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Regulation of Microphthalmia-Associated Transcription Factor MITF Protein Levels by Association with the Ubiquitin-Conjugating Enzyme hUBC9
Experimental Cell Research 255, 135–143 (2000) doi:10.1006/excr.2000.4803, available online at http://www.idealibrary.com on
Regulation of Microphthalmia-Associated Transcription Factor MITF Protein Levels by Association with the Ubiquitin-Conjugating Enzyme hUBC9 Weidong Xu,* ,† ,‡ Limin Gong,§ Maher M. Haddad,* ,† ,‡ Oliver Bischof, ¶ Judith Campisi, ¶ Edward T. H. Yeh,§ and Estela E. Medrano* ,† ,‡ ,1 *Roy M. and Phyllis Gough Huffington Center on Aging and †Department of Cell Biology and ‡Department of Dermatology, Baylor College of Medicine, One Baylor Plaza M320 and VAMC, Houston, Texas 77030; §Institute of Molecular Medicine, University of Texas– Houston Health Science Center, Houston, Texas 77030; and ¶Department of Cell and Molecular Biology, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720
The basic helix-loop-helix/leucine zipper (bHLH/ ZIP) microphthalmia-associated transcription factor (MITF) regulates transcription of genes encoding enzymes essential for melanin biosynthesis in melanocytes and retinal pigmented epithelial cells. To determine how MITF activity is regulated, we used the yeast two-hybrid system to identify proteins expressed by human melanoma cells that interact with MITF. The majority of clones that showed positive interaction with a 158-amino-acid region of MITF containing the bHLH/ZIP domain (aa 168 –325) encoded the ubiquitin conjugating enzyme hUBC9. The association of MITF with hUBC9 was further confirmed by an in vitro GST pull-down assay. Although hUBC9 is known to interact preferentially with SENTRIN/ SUMO1, in vitro transcription/translation analysis demonstrated greater association of MITF with ubiquitin than with SENTRIN. Importantly, cotransfection of MITF and hUBC9 expression vectors resulted in MITF protein degradation. MITF protein was stabilized by the proteasome inhibitor MG132, indicating the role of the ubiquitin–proteasome system in MITF degradation. Serine 73, which is located in a region rich in proline, glutamic acid, serine, and threonine (PEST), regulates MITF protein stability, since a serine to alanine mutation prevented hUBC9-mediated MITF (S73A) degradation. Furthermore, we identified lysine 201 as a potential ubiquitination site. A lysine to arginine mutation abolished MITF (K201R) degradation by hUBC9 in vivo. Our experiments indicate that by targeting MITF for proteasome degradation, hUBC9 is a critical regulator of melanocyte differentiation. © 2000 Academic Press 1 To whom correspondence and reprint requests should be addressed at VA Medical Center, Research Building 110, 2002 Holcombe Boulevard, Houston, TX 77030. Fax: (713) 794-7938. E-mail: [email protected].
Key Words: MITF; ubiquitin; SUMO; melanocytes; differentiation.
INTRODUCTION
The mitf gene, originally cloned from a transgenic insertional mutation at the mouse microphthalmia locus [1, 2], encodes a basic-helix-loop-helix/leucine zipper protein. MITF is believed to promote the entrance of neural crest cells into the melanocyte differentiation pathway [3]. Melanin production is restricted to differentiated melanocytes and retinal pigmented epithelial cells, derived from the neural crest and neuroectoderm, respectively [4, 5]. MITF regulates expression of the Kit receptor [3] and the tyrosinase and tyrosinaserelated protein 1 (TRP1) enzymes [6], which are essential for melanin production. Tyrosinase is the ratelimiting enzyme in melanin biosynthesis, catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and then to DOPA quinone [7–9]. TRP1 catalyzes the conversion of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) to indole-5,6-quinonecarboxylic acid [10, 11]. Mutations in MITF are responsible for Waardenburg syndrome type 2 [12], a dominantly inherited disorder characterized by sensorineural hearing loss, heterochromia iridis, and abnormal pigmentation of the skin and hair. Recent evidence implicates the ubiquitin (Ub) proteolytic system in the degradation of many short-lived key regulatory proteins including c-Fos, c-Jun, MAT-2, GCN4, and ATF-2 [13, 14]. Ubiquitin is covalently attached to cellular proteins through an enzymatic cascade that begins with the ATP-dependent activation of Ub-activating enzyme (E1) to form a high-energy E1–Ub thiolester. Activated Ub is then transferred to the SH group of the active-site cysteine residue of an Ub-conjugating enzyme (E2 or Ubc). Finally, in the presence of an Ub–protein ligase (E3), the C-terminal
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0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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glycine residue of Ub is conjugated (via an isopeptide bond) to the ⑀-amino group of a lysine residue on the protein substrate. Progressive conjugation of Ub, by linkage between a lysine residue on one Ub to the C-terminus of another Ub, results in formation of multiubiquitin chains [15, 16]. Multiubiquitination has been hypothesized to be necessary, but not sufficient, to target proteins for degradation by the 26S proteasome [17]. This pathway of rapid protein modification, translocation, and degradation controls a variety of cellular events, including DNA repair, cell cycle progression, transcription, stress responses, and immunological responses [13, 15, 16, 18]. Despite the importance of the MITF protein in growth and differentiation of pigment cells, little is known about MITF–protein associations and how such associations could affect MITF transcriptional activity. Therefore, we used the yeast two-hybrid system to identify proteins that interact with MITF. hUBC9 was found to specifically associate with MITF and to facilitate MITF–protein degradation. To determine sequences in the MITF molecule associated with its degradation, several strategies were employed. Serine 73 phosphorylation was previously demonstrated to selectively enhance MITF association with the transcriptional coactivator p300 [19]. We demonstrate that S73 is also essential for ubiquitination. We further show that lysine 201 is a potential ubiquitination site. We propose that hUBC9 plays an important role in the control of ubiquitin proteasome-mediated degradation of MITF. MATERIALS AND METHODS Cell culture. The human melanoma cell line IIB-Mel-J was grown and subcultured as previously described [20]. COS7 cells were cultured in Dulbecco’s medium supplemented with 10% fetal bovine serum. Construction of the yeast two-hybrid cDNA library. Poly(A) ⫹ mRNA (5 g) from IIB-Mel-J cells was used to prepare cDNAs fragments with an EcoRI site at their 5⬘ ends and an XhoI site at their 3⬘ ends by standard procedures. The cDNA fragments of 0.5– 3.0 kb were cloned into the HybriZAP, yielding 3 ⫻ 10 6 independent plaques, more than 90% of which contain a cDNA insert. A phagemid cDNA library with the cDNAs fused to the Gal4 activation domain was isolated from the lambda HybriZAP library by in vivo excision with Exassist helper phages, according to the manufacturer’s recommendation (Stratagene, Inc.). Plasmids. The full-length MITF coding region [21] was amplified from the IIB-Mel-J cDNA library using the polymerase chain reaction (PCR) and synthetic oligonucleotides. It was cloned into the Gal4 DNA-binding domain vector pGBT9 (Clontech, Inc.), yielding pGBT9 –MITF. The Gal4 –MITF fusion junction and the full length of MITF were confirmed by DNA sequencing. The truncated MITF bait clone was generated by cutting the full-length MITF cDNA with StuI and PstI. The resulting fragment, which encoded the basic helix-loophelix/leucine zipper (bHLH/ZIP) domain, was cloned into pGBT9 to generate pGBT9 –MITF(S,P). The epitope-tagged MITF mammalian expression vector was constructed by cloning the HindIII/XbaI fulllength MITF (from pBluescript–MITF) into pFLAG-CMV2 (Eastman
