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Molecular and Cellular Biology, March 2008, p. 1606-1615, Vol. 28, No. 5
0270-7306/08/$08.00+0     doi:10.1128/MCB.01567-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Deubiquitylating Enzyme UBP64 Controls Cell Fate through Stabilization of the Transcriptional Repressor Tramtrack{triangledown}

Prashanth Kumar Bajpe,1 Jan A. van der Knaap,1 Jeroen A. A. Demmers,2 Karel Bezstarosti,2 Andrew Bassett,4 Heleen M. M. van Beusekom,3 Andrew A. Travers,4 and C. Peter Verrijzer1*

Department of Biochemistry, Centre for Biomedical Genetics,1 Proteomics Center,2 Department of Cardiology, Erasmus University Medical Center, P.O. Box 1738, 2040 CA Rotterdam, The Netherlands,3 MRC Laboratory of Molecular Biology, Cambridge, United Kingdom4

Received 27 August 2007/ Returned for modification 30 September 2007/ Accepted 15 December 2007


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ABSTRACT
 
Protein ubiquitylation plays a central role in multiple signal transduction pathways. However, the substrate specificity and potential developmental roles of deubiquitylating enzymes remain poorly understood. Here, we show that the Drosophila ubiquitin protease UBP64 controls cell fate in the developing eye. UBP64 represses neuronal cell fate but promotes the formation of nonneuronal cone cells. Using a proteomics approach, we identified the transcriptional repressor Tramtrack (TTK) as a primary UBP64 substrate. In common with TTK, reduced UBP64 levels lead to a loss of cone cells, supernumerary photoreceptors, and mechanosensory bristle cells. Previously, it was demonstrated that the blockade of neuronal cell fate was relieved by SINA-dependent ubiquitylation and degradation of TTK. We found that UBP64 counteracts SINA function by deubiquitylating TTK, leading to its stabilization and thereby promoting a nonneuronal cell fate. Mass spectrometric mapping revealed that SINA ubiquitylates multiple sites dispersed throughout TTK, which are duly deubiquitylated by UBP64. This observation suggests that both E3 SINA and UBP64 use a scanning mechanism to (de)ubiquitylate TTK. We conclude that the balance of TTK ubiquitylation by SINA and deubiquitylation by UBP64 constitutes a specific posttranslational switch controlling cell fate.


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INTRODUCTION
 
Protein ubiquitylation is involved in virtually all aspects of eukaryotic cell biology, regulating processes including cell cycle progression, organelle biogenesis, apoptosis, protein transport, transcription, and DNA repair (10, 14, 18, 20, 21, 30, 36). Ubiquitin is a 76-amino-acid polypeptide that can be linked covalently to a target protein via an isopeptide bond of its C-terminal glycine to an internal lysine (K) of the substrate. Monoubiquitylation typically functions as a signaling modification in a manner analogous to that of phosphorylation. Ubiquitin molecules can also form polyubiquitin chains in which the use of different internal lysine residues determines the functional outcome. K48-linked polyubiquitylation chains constitute a tag for proteasomal degradation, whereas K63-linked polyubiquitin chains play a role in endocytosis and lysosomal sorting (19, 40). The enzymes that catalyze protein ubiquitylation have been extensively studied (18, 30, 31). However, the potential regulatory roles and substrate selectivities of deubiquitylating enzymes (DUBs) that remove mono- or polyubiquitin chains from proteins remain less well understood.

The ubiquitin-specific processing proteases (USPs) or ubiquitin-processing proteases (UBPs) form the largest subclass of DUBs (27). The enzymatic domains of USPs/UBPs comprise two well-conserved cysteine and histidine boxes, harboring the key catalytic residues, separated by large, variable protein sequences (43). Outside their catalytic domains, USPs/UBPs are highly variable, suggestive of a wide variety of cellular functions. Originally, DUBs were thought to function mainly nonspecifically during ubiquitin recycling. However, recent research uncovered an increasing number of DUBs that target specific substrates (27). For example, during Drosophila eye development, fat facets deubiquitylate and stabilize liquid facets (LQF). LQF, in turn, mediate endocytosis of the Delta receptor, which is critical for cell patterning (9, 29).

Regulated protein ubiquitylation and destruction can control critical developmental switches, as exemplified by studies on Drosophila eye development. The adult fly eye consists of ~800 repeating units or ommatidia, each comprising eight photoreceptor cells (R1 to R8), four lens secreting nonneuronal cone cells, six pigment cells, and one mechanosensory bristle cell at alternate ommatidial vertices (45). The developmental program underlying the formation of the Drosophila compound eye is orchestrated by intercellular signaling. A wave of pattern formation and cell type determination, referred to as "morphogenetic furrow," sweeps across the presumptive eye epithelium. This process results in evenly spaced cell clusters that will eventually form the adult ommatidia. Importantly, dynamic cell-cell interactions, rather than lineage, determine cell fate specification and differentiation (22, 45). For example, differentiation of the R7 photoreceptor requires signaling through epidermal growth factor receptor and Sevenless (SEV) receptor tyrosine kinases, activating the Ras-mitogen-activated protein (MAP) kinase pathway in precursor cells (13). The transcription factors YAN, Pointed (PNT), and TTK are the downstream effectors of this signaling pathway.

YAN and TTK each form a distinct blockade to cell differentiation. In undifferentiated precursor cells, the transcriptional repressor YAN forms a general barrier to differentiation. However, the repressive activity of YAN can be antagonized by activated PNT, which stimulates transcription. Following Ras-MAP kinase signaling, both YAN and PNT are phosphorylated but with opposite effects. Phosphorylation relieves YAN's transcriptional repression, allowing phosphorylated PNT to activate the same set of genes, thereby promoting cell differentiation (7, 28). Once YAN's block of cell differentiation is removed, the transcriptional repressor TTK forms a second, more selective differentiation barrier.

