Payments and Quality of Ante-NatalCare in Two Rural Districts ofTanzania Paper 4 from the Ethics, Payments, and P.O. Box 33223, Dar es Salaam, Tanzania Maternal Survival Project 157 Mgombani Street, Regent EstateTel: +255 (0) 22 2700083 / 2772556Fax: +255 (0) 22 2775738 Paula Tibandebage, Maureen Mackintosh, Tausi Kida,
Pq089603275pProc. Natl. Acad. Sci. USA Vol. 93, pp. 3275–3279, April 1996 DUB-1, a deubiquitinating enzyme with growth-
YUAN ZHU*, MARTIN CARROLL*, FEROZ R. PAPA†, MARK HOCHSTRASSER†, AND ALAN D. D'ANDREA*‡*Divisions of Pediatric Oncology and Cellular and Molecular Biology, Dana–Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; and †Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637 Communicated by David M. Livingston, Dana–Farber Cancer Institute, Boston, MA, November 29, 1995 (received for review October 3, 1995) Cytokines regulate cell growth by inducing
substrate. These enzymes are ubiquitin specific but share the expression of specific target genes. Using the differential
certain properties with other thiol proteases. Genes for at least display method, we have cloned a cytokine-inducible imme-
15 deubiquitinating enzymes were identified from the yeast diate early gene, DUB-1 (for deubiquitinating enzyme).
genome, making them the largest known gene family in the DUB-1 is related to members of the UBP superfamily of
ubiquitin system. Several proteins implicated in growth and deubiquitinating enzymes, which includes the oncoprotein
development, including the mammalian proteins Tre-2 and Tre-2. A glutathione S-transferase–DUB-1 fusion protein
Unp and the Drosophila fat facets protein, were either shown cleaved ubiquitin from a ubiquitin-b-galactosidase protein.
to be deubiquitinating enzymes or to have sequence similarity When a conserved cysteine residue of DUB-1, required for
to such enzymes (21).
ubiquitin-specific thiol protease activity, was mutated to
In the current study, we used the strategy of differential serine (C60S), deubiquitinating activity was abolished. Con-
display (22, 23) to clone an immediate early cDNA (DUB-1) tinuous expression of DUB-1 from a steroid-inducible pro-
that is specifically induced by IL-3. The DUB-1 cDNA encodes moter induced growth arrest in the G1 phase of the cell cycle.
a 526-aa protein that has deubiquitinating activity. Interest- Cells arrested by DUB-1 expression remained viable and
ingly, misregulated expression of DUB-1 induces cell cycle resumed proliferation upon steroid withdrawal. Our results
suggest that DUB-1 regulates cellular growth by modulating
arrest in the G1 phase of the cell cycle. Our results support the either the ubiquitin-dependent proteolysis or the ubiquitina-
hypothesis that protein ubiquitination is important in growth- tion state of an unknown growth regulatory factor(s).
factor–mediated cellular proliferation. They also implicate deubiquitinating enzymes as regulatory enzymes that couple Interleukin 3 (IL-3) is a glycoprotein hormone that regulates extracellular signaling to cell growth.
growth of hematopoietic progenitor cells (1). IL-3, like other cytokines, acts during the G1 phase of the cell cycle to drive MATERIALS AND METHODS
cells into S phase. IL-3 exerts its biologic function through a specific receptor (IL-3R) that is expressed on its target cells (2, Cells and Cell Culture. BayF3 is an IL-3-dependent murine
3). The IL-3R activates multiple signal transduction pathways, pro-B cell line (24). BayF3 cells were maintained in RPMI including the Ras–Raf–mitogen activating protein kinase path- 1640 medium supplemented with 10% fetal calf serum (FCS) way and the JAK–STAT pathway, resulting in the induction of and 10% conditioned medium from WEHI-3B cells as a source immediate early genes. How these immediate early genes of IL-3 (25).
couple IL-3R activation to the biochemical machinery of cell Differential Display and Cloning of DUB-1 cDNA. Total
growth and cell cycle progression is poorly understood.