Kodak, Inc.) to create pFLAG-CMV2–MITF. The HindIII/XhoI fragment of hUBC9 was generated by PCR and cloned into pFLAGCMV2 to generate pFLAG-CMV2– hUBC9. The EcoRI/XhoI fragment of hUBC9 was inserted into pGEX-4T-1 (Pharmacia, Inc.) to create pGEX-hUBC9. pGEX-hUBC9 expresses an IPTG-inducible glutathione S-transferase (GST) hUBC9 fusion protein in Escherichia coli. The fusion junctions in these vectors were confirmed by DNA sequencing. Yeast two-hybrid system. The Gal4 yeast two-hybrid system was used to detect protein–protein interactions. The yeast strain PJ-X, which carries HIS3, ADE2, and Lac Z reporter genes under the control of Gal4-responsive elements, was used for library screening. The bait clone pGBT9 –MITF(S,P) was used because (unlike pGBT9 – MITF) it did not activate transcription of the reporter genes when expressed alone or with the Gal4 transactivation domain. The bait fusion protein (Gal4 BD–MITF168 –325) was expressed in PJ-X, as assessed by Western blotting using a monoclonal anti-Gal4 DNAbinding domain antibody (Clontech, Inc.). PJ-X expressing pGBT9 – MITF(S,P) was transformed with the IIB-Mel-J two-hybrid cDNA library using lithium acetate, as described by Gietz et al. [22]. Transformants were plated on selective synthetic dextrose-containing medium lacking adenine, tryptophan, and leucine (SD-ATL). Colonies that grew on this medium were tested further for growth on synthetic dextrose medium either lacking histidine, adenine, tryptophan, and leucine (SD-HATL) or lacking histidine, tryptophan, and leucine (SD-HTL) and supplemented with 5 mM 3-aminotriazole. Colonies that grew in the absence of adenine and histidine (⫹5 mM 3-aminotriazole) were analyzed for -galactosidase activity using a liquid assay [23]. cDNA inserts from plasmids that conferred triplepositive (Ade⫹, His⫹, Lac Z⫹) yeast colonies were tested for bait specificity. This was accomplished by retransformation with different Gal4 DNA-binding fusion plasmids and with pGBT9 that lacked an insert. In vitro translation. For in vitro translation of MITF and Ub, we constructed pcDNA3–Ub and pBS–MITF plasmids by inserting fulllength Ub and MITF into the pcDNA3 and pBS vectors, respectively. The resulting plasmids were used as templates for in vitro transcription and translation using TNT T7-coupled rabbit reticulocyte lysates (Promega) under conditions recommended by the supplier. Reaction mixtures contained 25 l of lysate, 40 Ci of [ 35S]methionine (1000 Ci/mmol), 15 units of RNasin (Promega), 50 M amino acid mixture (minus methionine), and 1 g of DNA in a final reaction volume of 50 l. The reactions were incubated for 60 min at 30°C. In vitro binding assays. Bacterially expressed GST– hUBC9, GST–Ub, and GST–sentrin were produced in the E. coli strain BL21 (DE3) upon induction by 1 mM IPTG for 3–5 h at 37°C. Proteins were purified on glutathione–Sepharose 4B beads (Pharmacia, Inc.), according to the manufacturer’s instructions. Purified proteins were dialyzed against 1⫻ PBS (10 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl) overnight at 4°C. Equal amounts (3 g) of purified GST– hUBC9, GST–Ub, and GST–sentrin proteins were incubated with 0.1% NP-40 PBS-buffered 50 l glutathione–Sepharose 4B beads at 4°C for 30 min with gently agitation. 35S-labeled in vitro transcription/translation proteins (5 l, 10% of the total reaction) were added to the GST– hUBC9-, GST– ubiquitin-, and GST–sentrinsaturated glutathione–Sepharose 4B beads at 4°C for 1 h with gentle agitation. The beads were then washed three times with 0.1% NP-40 PBS and boiled in 50 l SDS–PAGE sample buffer. The proteins were separated by 12% SDS–PAGE and exposed to X-ray film. Covalent conjugate formation assay. For covalent conjugate formation between MITF and Ub, 10 l of [ 35S]methionine-labeled MITF and 10 l of in vitro translated Ub were incubated in a solution of 20 mM Tris–HCl, pH 7.52, 50 mM NaCl 2, 10 mM MgCl 2, 0.1 mM DTT, 4 mM ATP, 5 U/ml inorganic pyrophosphatase for 1 h at 37°C. The reaction was stopped by addition of SDS–PAGE sample buffer. Samples were loaded onto a 12% SDS–polyacrylamide gel. Gels were
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UBIQUITINATION OF MITF PROTEIN fixed, treated in an amplification solution (Amersham), dried, and visualized by autoradiography. DNA transfection and Western blotting. Proliferating COS7 cells were transfected with pFLAG-CMV2–MITF, and mutants pFLAGCMV2–MITF(S73A), pFLAG-CMV2–MITF(K201R), and pFLAGCMV2– hUBC9 vectors, using LIPOFECTAMINE reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. After 48 h, cells were treated with or without 50 M MG132 for 2 h. Transfected cells were then lysed with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris–HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 mM dithiothreitol, and 2.5 g/ml aprotinin), passed through a 27-gauge needle, and centrifuged at 14,000g for 10 min. Equal amounts of supernatant protein (20 g) were separated by 12% SDS–PAGE and blotted onto nitrocellulose membranes. The blots were washed with T-TBS buffer (20 mM Tris, 150 mM NaCl, 0.2% Tween 20) and blocked with 5% nonfat milk in T-TBS for 1 h at room temperature. Membranes were then incubated with 3 g/ml of an anti-FLAG M2 antibody (Eastman Kodak, Inc.) for 1 h at room temperature. After washing with T-TBS buffer, the membranes were incubated with sheep anti-mouse IgG conjugated to horseradish peroxidase (1:3000) in T-TBS containing 5% nonfat milk for 1 h at room temperature. After washing, antibody-bound proteins were detected using the ECL Western blotting detection kit (Amersham, Inc.). Site-directed mutagenesis of MITF. Site-directed mutagenesis was performed using deoxyoligonucleotides and a QuikChange sitedirected mutagenesis kit (Stratagene), according to the manufacturer’s specifications. Plasmid pBS–MITF was used as a template. Mutations including S73 to A, K201 to R, K182 to R, and K205 to R were confirmed by DNA sequencing.
RESULTS
Identification of hUBC9 as an MITF Binding Protein by the Yeast Two-Hybrid System and GST Pull-Down Assays Initial experiments showed that MITF fused to the Gal4 DNA-binding domain (pGBT9 –MITF) was a strong transactivator of reporter genes in the yeast two-hybrid system, as previously reported [24]. Thus, the full-length MITF was not suitable as bait in the yeast system. We therefore constructed a series of MITF fragments fused to the Gal4 DNA-binding domain and tested them for intrinsic transactivation ac-
FIG. 1. Schematic diagram of the structure of MITF protein. (A) Wild-type basic-helix-loop-helix/leucine zipper MITF protein. (B) Truncated MITF (MITF 168 –325) that was fused to the Gal4 DNAbinding domain and used as bait in the yeast two-hybrid system (see Materials and Methods for construction).