TTK blocks neuronal cell identity but promotes specific nonneuronal fates. TTK is a POZ domain zinc finger transcriptional repressor (6, 15), which, due to differential splicing, is expressed as two distinct polypeptides: TTK69 and TTK88 (33). TTK69 recruits the NuRD corepressor complex to silence target genes (26). TTK88 acts as a silencer by recruiting another transcriptional corepressor, CoREST, to genes critical for neuronal cell fate (12). TTK expression levels are regulated largely posttranslationally. During eye development, polyubiquitylation and the destruction of TTK allow the differentiation of precursor cells into R7 photoreceptors. TTK polyubiquitylation is mediated by the Ring domain protein SINA, an E3 ubiquitin ligase that associates with Phyllopod (PHYL) (24, 38). Upon Ras activation, PHYL expression is induced, promoting TTK ubiquitylation and proteasome-mediated degradation, thus removing the second blockade to R7 photoreceptor differentiation. Whereas TTK needs to be degraded to allow R7 neuronal cell differentiation, the formation of nonneuronal cone and pigment cells depends on its presence (24, 38). Finally, PNT and TTK function antagonistically in establishing an epidermal growth factor receptor-dependent transcriptional switch to regulate mitosis in developing eye discs (2).

UBP64 is a UBP/USP that was identified as being a dominant enhancer of position effect variegation (16). We noted that new hypomorphic mutants that we generated displayed a defective eye phenotype. We therefore set out to determine the potential regulatory role and mechanism of action of UBP64 during eye development. Through an unbiased proteomic approach, we found that UBP64 specifically targets TTK. Genetic analysis established that UBP64 is a positive regulator of TTK and that these factors similarly affect cell fate decisions during development. UBP64 inhibits neuronal differentiation but promotes nonneuronal cone cell differentiation. Next, we employed biochemical experiments to investigate UBP64's mechanism of action. Our results showed that UBP64 deubiquitylates and stabilizes TTK. We conclude that UBP64 antagonizes the E3 ubiquitin ligase SINA, constituting a posttranslational developmental switch controlling cell fate.


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MATERIALS AND METHODS
 
DNA constructs, fly stocks, and genetics. Details of the cloning procedures are available upon request. The cDNA clone LD26783 (Drosophila Genome Research Centre) contains the full-length dUbp64 cDNA within the pOT2 plasmid. The 4.6-kb cDNA was amplified in two fragments and cloned into vector pGEX 2TKN, generating pGEX2TKN-Ubp64, which was verified by sequencing of the complete insert. The substitution mutation to create UBP64C405A was generated using DpnI-based site-directed mutagenesis and verified by sequencing. The upstream activation sequence (UAS) and mammalian expression constructs were generated by subcloning the wild-type (WT)- and UBP64C405A-encoding cDNAs into pUAST (4) and pcDNA 3.1(+) (Invitrogen). pRMHA3-Ttk69 and Sina (38) S2 cell expression plasmids were obtained from Travers laboratory stocks. Both cDNAs were subcloned into pcDNA 3.1, creating pcDNA-Ttk69 and pcDNA-Sina. The His6-ubiquitin expression plasmid was a gift from S. Mittnacht. All fly stocks were maintained under standard conditions, and crosses were performed using standard procedures. Additional details will be provided upon request. P(EP)ubp64 was purchased from GenExel (strain 28938). Sequence analysis revealed that the enhancer promoter (EP) element is inserted into the ubp64 5' untranslated region (5'-AATTTCTAGCGCAGTCAGGAC[P element]GCAGGAGCAGCCGAGCAGGGCC-3'). ubp64{Delta}1, ubp64{Delta}2, and revertant strains were generated by imprecise and precise P-element excisions of P(EP)ubp64. In the ubp64{Delta}1 and ubp64{Delta}2 lines, about 2.8 kb of the EP element was deleted from its 5' end, likely removing a promoter driving Ubp64 transcription. This resulted in stronger hypomorphic alleles, as revealed by Western immunoblotting. Currently, no full Ubp64 null mutant is available. The UAS-Ttk69 line was described previously (1). The GMR>Ttk69 strain was generated by recombining UAS-Ttk69 with GMR-Gal4 (both P elements were inserted into the second chromosome). To generate transgenic UAS-Ubp64 and UAS-Ubp64C405A, P-element-mediated germ line transformation was carried according to standard procedures (4).

SEM. Specimens for scanning electron microscopy (SEM) were prepared as described previously (3). Briefly, adult flies were fixed for a few hours in a fixative containing 4% formaldehyde and 1% glutaraldehyde in sodium phosphate buffer (pH 7.2). Subsequently, the flies were dehydrated in graded ethanol and incubated in hexamethyldisilazane. After drying, the specimens were mounted onto aluminum stubs with adhesive carbon tabs and sputter coated for 1 min using an Agar automatic sputter coater (Agar Scientific Ltd., Essex, United Kingdom). The specimen was then ready to view on a JEOL JSM5200 scanning electron microscope (Jeol-Europe, Nieuw Vennep, The Netherlands).