cellular RNA was isolated from starved or IL-3-stimulated Cell growth and cell cycle progression are controlled, at least BayF3 cells by the guanidinium isothiocyanate procedure (26) in part, by ubiquitin-mediated proteolysis (4, 5). Ubiquitin- and subjected to the differential display analysis (22) (Gene mediated proteolysis requires ATP and results in covalent Hunter, Boston). A partial cDNA fragment that was specifi- conjugation of target proteins with multiple ubiquitin mole- cally induced by IL-3 was isolated using a 59 primer (59- cules (6–9). Multiubiquitinated proteins are rapidly degraded TCTGTGCTGG-39) and a 39 primer (59-TTTTTTTTTTT- by the 26S proteasome, a multicatalytic protease complex (10, TGT-39) and subcloned into pCRII (Invitrogen). This partial 11). Recent evidence shows that intracellular levels of cyclins cDNA (298 bp) was shown by dideoxy DNA sequencing to and cyclin dependent kinase inhibitors (12, 13), as well as other contain the 59 and 39 primers. A cDNA library, from BayF3 growth regulatory proteins, such as p53 (14, 15), c-Jun (16), cells growing in IL-3, was constructed in the phage vector and IkBa (17), are regulated by ubiquitin-mediated proteol- lZAP (Stratagene). Poly(A)1 mRNA used for library con- ysis. It is also possible that ubiquitination alters a protein's struction was prepared by the Fast Track mRNA Isolation Kit function without affecting its metabolic stability (18).
(Invitrogen). The partial cDNA isolated by differential display Little is known about the regulatory enzymes that determine was labeled with [32P]dCTP by random prime labeling (27) and which cellular proteins are specifically destroyed by ubiquitin- used to screen 1 3 106 plaque-forming units from the library.
mediated proteolysis. Most evidence suggests that substrate Three independent positive clones of different lengths that specificity is determined by ubiquitin-conjugating enzymes hybridized with the probe were isolated, and the corresponding (19, 20). Recently, a large superfamily of genes encoding plasmids were isolated from the phage clones. The longest deubiquitinating enzymes was identified (21). Deubiquitinat- cDNA clone was sequenced on both strands by the dideoxy ing enzymes remove ubiquitin from intracellular protein con- jugates by cleaving the amide linkage between the C terminus DNA sequencing method (United States Biochemical).
of ubiquitin and either a-amino or «-amino groups of the Abbreviations: IL-3, interleukin 3; IL-3R, IL-3 receptor; GST, gluta- thione S-transferase; ORF, open reading frame.
The publication costs of this article were defrayed in part by page charge ‡To whom reprint requests should be addressed at: Division of payment. This article must therefore be hereby marked ‘‘advertisement'' in Pediatric Oncology, Dana–Farber Cancer Institute, 44 Binney Street, accordance with 18 U.S.C. §1734 solely to indicate this fact.
Boston, MA 02115.
Cell Biology: Zhu et al. Proc. Natl. Acad. Sci. USA 93 (1996) Northern Blot Analysis. RNA samples (10–30 mg) were
electrophoresed on denaturing formaldehyde gels and blotted onto Duralon-UV membranes (Stratagene). The cDNA in- serts, purified from agarose gels (Qiagen, Chatsworth, CA), were radiolabeled (27) and hybridized for 1 hr to the filters in a 68°C oven. Hybridized filters were finally washed at room temperature in 0.13 SSC (13 SSC 5 0.15 M sodium chloridey 0.015 M sodium citrate, pH 7) and 0.1% SDS.
Deubiquitination Assay. The deubiquitination assay of ubiq-
uitin–b-galactosidase fusion proteins has been previously de- scribed (21). A 1578-bp fragment from the wild-type DUB-1 cDNA (corresponding to aa 1 to 526) and a cDNA containing a missense mutation (C60S) were generated by polymerase chain reaction (PCR) and inserted, in frame, into pGEX-2TK (Pharmacia) downstream of the glutathione S-transferase (GST) coding element. Ub–Met–bgal was expressed from a pACYC184-based plasmid. Plasmid-bearing Escherichia coli MC1061 cells were lysed and analyzed by immunoblotting with anti-bgal antibodies (Cappel) and the enhanced chemilumi- nescence system (Amersham).
Generation of Anti-DUB-1 Antiserum and Analysis of the
DUB-1 Polypeptide. A DUB-1 antiserum was raised by inject-
ing a full-length GST–DUB-1 fusion protein into a New Zealand White rabbit and was affinity purified with a GST– DUB-1 affinity matrix, as previously described (28). In vitro translation of the full length DUB-1 polypeptide was per- formed by standard procedures (Promega). Immunoblotting was performed as previously described (29) using the affinity- purified anti-DUB-1 antiserum and enhanced chemilumines- cence technology.