TABLE 1 Retransformation Assay of Yeast (Strain PJ-X) with MITF, hUBC9, and Control Plasmids Growth
Gal4 DBD
Gal4 TD
SD-TL
SD-HATL
SD-HTL (5 mM 3-AT)
p53 Lamin C p53 pGBT9–MITF(S,P) pGBT9–MITF(S,P) None pGBT9–MITF(S,P)
SV40T SV40T hUBC9 SV40T None hUBC9 hUBC9
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹⫹
Note. (⫺) indicates failure to grow and (⫹⫹⫹⫹) indicates robust growth. SD-TL, SD-HATL, and SD-HTL (5 mM 3-AT) are synthetic dextrose (SD) medium lacking tryptophan (T), leucine (L), histidine (H), and adenine (A) or supplemented with 5 mM 3-aminotriazole. DBD, DNA-binding domain fusion protein; TD, transactivation domain fusion protein.
tivity. The largest MITF fragment that failed to activate the reporter genes encompassed amino acids 168 – 325, which included the bHLH/ZIP domain. Figure 1 shows the structure of the full-length and bait MITF proteins. The plasmid containing the truncated MITF fragment (amino acids 168 –325) fused to the Gal4 DNA-binding domain [pGBT9 –MITF(S,P)] was used to screen a two-hybrid library in which cDNAs from human melanoma IIB-Mel-J cells were fused to the Gal4 transactivation domain. Approximately 8 ⫻ 10 6 yeast transformants were tested for growth on synthetic dextrose medium (SDATL), lacking tryptophan and leucine, to select for the presence of bait and library plasmids, and lacking adenine, to select for plasmids encoding polypeptides that interact with the Gal4 –MITF 168 –325 bait protein. Of 32 large colonies, 25 grew on selective synthetic dextrose medium lacking histidine, adenine, tryptophan, and leucine (SD-HATL) and containing 5 mM 3-aminotriazole, indicating expression of the ADE2 and HIS3 reporter genes. These colonies also expressed the Lac Z reporter, as assessed by -galactosidase activity assays. Library plasmids were rescued from these colonies, and the cDNA inserts from 20 of them were sequenced. Sequence analysis showed that 15 of the inserts were from the same gene. A GenBank database search showed that the insert sequences were identical to the human ubiquitin-conjugating enzyme hUBC9 [25–32]. Retransformation assays showed that the hUBC9 fusion protein interacted specifically with MITF, because no interaction was detected with either the Gal4-DNA-Binding Domain (DBD) vector alone or the Gal4-DBD-p53, which produces a p53 fusion protein (Table 1). From these experiments, we conclude
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Association of MITF with hUBC9 Causes Its Degradation through the Ubiquitin-Dependent Proteasome Pathway
FIG. 2. MITF interacts with hUBC9 and forms ubiquitin/MITF conjugates. (A) Equal amounts (3 g) of purified GST (lane 2) or GST– hUBC9 (lane 3) protein were bound to glutathione–Sepharose 4B beads and incubated with [ 35S]methionine-labeled MITF produced by in vitro transcription/translation. Proteins bound to the washed beads were boiled in SDS–PAGE sample buffer, analyzed by SDS–PAGE, and exposed to X-ray film overnight. Lane 1 is 10% of the MITF in vitro translation product. (B) Equal amounts (3 g) of purified GST, GST– ubiquitin, or GST–sentrin proteins were bound to glutathione–Sepharose 4B beads and incubated with [ 35S]methionine, in vitro translated MITF at 4°C for 60 min with gentle agitation. The beads were then washed three times with 0.1% NP-40 PBS and boiled in 50 l SDS–PAGE sample buffer. The proteins were separated by 12% SDS–PAGE and exposed to X-ray film.
that the MITF– hUBC9 interaction is specific in the yeast-two hybrid system. To confirm the interaction between MITF and hUBC9, we subcloned the hUBC9 cDNA into the pGEX-4T-1 vector to produce a GST– hUBC9 fusion protein after IPTG induction in E. coli cells. Bacterially expressed GST– hUBC9 was purified, immobilized on glutathione–Sepharose 4B beads, and tested for its ability to bind [ 35S]methionine-labeled, in vitro translated MITF protein. As expected, MITF could be precipitated by GST– hUBC9 (Fig. 2A, lane 3) but not with GST alone (Fig. 2A, lane 2), confirming that the interaction between hUBC9 and MITF is specific. hUBC9 is believed to act as an E2-conjugating enzyme for the small ubiquitin-like protein sentrin/ SUMO in the sentrinization pathway [33–37]. However, hUBC9 is also involved in the ubiquitination/ degradation of transcription factors such as E2A [25] and ATF2 [14]. In vitro transcription/translation analysis demonstrated greater association of MITF with GST– ubiquitin than with GST–sentrin (Fig. 2B, lanes 3 and 4). In addition, in vitro translated MITF protein showed that it preferentially formed conjugates with ubiquitin, rather than sentrin/SUMO (data not shown).
Association of MITF with hUBC9 raised the possibility that MITF could be degraded through an hUBC9dependent, proteasome degradation pathway. To investigate this possibility, COS7 cells were transfected with pFLAG-CMV2–MITF alone or together with pFLAG-CMV2– hUBC9. Forty-eight hours after transfection, the cells were treated for 2 h with either the proteasome inhibitor MG132 or the solvent DMSO as a control. Cells were then solubilized and analyzed by SDS–PAGE and Western blotting with an anti-FLAG antibody. In the absence of exogenously expressed hUBC9, FLAG-tagged MITF levels showed a small increase upon addition of the proteasome inhibitor (Fig. 3A, lanes 1 and 2). By contrast, almost no FLAGtagged MITF was detected in the presence of hUBC9 (Fig. 3A, lane 3). This striking hUBC9-dependent de-
FIG. 3. hUBC9 alters MITF levels in a proteasome-dependent manner. (A) COS7 cells were transfected with either 4 g of pFLAGCMV2–MITF only (lanes 1 and 2) or 4 g pFLAG-CMV2–MITF together with 2 g pFLAG-CMV2– hUBC9 (lanes 3 and 4). Fortyeight hours after transfection, cells were treated for 2 h with 50 M of the proteasome inhibitor MG132 or with DMSO (MG132 solvent). Whole-cell extracts were then prepared as described under Materials and Methods. Equal amounts of supernatant proteins were analyzed by SDS–PAGE and Western blotting using the anti-FLAG antibody M2. The FLAG-tagged MITF and FLAG-tagged hUBC9 protein bands are indicated by arrowheads. Positions of protein molecular mass standards (in kDa) are indicated on the left. (B) COS7 cells were transfected with 4 g pFLAG-CMV2–MITF (lane 1) and the indicated amount of pFLAG-CMV2– hUBC9 (lanes 2– 8). After 48 h, one sample (lane 7) was treated for 2 h with the proteasome inhibitor MG132. Cell extracts were prepared and analyzed by SDS–PAGE and Western blotting using an anti-FLAG antibody. The FLAGtagged MITF and FLAG-tagged hUBC9 protein bands are indicated by arrowheads.