Antibodies, immunoprecipitations, and immunostaining. All immunological procedures were performed by using standard procedures. Antisera directed against UBP64 were generated by the immunization of guinea pigs with a purified glutathione S-transferase (GST)-tagged polypeptide spanning amino acids 309 to 743. Rabbit TTK69 and rat TTK88 antibodies were generated in the Travers laboratory (26). Anti-Cut antibody (2B10) and Elav (7E8A10) were procured from the Developmental Studies Hybridoma bank. Anti-UBP64 antibodies were affinity purified using GST-UBP64 (amino acids 309 to 743) immobilized on an Affi-gel15 (Bio-Rad) matrix as described previously (8). UBP64 was purified using affinity-purified UBP64 antibodies coupled to protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) essentially as described previously (8). Briefly, 80 µl of beads coated with anti-UBP64 antibodies was incubated with 1 ml of embryo nuclear extract (10-mg/ml protein concentration) in 50 mM HEPES-KOH-0.2 MEDTA-25 mM MgCl2-20% glycerol (HEMG) buffer containing 100 mM KCl (HEMG/100), 0.1% NP-40, 0.1 mg/ml insulin, and a cocktail of protease inhibitors (8). After 2 h at 4°C, the beads were washed extensively with HEMG/600-0.1% NP-40. Following two washes with HEMG/200, bound proteins were eluted with 100 mM glycine (pH 2.5), resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and visualized by Coomassie staining. Approximately 0.03% of the input was loaded onto the input lane. For coimmunoprecipitations, 25 µl of nuclear extract (input) diluted in HEMG/100 was incubated with 5 µl protein A-Sepharose beads coated with anti-UBP64 antibodies. After extensive washes with HEMG/300-0.1% NP-40, bound proteins were resolved by SDS-PAGE and analyzed by Western immunoblotting with antibodies directed against either UBP64, TTK88, or TTK69. For mock immunoprecipitations, 5 µl protein A beads was directly added to 25 µl nuclear extract. Ten percent of the input was used for loading in the input lane. For immunostaining of pupal retinae, recently formed pupae were collected and allowed to age for an additional 40 h. Retinal discs were dissected and fixed for a few hours using Brower's fixative (5) prior to immunostaining with the appropriate antibodies, as described previously (44). For Western blotting, 10 retinae were isolated and directly lysed in SDS sample buffer, resolved by SDS-PAGE, and visualized by immunoblotting. Typically, the equivalent of approximately one disc was loaded per lane.

Mass spectrometric analysis. One-dimensional SDS-PAGE gel lanes were cut into 2-mm slices using an automatic gel slicer and subjected to in-gel reduction with dithiothreitol, alkylation with iodoacetamide, and digestion with trypsin (sequencing grade; Promega), essentially as described previously (42). Nanoflow liquid chromatography-tandem mass spectrometry was performed on an 1100 series capillary liquid chromatography system (Agilent Technologies) coupled to an LTQ-Orbitrap mass spectrometer (Thermo) operating in positive mode and equipped with a nanospray source. Peptide mixtures were trapped on a ReproSil C18 reversed-phase column (column dimensions, 1.5 cm by 100 µm, packed in-house; Dr Maisch GmbH) at a flow rate of 8 µl/min. Peptide separation was performed on a ReproSil C18 reversed-phase column (column dimensions, 15 cm by 50 µm, packed in-house; Dr Maisch GmbH) using a linear gradient from 0 to 80% B buffer (A buffer is 0.1 M acetic acid, and B buffer is 80% [vol/vol] acetonitrile-0.1 M acetic acid) for 70 min and at a constant flow rate of 200 nl/min using a splitter. The column eluent was directly sprayed into the electrospray ionization source of the mass spectrometer. Mass spectra were acquired in continuum mode; fragmentation of the peptides was performed in data-dependent mode. Peak lists were automatically created from raw data files using Mascot Distiller software (version 2.0; MatrixScience). The Mascot search algorithm (version 2.0; MatrixScience) was used for searching against FlyBase (version FB2006_01, released 8 December 2006). Peptide tolerance was typically set to 10 ppm, and the fragment ion tolerance was set to 0.8 Da. A maximum number of two missed cleavages by trypsin were allowed, and carbamido-methylated cysteine and oxidized methionine were set as fixed and variable modifications, respectively. The Mascot score cutoff value for a positive protein hit was set to 60. Individual peptide tandem mass spectra with Mowse scores below 40 were checked manually and either interpreted as being valid identifications or discarded. Typical contaminants, also present in immunopurifications using beads coated with preimmune serum or antibodies directed against irrelevant proteins, were omitted from the table. These common contaminants included actins (Act5C, Act79B, and Act88F), tubulins (alphaTub67C and betaTub56), translation factors (Ef1beta, Ef1gamma, eIF2B-gamma, eIF-3p40, eIF3-S8, eIF3-S9, and eIF-4E), fibrillarin, histones, heat shock proteins (Hsc70-3, Hsc70-4, Hsc70-5, Hsp26, and Hsp27), pontin, reptin, ribosomal proteins (RpL11, RpL12, RpL23, RpL40, RpLP0, RpS14b, RpS16, RpS27A, RpS3, RpS7, and RpS8), and yolk proteins (Yp1, Yp2, and Yp3).

Deubiquitylation assays. H1299 cells were cultured in RPMI medium with 10% fetal bovine serum. 293T cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. For TTK stability assays, the appropriate expression vectors were cotransfected into H1299 cells using Fugene 6 transfection reagent (Roche Molecular Diagnostics) according to the manufacturer's protocol. In vitro deubiquitylation assays were performed essentially as described previously (39). To purify high amounts of Ub-TTK69, 10 µg of pcDNA-Ttk69, 10 µg His6-ubiquitin, and 0.125 µg of pcDNA-Sina expression plasmids were cotransfected into 293T cells in a 10-cm dish using polyethyleneimine (Sigma). To enhance the ubiquitylation levels of TTK69, 50 µM of MG-132 (Calbiochem) was added 4 h before the cells were harvested. Ub-TTK69 was purified under denaturing conditions by Ni2+-nitrilotriacetic acid (NTA) agarose chromatography (Qiagen) according to instructions provided by the manufacturer. Recombinant GST-tagged UBP64 and UBP64C405A were purified from Escherichia coli under native conditions as described previously (8). Deubiquitylation reaction mixtures were assembled on ice in reaction buffer containing 60 mM HEPES (pH 7.6), 5 mM MgCl2, and 4% glycerol. Reaction mixtures were incubated for 1 h at 30°C, after which they were stopped by adding SDS-PAGE loading buffer and analyzed by SDS-PAGE followed by immunoblotting with anti-hemagglutinin (HA) antibody.