Heterologous Expression of DUB-1 in BayF3 Cells and Cell
Growth Analysis. The open reading frame (ORF) of DUB-1
[or DUB-1(C60S)] was generated by PCR using the following 39 (219 to 1) and 59-ATCTCGAGGTGTCCACAGGAGCCT- GTGT-39 (1802 to 1781). The fragments (1637 bp) were subcloned into the Sma IyXho I cloning sites of pMSG (Pharmacia), which contains a mouse mammary tumor virus– long terminal repeat inducible promoter and a gpt selection marker. Parental BayF3 cells were electroporated with vector alone or with pMSG–DUB-1 as previously described (25).
After 3 days in IL-3 medium, the cells were selected in IL-3 medium containing 250 mgyml xanthine, 15 mgyml hypoxan- thine, 10 mgyml thymidine, 2 mgyml aminopterin, and 25 mgyml mycophenolic acid. Gpt-resistant subclones were iso- lated by limiting dilution. DUB-1 expression was induced by adding 0.1 mM dexamethasone (diluted from 10 mM stock in ethanol). Cell proliferation and cell viability were measured by trypan blue exclusion (25).
Analysis of Cell Cycle. Cell cycle analysis was performed by
fluorescence-activated cell sorter, as previously described (30).
The percentage of cells in each phase of the cell cycle was determined by analyzing data with the computer programCELLFIT (Becton Dickinson).
DUB-1 Is a Hematopoietic-Specific Immediate Early Gene
Encoding a Deubiquitinating Enzyme. BayF3 is a murine
FIG. 1. Sequence and homologies of the DUB-1 cDNA. (A) Nucle- lymphocyte cell line that depends on IL-3 for growth and otide and predicted amino acid sequence of DUB-1. Underlined se- viability (24, 30, 31). By comparing mRNA from IL-3-deprived quences are copies of a conserved motif shown by Shaw and Kamen (32) and IL-3-stimulated BayF3 cells (22, 23), we initially isolated to confer message instability and which are found in the 39 untranslated an IL-3 inducible, immediate early cDNA fragment (DUB-1).
regions of many mitogen-induced, immediate early mRNAs. A consensus The full-length 2674-bp DUB-1 cDNA was subsequently iso- polyadenylylation signal is double underlined. The sequence of the lated and found to contain a 1581-bp ORF (Fig. 1 A). There are murine DUB-1 cDNA has been assigned GenBank no. 24133 U41636. (B) two stop codons within the 183 bp of 59 untranslated region.
Sequence homologies between yeast Doa4 (21), human Tre-2 (33), murine Unp (34), and murine DUB-1. Alignment of DUB-1 with human In addition, we isolated a murine genomic clone that contains c-myc is also shown. The homologous domain of c-myc contains the a TATA box at position 2321 and an IL-3 inducible enhancer nuclear localization sequence PAAKRAKLD (35) but not the c-myc (Y.Z., unpublished data).
DNA binding domain.
Cell Biology: Zhu et al. Proc. Natl. Acad. Sci. USA 93 (1996) The DUB-1 ORF is predicted to encode a polypeptide of 526 aa (59 kDa). Comparison of the DUB-1 protein sequence with entries in GenBank data base (3y96) detected significant similarity with several deubiquitinating enzymes, including Tre-2 (33, 36), Unp (34), and Doa4 (21). The sequence similarity was largely restricted to the conserved Cys and His boxes previously identified for this enzyme superfamily (Fig.
1B) (21). These elements probably help form the enzyme active site (21). The likely active site nucleophile is a cysteine residue in the Cys box that is found in all known family members (21) and is also present in DUB-1 (Cys60). The 39 untranslated region of the DUB-1 cDNA contained two AT- TTA sequences, located in A 1 T rich domains. The AUUUA sequence, found in the 39 untranslated regions of many im- mediate early mRNAs, may play a role in DUB-1 mRNA turnover (32). The DUB-1 mRNA was detected in multiple hematopoietic cell lines, but not in nonhematopoietic cell lines or tissues from adult mice (data not shown).
DUB-1 Encodes a Functional Deubiquitinating Enzyme. In
order to determine whether DUB-1 has deubiquitinating ac- tivity, we expressed DUB-1 as a GST fusion protein. The DUB-1 ORF was subcloned into the bacteria expression vector, pGEX. pGEX–DUB-1 was co-transformed into E. coli FIG. 3. DUB-1 mRNA levels are induced by IL-3 in early G1 phase, (MC1061) with a plasmid expressing the protein Ub–Met–b followed by a rapid decline. BayF3-EPO-R cells were arrested in early gal, in which ubiquitin is fused to the N terminus of b-galac- G1 phase by growth factor starvation for 12 hr and were restimulated tosidase. As shown by immunoblot analysis (Fig. 2), two with IL-3 to enter the cell cycle synchronously. Total RNA (10 mg per independent cDNA clones encoding GST–DUB-1 fusion pro- lane) extracted from cells at the indicated time (in hours) was tein resulted in cleavage of Ub–Met–b gal (lanes 3, 4, and 7) subjected to Northern blot analysis with the indicated cDNA probes.