UBIQUITINATION OF MITF PROTEIN
cline in MITF levels was partially rescued by addition of the proteasome inhibitor MG132 (Fig. 3A, lane 4). These results were further confirmed by cotransfecting increasing amounts of a FLAG-tagged hUBC9 plasmid and a fixed amount of FLAG-tagged MITF plasmid (Fig. 3B). The steady-state level of FLAG-tagged MITF declined with increasing hUBC9 protein levels in a dose-dependent manner. In COS7 cells transfected with FLAG-tagged MITF (Fig. 3B), the anti-FLAG antibody detected two proteins (molecular weights of 55 and 59 kDa). These proteins corresponded in molecular weight to two differentially phosphorylated MITF species previously identified in human melanoma cells [19]. Interestingly, the 59-kDa protein, which can transactivate a luciferase reporter driven by the tyrosinase promoter [19], appeared to be more susceptible to degradation by hUBC9 than the 55-kDa protein, especially at low levels of hUBC9 (Fig. 3B, lanes 2– 6). Higher levels of hUBC9 eventually resulted in loss of both the 55- and 59-kDa proteins. As observed in Fig. 3A, addition of the proteasome inhibitor MG132 almost completely rescued MITF. Taken together with the stabilization of MITF observed with proteasome inhibitor MG132, these results strongly suggest that phosphorylated MITF is targeted for proteolysis by the ubiquitin-dependent proteasome pathway through association with hUBC9. The Serine 73 (S73) Phosphorylation Site, Located in a PEST-like Sequence, Is Required for MITF Ubiquitination and hUBC9-Dependent Degradation Short-lived proteins generally contain regions enriched with Pro, Glu, Ser, and Thr residues (PEST sequences) [38]. PEST elements rich in S/TP sequences are the minimum consensus phosphorylation sites for some protein kinases, such as the cyclin-dependent kinase [39]. MITF has been shown to be phosphorylated at S73, which is within the classical PEST sequence PPVPGSSAPNSP. We used point mutation analysis to investigate the role of this phosphorylation site in MITF. In vitro transcription/translation demonstrated that a serine to alanine mutation at amino acid 73 (S73A) almost completely abolished MITF ubiquitination (Fig. 4A). This suggests that S73 phosphorylation may be essential for MITF ubiquitination. To investigate this possibility, COS7 cells were transfected with a MITFS73A mutant alone or together with pFLAG-CMV2– hUBC9. In contrast to what was observed with the wild-type MITF protein (Fig. 3B), the anti-FLAG antibody detected only one MITFS73A protein band, which was resistant to UBC9-mediated degradation (Fig. 4B). Consistent with these results and in contrast to that observed with the wild-type MITF (Fig. 3, lane 7), addition of MG132 did not increase MITF(S73A) levels (Fig. 4, lane 5). These results
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FIG. 4. The serine 73 (S73) phosphorylation site is essential for MITF ubiquitination and hUBC9-dependent degradation. (A) In vitro translated wild-type (lane 1) and S73 mutant MITF (MITFS73A) (lane 2) were incubated with in vitro translated ubiquitin for 60 min at 30°C. The samples were then washed and separated by SDS–PAGE and visualized by autoradiography. (B) FLAGMITFS73A mutant was transfected in COS7 cells with (lanes 2– 4) or without (lane 1) increasing amounts of hUBC9, treated with (lane 5) or without (lanes 1– 4) MG132. Whole-cell extracts were made and Western blotted with an anti-FLAG antibody.
strongly suggest that S73 is critical for MITF ubiquitination in vivo. Identification of MITF Lysine 201 (K201) as a Potential Ubiquitination Site Protein modification by ubiquitination targets proteins for degradation by the 26S proteasome. Initially, ubiquitin is activated by an ATP-dependent pathway in a reaction catalyzed by the enzyme E1. Activated ubiquitin is then transferred to the E2 ubiquitin-conjugating enzyme, which catalyzes formation of an isopeptide bond between the C-terminal glycine of ubiquitin and the ⑀-amino group of a lysine residue on the target protein [15]. MITF contains 21 lysine residues that are distributed throughout the entire molecule. We attempted to identify the ubiquitination site(s) by constructing lysine to arginine substitutions spanning the MITF molecule. Wild-type and mutated MITF proteins were translated in vitro and incubated with ubiquitin. Ubiquitinated products were observed from wild-type MITF and from K182R and K205R mutant proteins (Fig. 5A). However, ubiquitination was almost completely abolished when the lysine residue at position 201 was replaced by arginine (Fig. 5A, lane 3), suggesting that this lysine residue is the target for ubiquitin conjugation. These results were confirmed in vivo by cotransfecting COS7 cells with the
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FIG. 5. Identification of MITF lysine 201 (K201) as a potential ubiquitination site. (A) In vitro translated wild-type (lane 1) and MITF mutant proteins (K182R, K201R, K205R) (lanes 2, 3, and 4) were incubated with in vitro translated ubiquitin for 60 min at 30°C. The samples were then separated by SDS–PAGE and visualized by autoradiography. Lane 5 shows in vitro translated wild-type MITF in the absence of ubiquitin. (B) FLAG-MITFK201R mutant was transfected in COS7 cells with (lanes 2– 4) or without (lane 1) increasing amounts of hUBC9, treated with (lane 5) or without (lanes 1– 4) MG132. Whole-cell extracts were made and Western blotted with an anti-FLAG antibody.