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RESULTS
 
UBP64 is required for normal eye development. To analyze UBP64 function, we set out to generate additional mutant lines through the mobilization of a P element inserted into the 5' untranslated region of the UBP64 gene. This resulted in the isolation of two novel hypomorphs, named ubp64{Delta}1 and ubp64{Delta}2. In the ubp64{Delta}1 and ubp64{Delta}2 lines, about 2.8 kb of the EP element was deleted from its 5' end, likely removing a promoter driving Ubp64 transcription. Both ubp64{Delta}1 and ubp64{Delta}2 are stronger mutants than the parental line but are still homozygous viable. All experiments discussed below were performed with both alleles, but because they yielded similar results, only those obtained with ubp64{Delta}1 are shown. Whole-mount immunostaining revealed a strong reduction of UBP64 levels in ubp64{Delta}1 embryos but not in a revertant line (Rev) generated by precise excision of the P element (Fig. 1A to C). These experiments also showed that at this developmental stage, UBP64 is widely expressed and present in both the cytoplasm and nucleus. In spite of the reduced UBP64 levels in ubp64{Delta}1 animals, we did not detect any obvious developmental defects at the embryonic stages. However, inspection of adult ubp64{Delta}1 flies revealed a "rough eye" phenotype, characterized by distorted eye morphology, an irregular facet arrangement, and disorganized bristles, visualized by SEM (Fig. 1D to F). Interestingly, we noted that ubp64{Delta}1 flies have an additional mechanosensory bristle at multiple ommatidial vertices (Fig. 1E, bottom). Western immunoblotting confirmed the reduced UBP64 levels in extracts prepared from dissected eye discs from ubp64{Delta}1 flies (Fig. 1G, top). Antibodies directed against histone H3 were used as a loading control (Fig. 1G, bottom). Next, we employed immunostaining of eye discs using anti-UBP64 antibodies to determine the expression pattern during eye development. These experiments revealed that UBP64 is expressed ubiquitously in the developing eye and not in a cell type-selective manner (Fig. 1H to J). UBP64 levels are clearly reduced in ubp64{Delta}1 but not in revertant eye discs. We conclude that UBP64 activity is required for normal eye development.


Figure 1
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  		FIG. 1. ubp64 mutants have defective eye development. (A to C) UBP64 expression was monitored by whole-mount immunostaining of WT (A), ubp64{Delta}1 (ubp64{Delta}1/ubp64{Delta}1) (B), and revertant (Rev) (C) embryos using affinity-purified antibodies directed against UBP64. UBP64 is widely expressed and present in the cytoplasm and nucleus. UBP64 levels are severely reduced in ubp64{Delta}1 embryos compared to WT and Rev embryos. (D to F) Scanning electron micrographs of adult eyes of WT (D), ubp64{Delta}1 (E), and Rev (F) flies. An enlarged zoom image is shown at the bottom of each panel. In contrast to WT or Rev eyes, ubp64{Delta}1 eyes have disorganized facets and bristles, which gives the external surface a rough appearance. In addition, there are supernumerary bristles, three examples of which are indicated with arrows. (G) Western immunoblotting analysis of UBP64 protein levels in extracts prepared from isolated pupal retinae and resolved by SDS-PAGE. Three and nine microliters of extract prepared from 10 retinae dissolved in 50 µl sample buffer was loaded (indicated with a triangle). Detection of histone H3 served as a loading control. (H to J) UBP64 expression was monitored in the pupal retinae of WT (H), ubp64{Delta}1 (I), or Rev (J) animals. UBP64 is ubiquitously expressed in both the cytoplasm and nucleus of retina cells, but its expression level is severely diminished in ubp64{Delta}1 animals. (K and L) Scanning electron micrographs of adult eyes of GMR-Gal4/+ UAS-Ubp64/+ flies (abbreviated as GMR>Ubp64) (K) and GMR-Gal4/+ UAS-Ubp64C405A/+ (GMR>Ubp64C405A) (L) flies. The ectopic overexpression of UBP64 in precursor cells posterior to the morphogenetic furrow, directed by the GMR enhancer, strongly disrupts eye development and loss of bristles. In contrast, the overexpression of the catalytically dead UBP64C405A did not affect eye development. (M) Immunoblot analysis of extracts prepared from dissected eye discs confirmed the comparable levels of WT and mutant UBP64. Histone H3 serves as a loading control.

To investigate the spatiotemporal control of UBP64, we employed the GAL4/UAS system (4) to examine the effects of UBP64 misexpression during eye development. We used the glass multimer reporter (GMR) enhancer to drive GAL4 expression. Consequently, in animals harboring both GMR-Gal4 and UAS-Ubp64 (GMR>Ubp64), UBP64 is overexpressed in precursor cells posterior to the morphogenetic furrow. UBP64 overexpression also caused a rough eye phenotype (Fig. 1K). However, in addition to a distorted eye morphology and irregular arrangement of ommatidia, a clear reduction in the number of bristles was observed (Fig. 1K, bottom). Thus, whereas reduced UBP64 levels causes extra bristles, ectopic UBP64 expression leads to a loss of bristles. These observations support the notion that UBP64 inhibits the differentiation of a precursor cell into a bristle. The overexpression of a catalytically dead mutant in which a key cysteine in the UBP64 active site is replaced by an alanine (UBP64C405A) did not affect eye development (Fig. 1L). Thus, the effects of ectopic UBP64 expression critically depend on its enzymatic activity. Western immunoblot analysis of extracts prepared from dissected eye discs confirmed the approximately comparable levels of WT and mutant UBP64 (Fig. 1M). Taken together, these results established that UBP64 is required for normal eye development.

UBP64 targets TTK. To identify potential in vivo molecular targets of UBP64, we undertook an unbiased proteomic approach. Using affinity-purified, highly specific antibodies, we immunopurified UBP64 from Drosophila embryo nuclear extracts (Fig. 2A). Following extensive washes with a buffer containing 600 mM KCl and 0.1% NP-40, we resolved UBP64 and associated proteins by SDS-PAGE followed by Coomassie staining (Fig. 2A). Mass spectrometric analysis confirmed the presence of UBP64 and a range of associated proteins (Table 1). The most abundant UBP64-associated proteins were TTK88 and TTK69. The notion that UBP64 targets TTK was further strengthened by the copurification of the TTK69-associated NuRD corepressor complex. In addition to TTK and the NuRD complex, a number of other factors were identified. These included several transcription and chromatin-regulatory factors, the E3 ubiquitin ligase BRE1, and the F-box protein Slimb. None of these factors was identified in control immunopurifications using either mock beads or unrelated antibodies.