The different cell cycle phases were determined by flow-cytometric comparable to that observed with Ubp1, a known yeast analysis of cellular DNA content.
deubiquitinating enzyme (21) (lane 1). As controls, cells with the pGEX vector (lane 5) or pBluescript vector with a non- Continuous Expression of DUB-1 Arrests Cellular Growth.
transcribed DUB-1 insert (lane 2) failed to cleave Ub–Met–b Our initial attempts to obtain stable cell lines that consti- gal. A mutant DUB-1 polypeptide, containing a C60S muta- tutively express DUB-1 were unsuccessful. Because DUB-1 tion, was unable to cleave the Ub–Met–b gal substrate (lane 6).
expression is normally turned off after only a brief period of Expression of GST-DUB-1 in bacterial cells containing the synthesis (Fig. 3), we reasoned that continuous expression of Ub–Leu–b gal substrate showed greatly reduced levels of b gal DUB-1 mRNA might somehow interfere with cell growth activity (data not shown). The Leu–b gal product, unlike andyor viability. We therefore expressed DUB-1 in BayF3 Met–b gal or the respective Ub–b gal fusions, is short lived in cells using an inducible promoter (Fig. 4). Twelve gpt- E. coli (37). This result strongly suggests that DUB-1 cleaves resistant BayF3 subclones were generated after transfection Ub–Leu–b gal specifically at the C terminus of the ubiquitin with either pMSGyDUB-1 or mutant pMSGyDUB-1(C60S), moiety. Taken together, these results demonstrate that DUB-1 which encodes the inactive enzyme. Dexamethasone (0.1 has deubiquitinating activity and that Cys 60 is critical for its mM) induced DUB-1 mRNA in all transfected cells, but not thiol protease activity.
in parental or mock-transfected cells (data not shown).
DUB-1 mRNA Levels Are Induced by IL-3 in Early G1 Phase,
Dexamethasone induced expression of the DUB-1 protein Followed by a Rapid Decline. BayF3 cells arrest in early G1
(Fig. 4A, lane 2) or DUB-1(C60S) protein (lane 4) in trans- phase when deprived of IL-3 for 12 hr and can be induced to fected BayF3 cells to levels comparable with those observed reenter the cell cycle synchronously by readdition of growth during IL-3-induced expression from the endogenous DUB-1 factor (30). The 3.1-kb DUB-1 mRNA appeared 30 to 60 min gene (data not shown). These proteins had the same electro- after addition of IL-3 (Fig. 3) but rapidly decreased in abun- phoretic mobility (59 kDa) as full-length DUB-1 polypeptide dance before the completion of G1 phase. DUB-1 mRNA levels synthesized by in vitro translation (lane 5). After dexametha- were superinduced with IL-3 plus cycloheximide (data not sone induction, cells expressing DUB-1 failed to proliferate in shown), defining DUB-1 as an immediate early gene. Induction IL-3 medium (Fig. 4B). In contrast, dexamethasone-induced of DUB-1 mRNA was similar to that of c-myc, although c-myc cells expressing DUB-1 (C60S) proliferated normally in IL-3.
mRNA levels remained elevated throughout G1 phase. Cyclin Importantly, while dexamethasone induction of wild-type D2 mRNA accumulated later in G1 phase as previously DUB-1 inhibited cellular proliferation, as measured by total described (38).
cell number, it had little effect on cellular viability (Table 1).
The BayF3 subclones that were induced with dexamethasone to express either wild-type DUB-1 or DUB-1(C60S) remained viable in IL-3. Cells underwent apoptosis only after removal of IL-3.
