclear Proteins

<div><p>(A) Confocal microscopy studies on HeLa cells. The nuclear localization of delangin is illustrated in the top panels using a FITC-labeled secondary antibody detecting monoclonal rat anti-human delangin (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>). Panel on the right shows merging of DNA image (left panel, red) and delangin staining (center panel, green). Expression of a GFP-human MAU-2 fusion protein revealed by confocal fluorescence microscopy of transiently transfected HeLa cells is shown in the center and bottom panels. The fusion protein appears present in both the nucleus and the cytoplasm, sometimes with a strong nuclear localization, as shown in the center panels, but in other cells cytoplasmic expression predominated. Bottom panels show retention of the GFP-human MAU-2 protein in nuclei isolated from transiently transfected HeLa cells and subsequently extracted in 0.5% Triton-X. Center and bottom panels show DNA staining with TOPRO3 (left), the GFP fluorescence signal (center), and merged images (right). </p> <p>(B) Nuclear location for epitopes specific to human MAU-2. Top panel: Antisera against human MAU-2 cross reacted with four major bands in whole cell extracts from HeLa cells that had been treated with the negative (-ve) control siRNA oligonucleotide. The two bands indicated by the arrows were severely reduced in intensity (by ̃90%) when the same antisera was used to blot whole cell extracts from HeLa cells that had been subjected to human MAU-2 siRNA, using the M1 siRNA oligonucleotide (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>). Beta actin is shown as loading control. Bottom panel: HeLa cells were separated into cytoplasmic (C) and nuclear (N) fractions (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>). When these fractions were immunoblotted with human MAU-2 antisera, the bands specific to human MAU-2 were almost exclusively detected in the nuclear fraction, while the background bands appeared to be cytoplasmic. </p></div

Co-Immunoprecipitation Studies Support Interaction between Delangin and Human MAU-2

<div><p>(A) Delangin co-immunoprecipitates with a GFP-human MAU-2 fusion protein. HeLa cells were transiently transfected with a GFP-human MAU-2 fusion protein construct (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>) or, as a negative control, with the GFP vector on its own. Cells were fractionated into nuclear (N) and cytoplasmic (C) fractions, and aliquots of the nuclear fractions were used for immunoprecipitation with an anti-GFP antibody to generate immunoprecipitation fractions. Individual fractions were size fractionated by SDS-PAGE and immunoblotted, using antibodies against delangin (top panel) or against GFP (bottom panel) as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>. The immunoprecipitation fraction from immunoprecipitating the GFP-human MAU-2 fusion protein reveals a specific band at ̃100 kDa (arrow) that is not found in the negative control sample. This is the size expected for the fusion protein (̃70 kDa for human MAU-2 and ̃ 30 kDa for GFP). The upper panel shows that the delangin antibodies identify a band of ̃ 300 kDa that is co-precipitated in the GFP-human MAU-2 immunoprecipitation sample but not in the GFP vector-only control sample. </p> <p>(B) Delangin siRNA. In order to validate the specificity of the monoclonal antibody to delangin, HeLa cells were transfected with siRNA oligonucleotides designed to knock down delangin using individual oligonucleotides D1–D3 (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>) or all three combined, D123. D2 produced the most effective delangin knockdown. The same band that was recognized by the antibody in the immunoprecipitation fraction (see A) was knocked down by ̃ 90% in cells transfected with the D2 siRNA oligonucleotide, when referenced against negative (−ve) and mock-transfected HeLa cell (MT) controls. </p></div

Human MAU-2 Regulates Sister Chromatid Cohesion and Is Required for Loading Cohesins onto Chromatin