Figure 2
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  		FIG. 2. UBP64 stabilizes transcription factor TTK. (A) UBP64 and associated proteins immunopurified from 1 ml of Drosophila embryo nuclear extract (concentration of 10 mg/ml) using affinity-purified antibodies coupled to protein A-Sepharose CL-4B beads. Following extensive washes with HEMG buffer containing 600 mM KCl and 0.1% NP-40, bound proteins were eluted with 100 mM glycine (pH 2.5), resolved by SDS-PAGE, and visualized by Coomassie staining. Approximately 0.03% of the input material was loaded. Mass spectrometric analysis was used to identify UBP64 and associated proteins not present in the mock purification (see Table 1). Protein identities are indicated. MW, molecular weight (in thousands). (B) UBP64, TTK88, and TTK69 are associated in crude embryo nuclear extract. Nuclear extract (input) was incubated with protein A-Sepharose beads coated with anti-UBP64 antibodies. For mock immunoprecipitation (IP), protein A-Sepharose beads were directly added to the nuclear extract. After extensive washes with a buffer containing 300 mM KCl and 0.1% NP-40, bound proteins were resolved by SDS-PAGE and analyzed by Western immunoblotting with antibodies directed against either UBP64, TTK88, or TTK69. Ten percent of the input material was loaded. TTK69 levels are strongly reduced in ubp64{Delta}1 flies, as revealed by Western immunoblot analysis of protein extracts prepared from either WT or ubp64{Delta}1 eye discs. Anti-Moira ({alpha}-MOR) immunoblotting was performed as a loading control.


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  		TABLE 1. List of UBP64-associated proteins identified by nanoflow liquid chromatography-tandem mass spectrometry

Because it was the most predominant protein associated with UBP64 and has a well-established role in eye development, we decided to focus on TTK. First, we confirmed the association between UBP64 and TTK by coimmunoprecipitation and Western immunoblot analysis (Fig. 2B). Next, we investigated the effect of the reduced UBP64 levels in ubp64{Delta}1 flies on TTK. Western blot analysis of protein extracts prepared from dissected eye discs revealed a clear loss of TTK69 in ubp64{Delta}1 compared to WT flies (Fig. 2C). Unfortunately, our antibodies directed against TTK88 were not sufficiently sensitive to detect TTK88 in crude extracts. Anti-Moira immunoblotting was used as a loading control (Fig. 2C, bottom). Taken together, our results indicate that UBP64 associates with and affects the stability of TTK in vivo.

UBP64 and TTK interact genetically. Having established a biochemical and functional association between UBP64 and TTK, we next explored the role of these interactions during development. Here, we took advantage of the disruptive effect of TTK misexpression on eye development (24, 38, 41). Using the Gal4/UAS system, TTK was overexpressed under the control of the GMR enhancer. All genetic interaction experiments were performed with both Ttk88 and Ttk69. Because the results obtained for each form of Ttk were identical, we do not distinguish between Ttk69 and Ttk88 in our description. As shown previously (24, 38, 41), ectopic TTK expression (GMR>Ttk) caused a distorted eye morphology characterized by disorganized facets and a loss of bristles (Fig. 3B). The rough eye phenotype was strongly suppressed when TTK overexpression was combined with reduced UBP64 levels (GMR>Ttk/ubp64{Delta}1) (Fig. 3C). Thus, TTK overexpression-induced aberrant eye development is dependent on UBP64. The Ubp64 revertant did not modulate the GMR>Ttk phenotype, excluding possible genetic background effects (GMR>Ttk/Rev) (Fig. 3D). Consistent with a positive regulatory role for UBP64, the simultaneous ectopic expression of UBP64 and TTK (GMR>Ttk/GMR>Ubp64) caused dramatically defective eye development (Fig. 3E). In contrast, the combined overexpression of TTK and the UBP64 catalytic mutant (GMR>Ttk/GMR>Ubp64C405A) did not further enhance the GMR>Ttk phenotype (compare Fig. 3F with B and E), demonstrating the requirement of UBP64 enzymatic function. In agreement with the in vivo association and stabilization of TTK by UBP64 (Fig. 2), these results argue that TTK and UBP64 collaboratively regulate normal eye development.


Figure 3
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  		FIG. 3. Ubp64 interacts genetically with Ttk. (A and B) Scanning electron micrographs of adult eyes of WT flies (A) and GMR-Gal4 UAS-Ttk69/CyO flies (GMR>Ttk) (B) that ectopically express TTK under the control of the GMR enhancer. TTK misexpression during development causes a distorted eye morphology characterized by disorganized facets and loss of bristles. The TTK misexpression phenotype is suppressed when the UBP64 protein level is reduced in a ubp64{Delta}1 background (GMR-Gal4 UAS-Ttk69/+ ubp64{Delta}1/+, abbreviated as GMR>Ttk/ubp64{Delta}1) (C) but not in a Rev background (GMR-Gal4 UAS-Ttk69/+ Rev/+, abbreviated as GMR>Ttk/Rev) (D). (E) Ectopic expression of both TTK and UBP64 in GMR-Gal4 UAS-Ttk69/+ UAS-Ubp64/+ flies (GMR>Ttk/GMR>Ubp64) dramatically disrupts eye formation. (F) Combined overexpression of TTK and the enzymatically inactive UBP64C405A in GMR-Gal4/+ UAS-Ubp64C405A/+ GMR-Gal4 UAS-Ttk69/+ flies (GMR>Ttk/GMR>Ubp64C405A) did not affect the eye phenotype.