MITFK201R mutant alone or together with pFLAGCMV2– hUBC9. In contrast to that observed with the MITFS73A mutant (Fig. 4B), the anti-FLAG antibody detected two mutant MITFK201R bands which have an identical molecular weight as the wild-type MITF protein (Fig. 5B). Importantly, both MITFK201R isoforms were resistant to hUBC9-mediated degradation (Fig. 5B). Altogether, our results demonstrate that K201 is required for MITF ubiquitination. DISCUSSION
MITF regulates melanocyte differentiation and/or survival. Mutations at the mouse micropthalmia locus (mi) affect the development of melanocytes and several other cell types such as mast cells, osteoclasts, and retinal pigmented epithelial cells [40]. The mouse MITF protein interacts with the retinoblastoma tumor suppressor protein (pRb) [41] and with the coactivator p300 [42]. Using a cDNA library from human melanoma cells, we have identified hUBC9 as the only positive interacting protein for the human truncated micropthalmia protein MITF (MITF 168 –325). The yeast two-hybrid screen did not identify pRb or p300 as MITF 168 –325-interacting proteins. Because MITF and pRb have intrinsic Gal4 transactivation activities in the yeast system, we used the full-length
MITF coding region fused to the Gal4 transactivation domain and an N-terminal truncated pRb protein fused to the Gal4 DNA-binding domain to determine MITF–pRb interactions in yeast. No positive interaction was observed using these constructs, nor was interaction detected by coimmunoprecipitation (data not shown). These negative results may indicate either that MITF–pRb interact in a transient or weak manner or that such an interaction does not occur in human melanocytes. hUBC9 protein has been highly conserved throughout evolution. hUBC9 shares 75 and 82% amino acid similarity with the products of the yeast genes UBC9 (Saccharomyces cerevisiae) and hus5 (S. pombe), respectively [43, 44], and is 100% identical to the product of the mouse UBC9 gene [45]. A functional complementation assay showed that human UBC9 can substitute for the S. cerevisiae UBC9 gene, which is required for cell cycle progression [28]. In yeast, B-type-cyclin degradation is mediated by UBC9 [43]. Furthermore, repression of UBC9 prevents cell-cycle progression and causes yeast growth arrest in the G2 or early M phase [43]. The function of UBC9 in the G2/M phase could be to regulate levels of centromere proteins that direct chromosome segregation and mitosis through interaction with the centromere DNA-binding core complex CBF3 [27]. The crystal structure of the mammalian mUBC9 protein [46] revealed a number of unique features, including a protruding surface loop and a strong overall electrostatic dipole. Such features may have a role in allowing mUBC9 to interact with an exceptionally large variety of proteins. Nuclear magnetic resonance measurements show that two regions in murine/human UBC9 are likely to be important in protein–protein recognition in the ubiquitin pathway. The region near the C-terminus, which undergoes conformational change, may be important for UBC9 –substrate binding and catalytic activity [47]. Proteins that have been shown to interact with mUBC9 include the human papillomavirus type 16 E1 replication protein [28], the glucocorticoid receptor, c-Jun [30], the human Fas (CD95) protein [26], the Rad 51 recombination protein [31], the adenovirus E1A protein [45], the transcription factor ETS-1 [32], the helix-loop-helix protein E2A [25], and the transcription factor MITF (this communication). hUBC9-mediated MITF, E2A, and ATF2 [14, 25, 48] degradation stand in contrast to recent work showing that hUBC9 probably forms conjugates with ubiquitin-like proteins such as SUMO [33–37]. Modification of Ran GTPase protein by sentrin/SUMO requires UBC9 for nuclear transport [49]. However, using an in vitro ubiquitination assay, we demonstrated that MITF is preferentially ubiquitinized rather than sentrinized. Thereby, hUBC9 association with MITF ap-
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pears to be necessary for MITF recognition by the ubiquitin pathway. Phosphorylation of MITF does not alter its nuclear localization, DNA binding, or dimerization; however, phosphorylation facilitates association with the transcriptional coactivator p300 [19]. We have demonstrated that S73 phosphorylation favors MITF ubiquitination in vitro. Moreover, in vivo studies demonstrated that hUBC9 preferentially targets the 59-kDa MITF protein, previously demonstrated to be transcriptionally active, S73-phosphorylated MITF [19]. Recently, Ser 298, which locates downstream of the bHLH/ZIP, was found to be phosphorylated in melanoma cells [50]. However, S298 does not appear to be critical for MITF degradation since S73A mutation was sufficient to create a stable, 55-kDa protein. We propose that whereas S73 phosphorylation may trigger MITF activation, it may also provide a mechanism for degradation by active proteolysis. Phosphorylation and dephosphorylation have frequently been coupled with the ubiquitin-dependent proteasome pathway, since many substrates contain the acidic PEST sequences [39]. Activation of NFB by IB␣ ubiquitination and consequent degradation requires phosphorylation of S32 and S36 in the IB␣ molecule [51, 52]. Specific inhibition of the proteolytic activity results in accumulation of ubiquitinated forms of IB␣, indicating that IB␣ is targeted for degradation by a phosphorylation-dependent ubiquitination process [53]. The precise mechanism for how a PEST sequence is recognized by the ubiquitin-dependent proteasome pathway is not known. Protein phosphorylation may induce a conformational change that unmasks regions required for proteolysis [54]. Within this hypothesis, it is then likely that phosphorylation on S73 favors hUBC9 association and targets MITF protein for ubiquitination on K201. Importantly, K201 resides within the DNA-binding domain of the MITF protein. Thus, K201 ubiquitination may inhibit MITF transcriptional activity before it is recognized by the proteasome complex. Targeting phosphorylated MITF for proteasome degradation may not require its nuclear export since we have found that hUBC9 localizes in both the nuclear and cytoplasmic compartments (data not shown). This feature, in addition to the molecular structure [46], should explain hUBC9 multifunctionality and accessibility to a variety of cytoplasmic and nuclear partners. MITF overexpression results in apoptosis of mast cells [55]. Importantly, MITF protein levels increase during the commitment to differentiation but are almost undetectable in senescent human normal melanocytes in culture [56]. In these senescent melanocytes, absence of MITF protein was not accompanied by reduction in its mRNA levels, suggesting active degra-
dation of the MITF protein (unpublished results). It is thus likely that MITF levels are also critically regulated during development and differentiation of the melanocyte lineage. In addition, downregulation of melanin production and expression of a poorly or nondifferentiated phenotype is a common feature of metastatic human melanomas, both in vivo and in tissue culture. Our results raise the possibility that MITF– hUBC9 interaction and consequent MITF degradation may contribute to the poorly differentiated, nonpigmented phenotype of human metastatic melanoma tumors. We are grateful to Amey Herrin for assistance with tissue culture. This work was supported by Grants AG00594 and AG13663 (E.E.M) and AG09909 and AG11658 (J.C). M.M.H. was supported by a Training Grant (AG00183) from the National Institute on Aging.
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Regulation of Microphthalmia-Associated Transcription Factor MITF Protein Levels by Association with the Ubiquitin-Conjugating Enzyme hUBC9 Weidong Xu,* ,† ,‡ Limin Gong,§ Maher M. Haddad,* ,† ,‡ Oliver Bischof, ¶ Judith Campisi, ¶ Edward T. H. Yeh,§ and Estela E. Medrano* ,† ,‡ ,1 *Roy M. and Phyllis Gough Huffington Center on Aging and †Department of Cell Biology and ‡Department of Dermatology, Baylor College of Medicine, One Baylor Plaza M320 and VAMC, Houston, Texas 77030; §Institute of Molecular Medicine, University of Texas– Houston Health Science Center, Houston, Texas 77030; and ¶Department of Cell and Molecular Biology, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720
The basic helix-loop-helix/leucine zipper (bHLH/ ZIP) microphthalmia-associated transcription factor (MITF) regulates transcription of genes encoding enzymes essential for melanin biosynthesis in melanocytes and retinal pigmented epithelial cells. To determine how MITF activity is regulated, we used the yeast two-hybrid system to identify proteins expressed by human melanoma cells that interact with MITF. The majority of clones that showed positive interaction with a 158-amino-acid region of MITF containing the bHLH/ZIP domain (aa 168 –325) encoded the ubiquitin conjugating enzyme hUBC9. The association of MITF with hUBC9 was further confirmed by an in vitro GST pull-down assay. Although hUBC9 is known to interact preferentially with SENTRIN/ SUMO1, in vitro transcription/translation analysis demonstrated greater association of MITF with ubiquitin than with SENTRIN. Importantly, cotransfection of MITF and hUBC9 expression vectors resulted in MITF protein degradation. MITF protein was stabilized by the proteasome inhibitor MG132, indicating the role of the ubiquitin–proteasome system in MITF degradation. Serine 73, which is located in a region rich in proline, glutamic acid, serine, and threonine (PEST), regulates MITF protein stability, since a serine to alanine mutation prevented hUBC9-mediated MITF (S73A) degradation. Furthermore, we identified lysine 201 as a potential ubiquitination site. A lysine to arginine mutation abolished MITF (K201R) degradation by hUBC9 in vivo. Our experiments indicate that by targeting MITF for proteasome degradation, hUBC9 is a critical regulator of melanocyte differentiation. © 2000 Academic Press 1 To whom correspondence and reprint requests should be addressed at VA Medical Center, Research Building 110, 2002 Holcombe Boulevard, Houston, TX 77030. Fax: (713) 794-7938. E-mail: [email protected].