<div><p>(A) Assay for PSCS. HeLa cells were transfected with siRNA oligonucleotides (M1 and M2) designed to knock down human MAU-2 (hMAU-2). The cells were synchronized at the G2/M stage by addition of nocodazole. After 3 h, the cells in the supernatant were collected and knockdown efficiency was assayed by immunoblotting using specific antibodies against human MAU-2. Top panel, both M1 and M2 effectively knocked down human MAU-2 (̃90% and 80% knockdown, respectively) when referenced against a negative (-ve) control oligonucleotide supplied by the manufacturer; beta actin is shown as a loading control. Metaphase spreads were prepared (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>) from these synchronized cells and assayed for PSCS separation. Middle panel, an example of PSCS in a metaphase of HeLa cells transfected with M1. Bottom panel, a metaphase from HeLa cells transfected with a negative (-ve) control oligonucleotide (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>). </p> <p>(B) By cohesin loading assay. HeLa cells transfected with a negative control oligonucleotide or subjected to human MAU-2 knockdown using the M1 oligonucleotide were synchronised in G2/M by nocodazole treatment and then released to progress into the cell cycle. Chromatin fractions prepared from aliquots collected at 0, 1.5, 2.5, and 3.5 h were subjected to Western blotting with anti-SMC3 and anti-SCC1 antibodies to monitor loading of cohesin on the chromatin. Histone H3 was used as a loading control (lower panel). The intensity of the bands was quantified as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>. </p> <p>(C) Left panels: Western blotting of whole Hela cell extracts that had been subjected to M1 siRNA knockdown or controls shows that the effects seen in (B) did not result from non-specific knockdown of cohesins (SMC3 and SCC1). Panels to the right: to assess the integrity of the cohesin complexes present in the supernatant, SMC3 was immunoprecipitated (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040242#s4" target="_blank">Materials and Methods</a>) and co-immunoprecipitation of SCC1 was tested. The ratio of SMC3:SCC1 present in the immunoprecipitated samples is identical in negative control cells and cells subjected to human MAU-2 knockdown indicating that the integrity of the cohesin complexes is not affected. In the negative control there is a marked difference in the amount of immunoprecipitated SMC3 between time 0 and 3 h later, as in these cells cohesin gets loaded on the chromatin and there is fewer SMC3 available for immunoprecipitation in the supernatant (see B). In contrast, in cells subjected to human MAU-2 this difference is notably smaller, as in these cells cohesin fails to load on the chromatin to a comparable extent (see B). </p> <p>(D) Cohesin-loading defects observed in cells subjected to human MAU-2 knockdown are not the result of cell-cycle arrest. HeLa cells were treated as in (B) and aliquots were collected at 0 and 3.5 h. The cell-cycle profile of the cell population in each sample was analyzed by flow cytometry.</p></div

MAU-2 and PQN-85 Regulate Chromosome Segregation in Early C. elegans Embryos

<div><p>(A) Individual RNAi knockdowns. Chromosome-segregation defects are not obvious in the progeny of <i>histone::GFP</i> hermaphrodites injected with double-stranded <i>mau-2</i> RNA. However, lagging anaphase chromosomes were evident in the case of <i>pqn-85</i>(RNAi) and <i>scc-3</i>(RNAi) embryos (white arrows). </p> <p>(B) Double RNAi knockdowns. Early <i>mau-2</i> + <i>pqn-85</i> (RNAi) embryos showed chromosome lagging (white arrow) where some ensuing cells appear to be unaffected and others have multiple and misshapen nuclei (right image). Early <i>mau-2</i> + <i>scc-3</i> (RNAi) and <i>pqn-85</i> + <i>scc-3</i> (RNAi) embryos consistently showed severe chromosome segregation defects where the DNA does not appear to move to either pole. This results in the phenotype in which all cells either have multiple nuclei or have none at all. Precise assignment of cell-cycle stages was not possible because of the severity of the chromosomal phenotype. </p></div

A 71-kDa Protein Co-Purifies with FLAG-Nipped-B by Anti-FLAG Affinity Chromatography

<p>Nuclear extracts of <i>y w;</i> P[ <i>Chip-FLAG-Nipped-B, w</i>+] (FLAG) embryos and <i>y w</i> control embryos were bound to anti-FLAG beads, washed, and eluted with FLAG peptide as described in the text. FLAG-Nipped-B fusion protein was detected in the <i>y w;</i> P[ <i>Chip-FLAG-Nipped-B, w</i>+] eluate, but not in the <i>y w</i> eluate, by anti-FLAG Western blot (top panel). Other eluted proteins were detected by silver stain (bottom panel). A 71-kDa protein specific to the FLAG-Nipped-B eluate was identified by mass spectrometry as the product of the <i>Drosophila CG4203</i> gene. It is closely related to the human MAU-2 protein. Higher molecular weight bands specific to the FLAG-Nipped-B extract contain multiple proteins whose identities could not be established unambiguously. In the bottom panel, the p71 protein and several other proteins, including the markers, became doublets when the gel was dried for photography. All appeared as single bands before drying. </p