UBP64 controls eye cell differentiation through TTK. What developmental decisions are controlled by UBP64? To address this question, we sought to more precisely define the role of UBP64 in eye development by analyzing photoreceptor and cone cell differentiation. These specialized cell types can be identified by protein immunodetection of either CUT or ELAV, defining cone and photoreceptor cells, respectively. In contrast to WT pupal ommatidia, which contain four cut-positive cone cells (Fig. 4A), ubp64{Delta}1 ommatidia frequently contain three instead of four cone cells, suggesting that reduced UBP64 levels affect cone cell differentiation (Fig. 4B). Importantly, this phenotype closely mimics that of a Ttk69 loss-of-function mutation reported previously (24). The ommatidial arrangement in Rev was similar to that of the WT (Fig. 4C). In contrast to ubp64{Delta}1, ectopic UBP64 expression (GMR>Ubp64) leads to the presence of an additional, fifth cone cell (Fig. 4D). Thus, like TTK, UBP64 appears to promote cone cell formation.


Figure 4
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  		FIG. 4. UBP64 promotes cone cell formation. Eye discs isolated from 40-h-old pupae were analyzed and visualized by indirect immunofluorescence using antibodies directed against the cone cell-specific marker protein CUT. (A) Well-organized, repetitive pattern of ommatidia in WT pupal retina. Each ommatidium contains four CUT-expressing cone cells, as illustrated by the higher magnification of a representative ommatidium (bottom). (B) Ommatidia in homozygous ubp64{Delta}1/ubp64{Delta}1 mutant (ubp64{Delta}1) eye discs are less well organized and frequently contain fewer than four cone cells. (C) Rev retinae are similar to WT retinae. (D) Ectopic expression of UBP64 in GMR-Gal4/+ UAS-Ubp64/+ (abbreviated as GMR>Ubp64) pupal retinae disrupts the regular arrangement of ommatidia and leads to the appearance of extra cone cells. (E) Ectopic expression of TTK69 in GMR-Gal4 UAS-Ttk69/Rev pupae (GMR>Ttk69/Rev) strongly disrupts the ommatidial pattern. (F) A reduction of the UBP64 level in GMR-Gal4 UAS-Ttk69/+ ubp64{Delta}1/+ (GMR>Ttk69/ubp64{Delta}1) pupal retinae suppresses the TTK69 misexpression phenotype. Note that ubp64{Delta}1 and Rev have a common genetic background. (G) In contrast, the ectopic expression of both UBP64 and TTK69 in GMR-Gal4 UAS-Ttk69/+ UAS-Ubp64/+ (GMR>Ttk69/GMR>Ubp64) pupae enhances the phenotype further. (H) Ectopic expression of catalytically dead Ubp64C405A in GMR-Gal4 UAS-Ttk69/+ UAS-Ubp64C405A/+ pupae (GMR>Ttk69/GMR>Ubp64C405A) does not enhance the effects of TTK69 overexpression.

Analysis of the adult eye showed that reduced UBP64 levels suppress the effect of ectopic TTK expression, whereas the concomitant overexpression of UBP64 dramatically exacerbates the eye defect (Fig. 3). Here, we tested the interplay between TTK69 and UBP64 on cone cell differentiation. As described previously (41), ectopic TTK69 expression (GMR>Ttk69) severely disrupts the cone cell pattern in the ommatidium (Fig. 4E). This effect is suppressed strongly by reduced UBP64 levels in ubp64{Delta}1 mutants (Fig. 4F). The concomitant overexpression of both TTK69 and active UBP64 (Fig. 4G), but not an inactive UBP64 mutant (Fig. 4 H), enhances the defect. We conclude that TTK and UBP64 cooperate in promoting cone cell differentiation.

We also investigated the role of UBP64 in photoreceptor cell differentiation (Fig. 5). Photoreceptors were identified by immunofluorescence using antibodies directed against the neuronal marker ELAV. Although WT ommatidia harbor eight photoreceptor cells, only seven were visualized because the R8 photoreceptor lies below the focal plane selected (Fig. 5A). Again, ubp64{Delta}1 ommatidia are clearly disorganized and frequently contain supernumerary photoreceptors (Fig. 5B). Conversely, the ectopic expression of UBP64 (GMR>Ubp64) inhibits photoreceptor cell formation, leading to fewer than the normal complement of photoreceptors (Fig. 5D). In summary, the consequences of a loss of UBP64 expression or its ectopic expression clearly mirror those of corresponding changes in TTK levels reported previously by others (24, 25, 38, 41).


Figure 5
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  		FIG. 5. UBP64 inhibits photoreceptor differentiation. (A) To identify photoreceptor cells, eye discs from WT pupae were immunostained with antibodies directed against the neuronal marker ELAV. Note that although WT ommatidia harbor eight photoreceptor cells, only seven are visualized because the R8 photoreceptor lies below the selected focal plane. A higher magnification of a representative ommatidium is shown at the bottom. (B) Homozygous ubp64{Delta}1/ubp64{Delta}1 ommatidia are disorganized and frequently contain supranumerical photoreceptors. (C) Rev retina is similar to WT retina. (D) Ectopic expression of UBP64 in GMR-Gal4/+ UAS-Ubp64/+ pupal retinae (GMR>Ubp64) disrupts the regular arrangement of ommatidia and inhibits photoreceptor cell formation, leading to fewer than the normal complement of eight photoreceptors. (E and F) Ectopic expression of TTK69 in GMR-Gal4 UAS-Ttk69/Rev pupae (GMR>Ttk69/Rev) strongly disrupts the photoreceptor pattern (E), which is suppressed by the reduced UBP64 levels in GMR-Gal4 UAS-Ttk69/+ ubp64{Delta}1/+ (GMR>Ttk69/ubp64{Delta}1) pupae (F). (G and H) Ectopic expression of both UBP64 and TTK69 in GMR-Gal4 UAS-Ttk69/+ UAS-Ubp64/+ pupae (GMR>Ttk69/GMR>Ubp64) enhances this phenotype further (G), whereas the expression of catalytically dead Ubp64C405A in GMR-Gal4 UAS-Ttk69/+ UAS-Ubp64C405A/+ pupae (GMR>Ttk69/GMR>Ubp64C405A) does not enhance the effects of TTK69 overexpression (H).