Key Words: MITF; ubiquitin; SUMO; melanocytes; differentiation.
INTRODUCTION
The mitf gene, originally cloned from a transgenic insertional mutation at the mouse microphthalmia locus [1, 2], encodes a basic-helix-loop-helix/leucine zipper protein. MITF is believed to promote the entrance of neural crest cells into the melanocyte differentiation pathway [3]. Melanin production is restricted to differentiated melanocytes and retinal pigmented epithelial cells, derived from the neural crest and neuroectoderm, respectively [4, 5]. MITF regulates expression of the Kit receptor [3] and the tyrosinase and tyrosinaserelated protein 1 (TRP1) enzymes [6], which are essential for melanin production. Tyrosinase is the ratelimiting enzyme in melanin biosynthesis, catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and then to DOPA quinone [7–9]. TRP1 catalyzes the conversion of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) to indole-5,6-quinonecarboxylic acid [10, 11]. Mutations in MITF are responsible for Waardenburg syndrome type 2 [12], a dominantly inherited disorder characterized by sensorineural hearing loss, heterochromia iridis, and abnormal pigmentation of the skin and hair. Recent evidence implicates the ubiquitin (Ub) proteolytic system in the degradation of many short-lived key regulatory proteins including c-Fos, c-Jun, MAT-2, GCN4, and ATF-2 [13, 14]. Ubiquitin is covalently attached to cellular proteins through an enzymatic cascade that begins with the ATP-dependent activation of Ub-activating enzyme (E1) to form a high-energy E1–Ub thiolester. Activated Ub is then transferred to the SH group of the active-site cysteine residue of an Ub-conjugating enzyme (E2 or Ubc). Finally, in the presence of an Ub–protein ligase (E3), the C-terminal
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glycine residue of Ub is conjugated (via an isopeptide bond) to the ⑀-amino group of a lysine residue on the protein substrate. Progressive conjugation of Ub, by linkage between a lysine residue on one Ub to the C-terminus of another Ub, results in formation of multiubiquitin chains [15, 16]. Multiubiquitination has been hypothesized to be necessary, but not sufficient, to target proteins for degradation by the 26S proteasome [17]. This pathway of rapid protein modification, translocation, and degradation controls a variety of cellular events, including DNA repair, cell cycle progression, transcription, stress responses, and immunological responses [13, 15, 16, 18]. Despite the importance of the MITF protein in growth and differentiation of pigment cells, little is known about MITF–protein associations and how such associations could affect MITF transcriptional activity. Therefore, we used the yeast two-hybrid system to identify proteins that interact with MITF. hUBC9 was found to specifically associate with MITF and to facilitate MITF–protein degradation. To determine sequences in the MITF molecule associated with its degradation, several strategies were employed. Serine 73 phosphorylation was previously demonstrated to selectively enhance MITF association with the transcriptional coactivator p300 [19]. We demonstrate that S73 is also essential for ubiquitination. We further show that lysine 201 is a potential ubiquitination site. We propose that hUBC9 plays an important role in the control of ubiquitin proteasome-mediated degradation of MITF. MATERIALS AND METHODS Cell culture. The human melanoma cell line IIB-Mel-J was grown and subcultured as previously described [20]. COS7 cells were cultured in Dulbecco’s medium supplemented with 10% fetal bovine serum. Construction of the yeast two-hybrid cDNA library. Poly(A) ⫹ mRNA (5 g) from IIB-Mel-J cells was used to prepare cDNAs fragments with an EcoRI site at their 5⬘ ends and an XhoI site at their 3⬘ ends by standard procedures. The cDNA fragments of 0.5– 3.0 kb were cloned into the HybriZAP, yielding 3 ⫻ 10 6 independent plaques, more than 90% of which contain a cDNA insert. A phagemid cDNA library with the cDNAs fused to the Gal4 activation domain was isolated from the lambda HybriZAP library by in vivo excision with Exassist helper phages, according to the manufacturer’s recommendation (Stratagene, Inc.). Plasmids. The full-length MITF coding region [21] was amplified from the IIB-Mel-J cDNA library using the polymerase chain reaction (PCR) and synthetic oligonucleotides. It was cloned into the Gal4 DNA-binding domain vector pGBT9 (Clontech, Inc.), yielding pGBT9 –MITF. The Gal4 –MITF fusion junction and the full length of MITF were confirmed by DNA sequencing. The truncated MITF bait clone was generated by cutting the full-length MITF cDNA with StuI and PstI. The resulting fragment, which encoded the basic helix-loophelix/leucine zipper (bHLH/ZIP) domain, was cloned into pGBT9 to generate pGBT9 –MITF(S,P). The epitope-tagged MITF mammalian expression vector was constructed by cloning the HindIII/XbaI fulllength MITF (from pBluescript–MITF) into pFLAG-CMV2 (Eastman
Kodak, Inc.) to create pFLAG-CMV2–MITF. The HindIII/XhoI fragment of hUBC9 was generated by PCR and cloned into pFLAGCMV2 to generate pFLAG-CMV2– hUBC9. The EcoRI/XhoI fragment of hUBC9 was inserted into pGEX-4T-1 (Pharmacia, Inc.) to create pGEX-hUBC9. pGEX-hUBC9 expresses an IPTG-inducible glutathione S-transferase (GST) hUBC9 fusion protein in Escherichia coli. The fusion junctions in these vectors were confirmed by DNA sequencing. Yeast two-hybrid system. The Gal4 yeast two-hybrid system was used to detect protein–protein interactions. The yeast strain PJ-X, which carries HIS3, ADE2, and Lac Z reporter genes under the control of Gal4-responsive elements, was used for library screening. The bait clone pGBT9 –MITF(S,P) was used because (unlike pGBT9 – MITF) it did not activate transcription of the reporter genes when expressed alone or with the Gal4 transactivation domain. The bait fusion protein (Gal4 BD–MITF168 –325) was expressed in PJ-X, as assessed by Western blotting using a monoclonal anti-Gal4 DNAbinding domain antibody (Clontech, Inc.). PJ-X expressing pGBT9 – MITF(S,P) was transformed with the IIB-Mel-J two-hybrid cDNA library using lithium acetate, as described by Gietz et al. [22]. Transformants were plated on selective synthetic dextrose-containing medium lacking adenine, tryptophan, and leucine (SD-ATL). Colonies that grew on this medium were tested further for growth on synthetic dextrose medium either lacking histidine, adenine, tryptophan, and leucine (SD-HATL) or lacking histidine, tryptophan, and leucine (SD-HTL) and supplemented with 5 mM 3-aminotriazole. Colonies that grew in the absence of adenine and histidine (⫹5 mM 3-aminotriazole) were analyzed for -galactosidase activity using a liquid assay [23]. cDNA inserts from plasmids that conferred triplepositive (Ade⫹, His⫹, Lac Z⫹) yeast colonies were tested for bait specificity. This was accomplished by retransformation with different Gal4 DNA-binding fusion plasmids and with pGBT9 that lacked an insert. In vitro translation. For in vitro translation of MITF and Ub, we constructed pcDNA3–Ub and pBS–MITF plasmids by inserting fulllength Ub and MITF into the pcDNA3 and pBS vectors, respectively. The resulting plasmids were used as templates for in vitro transcription and translation using TNT T7-coupled rabbit reticulocyte lysates (Promega) under conditions recommended by the supplier. Reaction mixtures contained 25 l of lysate, 40 Ci of [ 35S]methionine (1000 Ci/mmol), 15 units of RNasin (Promega), 50 M amino acid mixture (minus methionine), and 1 g of DNA in a final reaction volume of 50 l. The reactions were incubated for 60 min at 30°C. In vitro binding assays. Bacterially expressed GST– hUBC9, GST–Ub, and GST–sentrin were produced in the E. coli strain BL21 (DE3) upon induction by 1 mM IPTG for 3–5 h at 37°C. Proteins were purified on glutathione–Sepharose 4B beads (Pharmacia, Inc.), according to the manufacturer’s instructions. Purified proteins were dialyzed against 1⫻ PBS (10 mM sodium phosphate buffer, pH 7.4, 150 mM NaCl) overnight at 4°C. Equal amounts (3 g) of purified GST– hUBC9, GST–Ub, and GST–sentrin proteins were incubated with 0.1% NP-40 PBS-buffered 50 l glutathione–Sepharose 4B beads at 4°C for 30 min with gently agitation. 