To test the possibility of nonspecific toxicity caused by pro- longed expression of active DUB-1 enzyme, we stopped DUB-1 synthesis in cells transfected with the wild-type DUB-1 construct by removal of dexamethasone at day 7. Cells resumed normal FIG. 2. DUB-1 encodes a functional deubiquitinating enzyme.
proliferation within 48 hr following dexamethasone with- Deubiquitination of ubiquitin-b-galactosidase (Ub–Met–bgal) fusion drawal (Fig. 4C). To provide further evidence against non- proteins expressed in bacteria. Shown is a Western blot using anti- specific toxicity, we induced DUB-1 expression in murine bgal antiserum. Co-expressed plasmids were pGEX–Ubp1 (lane 1) 3T3 fibroblasts (data not shown). Normal cell proliferation (21), pBluescriptyDUB-1 (DUB-1 is not expressed) (lane 2), pGEX– DUB-1.1 (lanes 3 and 7), pGEX–DUB-1.2 (lane 4), pGEX(vector) was observed for these cells, indicating that growth suppres- (lane 5), and pGEX–DUB-1(C68S) (lane 6).
sion by DUB-1 is cell-type specific.
Cell Biology: Zhu et al. Proc. Natl. Acad. Sci. USA 93 (1996) Table 1. Percent viable BayF3 cells DUB-1 (WT) DUB-1 (C60S) WT, wild type; DEX, dexamethasone.
Normally, DUB-1 mRNA levels rise soon after IL-3 addition during the early G1 phase of the cell cycle, followed by a rapid decline. When DUB-1 mRNA levels are maintained by con- tinuous synthesis from a dexamethasone-inducible promoter, BayF3 cells arrest in the G1 phase of the cell cycle. These data indicate that DUB-1 expression is tightly regulated and that DUB-1 may play a role in cytokine-induced cell proliferation.
Deubiquitinating enzymes studied in yeast have multiple functions (21). Some deubiquitinating enzymes, such as Ubp2, can apparently remove ubiquitin from ubiquitin-conjugated substrates prior to proteasome-substrate binding, thereby slowing the turnover of such proteins (39). Other deubiquiti- nating enzymes, such as Doa4, may remove ubiquitin from proteasome-bound degradation products, allowing recycling of ubiquitin and proteasomes and thereby promoting further protein degradation (21). Ubiquitin must also be cleaved from precursor forms by deubiquitinating enzymes. Finally, dynamic ubiquitination events may serve as reversible regulatory switches (40, 41).
Failure to turn off expression of DUB-1 presumably, as in our experiments, may cause G1 arrest by preventing the degradation of growth-inhibitory proteins, such as cyclin de- pendent kinase inhibitors, or by promoting the degradation of growth-permissive proteins, such as G1 cyclins. Alternatively, FIG. 4. Continuous expression of DUB-1 results in growth sup- pression. (A) Immunoblot analysis of steroid-induced DUB-1 polypeptide. Lysates (100 mg of total protein) from the indicated cells were electrophoresed in 10% SDS polyacrylamide gels and blotted with affinity-purified anti-DUB-1 antibody (1:1000). (B) BayF3-DUB-1 (open symbols) or BayF3-DUB-1(C60S) (filled sym- bols) were cultured in IL-3 medium with (‚, Œ) or without (h, ■) dexamethasone (0.1 mM). Cell number was calculated by the trypan blue exclusion technique. (C) BayF3 cells, transfected with wild-type DUB-1, were grown in IL-3 with (‚) or without (h) dexamethasone for 6 days. Dexamethasone-treated cells were washed, replated in IL-3 medium (without dexamethasone) on day 7 (arrow), and cultured for an additional 6 days (‚).
Cell cycle analysis demonstrated that the majority of BayF3 cells were arrested in the G1 phase of the cell cycle following dexamethasone induction of DUB-1 (Fig. 5). This concentration of dexamethasone (0.1 mM) slightly reduced IL-3 dependent proliferation of parental BayF3 cells or DUB-1(C60S)-expressing cells (to 80% of maximum), but it FIG. 5. Forced expression of DUB-1 results in growth arrest in the completely blocked proliferation of the wild-type DUB-1- G1 phase of the cell cycle. The indicated cell lines were grown for 48 expressing cells.
hr (5 3 105 cellsyml) with or without dexamethasone (0.1 mM). The cells were stained with propidium iodide and analyzed by flow cytometry. The percentage of cells in G1, S, and G2yM were: DUB-1 2 dexamethasone (32%, 61%, 7%), 1 dexamethasone (82%, 14%, 4%); DUB-1(C60S) 2 dexamethasone (35%, 57%, 8%), 1 dexameth- In the present work, we describe a novel murine immediate asone (35%, 57%, 8%). Data shown are representative of at least three early gene that encodes a deubiquitinating enzyme, DUB-1.
separate dexamethasone induction experiments.
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