The genetic interaction between Ttk69 and Ubp64 was also evident during photoreceptor differentiation. TTK69 overexpression (Fig. 5E) disrupted the photoreceptor pattern, but this was suppressed by reduced UBP64 levels (Fig. 5F). In contrast, combined ectopic TTK69 and UBP64 overexpression enhanced the defect (Fig. 5G), while coexpression with the inactive UBP64 mutant had no effect (Fig. 5H).

Combined with our biochemical and genetic results, these observations strongly suggest that UBP64 controls cell fate during eye development through the positive regulation of TTK.

UBP64 deubiquitylates and stabilizes TTK. TTK is regulated posttranslationally through SINA-mediated polyubiquitylation and proteasome-mediated proteolysis (24, 38). Therefore, an attractive mechanism for TTK stabilization by UBP64 is through deubiquitylation, counteracting proteasome targeting and destruction. To test whether UBP64 can directly deubiquitylate TTK, we reconstituted this reaction in vitro. First, we recapitulated SINA-dependent TTK ubiquitylation by the cotransfection of plasmids expressing His6-ubiquitin, HA epitope-tagged TTK69, and SINA in human H1299 cells, which lack endogenous TTK and Sina. Proteasome-mediated degradation was inhibited by the addition of the drug MG132. Following extract preparation and purification by nickel-NTA chromatography, TTK69 was visualized by immunoblotting. As illustrated by Fig. 6A, Sina-dependent TTK69 polyubiquitylation was readily detected.


Figure 6
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  		FIG. 6. UBP64 deubiquitylates TTK to counteract Sina-directed degradation. (A) Recapitulation of SINA-dependent TTK polyubiquitylation. Plasmids expressing HA-tagged TTK69, SINA, and His6-ubiquitin were cotransfected into H1299 cells in the indicated combinations. Proteasome-mediated degradation was inhibited by the addition of the drug MG132. After extract preparation, ubiquitylated proteins were purified under denaturing conditions by Ni2+-NTA chromatography, resolved by SDS-PAGE, and visualized by immunoblotting with antibodies directed against TTK69. (B) The relevant Coomassie-stained protein bands corresponding to the blot in A were excised and subjected to mass spectrometric analysis. The cartoon depicts the lysine residues that were covalently attached to the LRGG or GG ubiquitin remnants via an isopeptide bond. The sequences of the tryptic peptides and the ubiquitylated residue (red) are listed along with their mascot scores. (C) UBP64 stabilizes TTK69. A vector expressing HA-tagged TTK69 was cotransfected into H1299 with Sina and Ubp64 in the indicated combinations. In the absence of a proteasome inhibitor, the expression of Sina led to strongly reduced TTK69 levels. TTK69 degradation was effectively counteracted by the coexpression of UBP64. TTK69 was detected using anti-HA antibodies. Histone H3 and an anti-HA cross-reacting protein (*) serve as loading controls. (D) Recombinant GST-tagged UBP64 and UBP64C405A were purified from E. coli cell extracts by glutathione-Sepharose (fast flow) chromatography, resolved by SDS-PAGE, and visualized by Coomassie staining. MW, molecular weight (in thousands). (E) UBP64 deubiquitylates TTK. Ni2+-NTA-purified Ub-TTK69 was incubated in the absence or presence of either UBP64 or UBP64C405A, resolved by SDS-PAGE, and analyzed by Western immunoblotting using anti-HA antibodies.

Mass spectrometric analysis of the relevant protein bands on the gel confirmed the presence of both TTK69 and ubiquitin. Interestingly, we identified tryptic TTK69 peptides containing diglycine (GG) or leucine-arginine-glycine-glycine (LRGG) remnants of ubiquitin attached via an isopeptide bond to TTK69 lysine (K) residues (Fig. 6B). Strikingly, the nine mapped ubiquitylated K residues are not restricted to a single area but rather are distributed over most of the TTK69 sequence. We identified mainly K48-linked but also a minor amount of K63 polyubiquitylation chains.

As expected, the expression of SINA in the absence of a proteasome inhibitor led to strongly reduced TTK69 levels (Fig. 6C). However, the coexpression of UBP64 completely reversed the SINA-directed destruction of TTK69. The levels of histone H3 (Fig. 6C, bottom) and a cross-reacting protein (Fig. 6C, top) did not change, confirming that equal amounts of protein were loaded. Thus, our cell-based cotransfection system recapitulates the stabilization of TTK by UBP64 observed in the fly.

To investigate whether UBP64 might act through the direct removal of the polyubiquitin tags that mark TTK for proteasome-mediated destruction, we reconstituted TKK deubiquitylation with purified proteins. UBP64 and the UBP64C405A catalytic mutant were expressed and purified from E. coli (Fig. 6D). Next, purified polyubiquitylated TTK69 was incubated with UBP64, resolved by SDS-PAGE, and analyzed by Western immunoblotting (Fig. 6E). Recombinant WT UBP64, but not an equimolar amount of UBP64C405A, efficiently deubiquitylated TTK69. Interestingly, our mass spectrometric mapping of ubiquitylation sites indicated that SINA broadly ubiquitylates TTK69, which is duly removed by UBP64. Thus, neither E3 nor the DUB targets a single site on TTK69 but rather might scan most of the protein to target dispersed lysines. In conclusion, our results show that UBP64 can directly bind and deubiquitylate TTK.