35S-labeled in vitro transcription/translation proteins (5 l, 10% of the total reaction) were added to the GST– hUBC9-, GST– ubiquitin-, and GST–sentrinsaturated glutathione–Sepharose 4B beads at 4°C for 1 h with gentle agitation. The beads were then washed three times with 0.1% NP-40 PBS and boiled in 50 l SDS–PAGE sample buffer. The proteins were separated by 12% SDS–PAGE and exposed to X-ray film. Covalent conjugate formation assay. For covalent conjugate formation between MITF and Ub, 10 l of [ 35S]methionine-labeled MITF and 10 l of in vitro translated Ub were incubated in a solution of 20 mM Tris–HCl, pH 7.52, 50 mM NaCl 2, 10 mM MgCl 2, 0.1 mM DTT, 4 mM ATP, 5 U/ml inorganic pyrophosphatase for 1 h at 37°C. The reaction was stopped by addition of SDS–PAGE sample buffer. Samples were loaded onto a 12% SDS–polyacrylamide gel. Gels were
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UBIQUITINATION OF MITF PROTEIN fixed, treated in an amplification solution (Amersham), dried, and visualized by autoradiography. DNA transfection and Western blotting. Proliferating COS7 cells were transfected with pFLAG-CMV2–MITF, and mutants pFLAGCMV2–MITF(S73A), pFLAG-CMV2–MITF(K201R), and pFLAGCMV2– hUBC9 vectors, using LIPOFECTAMINE reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. After 48 h, cells were treated with or without 50 M MG132 for 2 h. Transfected cells were then lysed with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris–HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 mM dithiothreitol, and 2.5 g/ml aprotinin), passed through a 27-gauge needle, and centrifuged at 14,000g for 10 min. Equal amounts of supernatant protein (20 g) were separated by 12% SDS–PAGE and blotted onto nitrocellulose membranes. The blots were washed with T-TBS buffer (20 mM Tris, 150 mM NaCl, 0.2% Tween 20) and blocked with 5% nonfat milk in T-TBS for 1 h at room temperature. Membranes were then incubated with 3 g/ml of an anti-FLAG M2 antibody (Eastman Kodak, Inc.) for 1 h at room temperature. After washing with T-TBS buffer, the membranes were incubated with sheep anti-mouse IgG conjugated to horseradish peroxidase (1:3000) in T-TBS containing 5% nonfat milk for 1 h at room temperature. After washing, antibody-bound proteins were detected using the ECL Western blotting detection kit (Amersham, Inc.). Site-directed mutagenesis of MITF. Site-directed mutagenesis was performed using deoxyoligonucleotides and a QuikChange sitedirected mutagenesis kit (Stratagene), according to the manufacturer’s specifications. Plasmid pBS–MITF was used as a template. Mutations including S73 to A, K201 to R, K182 to R, and K205 to R were confirmed by DNA sequencing.
RESULTS
Identification of hUBC9 as an MITF Binding Protein by the Yeast Two-Hybrid System and GST Pull-Down Assays Initial experiments showed that MITF fused to the Gal4 DNA-binding domain (pGBT9 –MITF) was a strong transactivator of reporter genes in the yeast two-hybrid system, as previously reported [24]. Thus, the full-length MITF was not suitable as bait in the yeast system. We therefore constructed a series of MITF fragments fused to the Gal4 DNA-binding domain and tested them for intrinsic transactivation ac-
FIG. 1. Schematic diagram of the structure of MITF protein. (A) Wild-type basic-helix-loop-helix/leucine zipper MITF protein. (B) Truncated MITF (MITF 168 –325) that was fused to the Gal4 DNAbinding domain and used as bait in the yeast two-hybrid system (see Materials and Methods for construction).
TABLE 1 Retransformation Assay of Yeast (Strain PJ-X) with MITF, hUBC9, and Control Plasmids Growth
Gal4 DBD
Gal4 TD
SD-TL
SD-HATL
SD-HTL (5 mM 3-AT)
p53 Lamin C p53 pGBT9–MITF(S,P) pGBT9–MITF(S,P) None pGBT9–MITF(S,P)
SV40T SV40T hUBC9 SV40T None hUBC9 hUBC9
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹⫹
Note. (⫺) indicates failure to grow and (⫹⫹⫹⫹) indicates robust growth. SD-TL, SD-HATL, and SD-HTL (5 mM 3-AT) are synthetic dextrose (SD) medium lacking tryptophan (T), leucine (L), histidine (H), and adenine (A) or supplemented with 5 mM 3-aminotriazole. DBD, DNA-binding domain fusion protein; TD, transactivation domain fusion protein.
tivity. The largest MITF fragment that failed to activate the reporter genes encompassed amino acids 168 – 325, which included the bHLH/ZIP domain. Figure 1 shows the structure of the full-length and bait MITF proteins. The plasmid containing the truncated MITF fragment (amino acids 168 –325) fused to the Gal4 DNA-binding domain [pGBT9 –MITF(S,P)] was used to screen a two-hybrid library in which cDNAs from human melanoma IIB-Mel-J cells were fused to the Gal4 transactivation domain. Approximately 8 ⫻ 10 6 yeast transformants were tested for growth on synthetic dextrose medium (SDATL), lacking tryptophan and leucine, to select for the presence of bait and library plasmids, and lacking adenine, to select for plasmids encoding polypeptides that interact with the Gal4 –MITF 168 –325 bait protein. Of 32 large colonies, 25 grew on selective synthetic dextrose medium lacking histidine, adenine, tryptophan, and leucine (SD-HATL) and containing 5 mM 3-aminotriazole, indicating expression of the ADE2 and HIS3 reporter genes. These colonies also expressed the Lac Z reporter, as assessed by -galactosidase activity assays. Library plasmids were rescued from these colonies, and the cDNA inserts from 20 of them were sequenced. Sequence analysis showed that 15 of the inserts were from the same gene. A GenBank database search showed that the insert sequences were identical to the human ubiquitin-conjugating enzyme hUBC9 [25–32]. Retransformation assays showed that the hUBC9 fusion protein interacted specifically with MITF, because no interaction was detected with either the Gal4-DNA-Binding Domain (DBD) vector alone or the Gal4-DBD-p53, which produces a p53 fusion protein (Table 1). From these experiments, we conclude
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Association of MITF with hUBC9 Causes Its Degradation through the Ubiquitin-Dependent Proteasome Pathway
FIG. 2. MITF interacts with hUBC9 and forms ubiquitin/MITF conjugates. (A) Equal amounts (3 g) of purified GST (lane 2) or GST– hUBC9 (lane 3) protein were bound to glutathione–Sepharose 4B beads and incubated with [ 35S]methionine-labeled MITF produced by in vitro transcription/translation. Proteins bound to the washed beads were boiled in SDS–PAGE sample buffer, analyzed by SDS–PAGE, and exposed to X-ray film overnight. Lane 1 is 10% of the MITF in vitro translation product. (B) Equal amounts (3 g) of purified GST, GST– ubiquitin, or GST–sentrin proteins were bound to glutathione–Sepharose 4B beads and incubated with [ 35S]methionine, in vitro translated MITF at 4°C for 60 min with gentle agitation. The beads were then washed three times with 0.1% NP-40 PBS and boiled in 50 l SDS–PAGE sample buffer. The proteins were separated by 12% SDS–PAGE and exposed to X-ray film.