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DISCUSSION
 
In contrast to the extensive body of data on the function of ubiquitin ligases, detailed examples of the target selectivity and regulatory functions of DUBs are rare. Here, we analyzed the developmental function of the Drosophila DUB UBP64 and found the following. First, UBP64 controls cell fate decisions during eye development. Second, UBP64 represses neuronal cell fate but promotes the formation of nonneuronal cone cells. Reduced UBP64 levels lead to a loss of cone cells, supernumerary photoreceptors, and mechanosensory bristle cells. Conversely, ectopic UBP64 expression causes a loss of the neuronal photoreceptor and bristle cells but promotes the formation of extra cone cells. Third, UBP64 interacts biochemically and genetically with the transcription factor TTK. Fourth, UBP64 stabilizes TTK in selective cells in the developing eye, driving precursor cells toward a nonneuronal identity. Finally, UBP64 acts through the removal of the polyubiquitin degradation marks on TTK made by SINA. Thus, the balance between the activities of the E3 ubiquitin ligase Sina and the DUB UBP64 constitutes a specific posttranslational switch controlling cell fate.

Posttranslational control of cell fate determination. Cell fate determination is an intricately regulated process orchestrated by the combinatorial resultant of positive and negative signaling. At the end of signaling cascades are transcription factors, which control gene expression programs that drive cell differentiation. TTK forms a selective differentiation barrier by blocking neuronal cell identity and promoting specific nonneuronal fates, such as cone cells. The regulation of TTK itself is complex and occurs to a large extent at the posttranslational level. The activation of Ras-MAP kinase signaling through PHYL-mediated stimulation of SINA results in TTK polyubiquitylation and destruction, thus removing the second blockade to R7 photoreceptor differentiation (24, 38).

Through a series of biochemical, developmental, and genetic experiments, we found that UBP64 is a critical positive regulator of TTK. An unbiased proteomic search for potential UBP64-interacting proteins identified TTK. The physiological relevance of this physical interaction was established by the strong positive genetic interaction between UBP64 and TTK. Our results showed that UBP64 is required to maintain stable TTK levels in cells acquiring a nonneuronal cell identity. Consequently, a loss of UBP64 disrupts the cell differentiation process in the eye disc. Reduced UBP64 levels lead to a loss of cone cells but also supernumerary neuronal photoreceptors and bristle cells. Conversely, UBP64 overexpression results in missing photoreceptor and bristle cells but extra cone cells. Importantly, these developmental effects of the UBP64 dosage closely mimic those observed for TTK (24, 25, 38, 41). Cell-based assays and in vitro reconstitution experiments revealed that UBP64 stabilizes TTK through deubiquitylation, preventing its degradation by the proteasome. Mass spectrometric mapping revealed that SINA ubiquitylates a multitude of distinct lysines spread throughout the TTK69 sequence. Because UBP64 can efficiently deubiquitylate these sites, we conclude that both the E3 and the DUB are able to bind TTK69 and target multiple sites. We speculate that SINA and UBP64 might dock at a specific site of TTK69, followed by scanning the whole protein, rather than targeting only a restricted domain.

Interestingly, UBP64 expression appears to be ubiquitous and not cell type specific. The implication of this observation is that its activity might be under the control of developmental signaling. Our future research will be aimed at trying to identify the relevant signal transduction pathways. In conclusion, our results establish that the balance between ubiquitylation and deubiquitylation of a transcriptional repressor constitutes a specific posttranslational control point of a gene expression program orchestrating cell patterning.

Specific regulatory roles of DUBs. Recent research has revealed that DUBs play key roles in a multitude of biological processes including cancer, endocytosis, chromatin dynamics, epigenetic regulation, and neurodegeneration. The relatively few DUBs that have been analyzed in detail strongly support the notion that DUBs regulate specific processes through targeting of selective substrates. For example, the product of the cylindromatosis tumor suppressor gene is a negative regulator of NF-{kappa}B signaling (35). Fat facets stabilize LQF, which in turn mediates the endocytosis of the Delta receptor, critical for cell patterning (9, 29). Another DUB, USP7, plays a role in p53 tumor suppressor regulation. DNA damage signaling modulates the substrate specificity of USP7, promoting the deubiquitylation of either p53 or its E3 ubiquitin ligase MDM2 (11, 23). We recently showed that Drosophila USP7 deubiquitylates histone H2B and mediates developmental gene silencing. Interestingly, USP7 associates with a metabolic enzyme, GMP synthetase, which strongly stimulates its enzymatic activity, implicating a biosynthetic enzyme in the regulation of chromatin dynamics through the control of DUB activity (39). Finally, the DUB UBP8 is part of the SAGA coactivator complex and contributes to transcriptional activation (17). Recently, it was demonstrated that MYC stability in the nucleus is facilitated by USP28, which functions by antagonizing the FBW7{alpha} ubiquitin ligase complex (32). Likewise, the balance between CDC20 ubiquitylation by the anaphase-promoting complex and deubiquitylation by USP44 acts as a switch controlling anaphase entry (34, 37). Here, we provided an example of a DUB that controls developmental gene expression and cell fate through the selective stabilization of a transcriptional repressor. To the best of our knowledge, the SINA/UBP64-controlled switch described here constitutes the first detailed example of a developmental program regulated by a specific E3/UBP pair. We anticipate that further analysis of more DUBs will uncover their diverse regulatory roles in cell biology and that their functions will be equally intricate and as important as those of ubiquitin ligases.


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ACKNOWLEDGMENTS
 
We thank Karin Langenberg for help with the embryo immunofluorescence, HarmJan Vos for help with microinjections, and Jesper Svejstrup and David Baker for insightful comments on the manuscript.

This work was supported in part by a Bsik SCDD program grant (to C.P.V.) and a Medical Research Council scholarship (to A.B.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry, Erasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Phone: 10-4087461. Fax: 10-70-44747. E-mail: c.verrijzer{at}erasmusmc.nl Back

{triangledown} Published ahead of print on 26 December 2007. Back


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Molecular and Cellular Biology, March 2008, p. 1606-1615, Vol. 28, No. 5
0270-7306/08/$08.00+0     doi:10.1128/MCB.01567-07
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