that the MITF– hUBC9 interaction is specific in the yeast-two hybrid system. To confirm the interaction between MITF and hUBC9, we subcloned the hUBC9 cDNA into the pGEX-4T-1 vector to produce a GST– hUBC9 fusion protein after IPTG induction in E. coli cells. Bacterially expressed GST– hUBC9 was purified, immobilized on glutathione–Sepharose 4B beads, and tested for its ability to bind [ 35S]methionine-labeled, in vitro translated MITF protein. As expected, MITF could be precipitated by GST– hUBC9 (Fig. 2A, lane 3) but not with GST alone (Fig. 2A, lane 2), confirming that the interaction between hUBC9 and MITF is specific. hUBC9 is believed to act as an E2-conjugating enzyme for the small ubiquitin-like protein sentrin/ SUMO in the sentrinization pathway [33–37]. However, hUBC9 is also involved in the ubiquitination/ degradation of transcription factors such as E2A [25] and ATF2 [14]. In vitro transcription/translation analysis demonstrated greater association of MITF with GST– ubiquitin than with GST–sentrin (Fig. 2B, lanes 3 and 4). In addition, in vitro translated MITF protein showed that it preferentially formed conjugates with ubiquitin, rather than sentrin/SUMO (data not shown).
Association of MITF with hUBC9 raised the possibility that MITF could be degraded through an hUBC9dependent, proteasome degradation pathway. To investigate this possibility, COS7 cells were transfected with pFLAG-CMV2–MITF alone or together with pFLAG-CMV2– hUBC9. Forty-eight hours after transfection, the cells were treated for 2 h with either the proteasome inhibitor MG132 or the solvent DMSO as a control. Cells were then solubilized and analyzed by SDS–PAGE and Western blotting with an anti-FLAG antibody. In the absence of exogenously expressed hUBC9, FLAG-tagged MITF levels showed a small increase upon addition of the proteasome inhibitor (Fig. 3A, lanes 1 and 2). By contrast, almost no FLAGtagged MITF was detected in the presence of hUBC9 (Fig. 3A, lane 3). This striking hUBC9-dependent de-
FIG. 3. hUBC9 alters MITF levels in a proteasome-dependent manner. (A) COS7 cells were transfected with either 4 g of pFLAGCMV2–MITF only (lanes 1 and 2) or 4 g pFLAG-CMV2–MITF together with 2 g pFLAG-CMV2– hUBC9 (lanes 3 and 4). Fortyeight hours after transfection, cells were treated for 2 h with 50 M of the proteasome inhibitor MG132 or with DMSO (MG132 solvent). Whole-cell extracts were then prepared as described under Materials and Methods. Equal amounts of supernatant proteins were analyzed by SDS–PAGE and Western blotting using the anti-FLAG antibody M2. The FLAG-tagged MITF and FLAG-tagged hUBC9 protein bands are indicated by arrowheads. Positions of protein molecular mass standards (in kDa) are indicated on the left. (B) COS7 cells were transfected with 4 g pFLAG-CMV2–MITF (lane 1) and the indicated amount of pFLAG-CMV2– hUBC9 (lanes 2– 8). After 48 h, one sample (lane 7) was treated for 2 h with the proteasome inhibitor MG132. Cell extracts were prepared and analyzed by SDS–PAGE and Western blotting using an anti-FLAG antibody. The FLAGtagged MITF and FLAG-tagged hUBC9 protein bands are indicated by arrowheads.
UBIQUITINATION OF MITF PROTEIN
cline in MITF levels was partially rescued by addition of the proteasome inhibitor MG132 (Fig. 3A, lane 4). These results were further confirmed by cotransfecting increasing amounts of a FLAG-tagged hUBC9 plasmid and a fixed amount of FLAG-tagged MITF plasmid (Fig. 3B). The steady-state level of FLAG-tagged MITF declined with increasing hUBC9 protein levels in a dose-dependent manner. In COS7 cells transfected with FLAG-tagged MITF (Fig. 3B), the anti-FLAG antibody detected two proteins (molecular weights of 55 and 59 kDa). These proteins corresponded in molecular weight to two differentially phosphorylated MITF species previously identified in human melanoma cells [19]. Interestingly, the 59-kDa protein, which can transactivate a luciferase reporter driven by the tyrosinase promoter [19], appeared to be more susceptible to degradation by hUBC9 than the 55-kDa protein, especially at low levels of hUBC9 (Fig. 3B, lanes 2– 6). Higher levels of hUBC9 eventually resulted in loss of both the 55- and 59-kDa proteins. As observed in Fig. 3A, addition of the proteasome inhibitor MG132 almost completely rescued MITF. Taken together with the stabilization of MITF observed with proteasome inhibitor MG132, these results strongly suggest that phosphorylated MITF is targeted for proteolysis by the ubiquitin-dependent proteasome pathway through association with hUBC9. The Serine 73 (S73) Phosphorylation Site, Located in a PEST-like Sequence, Is Required for MITF Ubiquitination and hUBC9-Dependent Degradation Short-lived proteins generally contain regions enriched with Pro, Glu, Ser, and Thr residues (PEST sequences) [38]. PEST elements rich in S/TP sequences are the minimum consensus phosphorylation sites for some protein kinases, such as the cyclin-dependent kinase [39]. MITF has been shown to be phosphorylated at S73, which is within the classical PEST sequence PPVPGSSAPNSP. We used point mutation analysis to investigate the role of this phosphorylation site in MITF. In vitro transcription/translation demonstrated that a serine to alanine mutation at amino acid 73 (S73A) almost completely abolished MITF ubiquitination (Fig. 4A). This suggests that S73 phosphorylation may be essential for MITF ubiquitination. To investigate this possibility, COS7 cells were transfected with a MITFS73A mutant alone or together with pFLAG-CMV2– hUBC9. In contrast to what was observed with the wild-type MITF protein (Fig. 3B), the anti-FLAG antibody detected only one MITFS73A protein band, which was resistant to UBC9-mediated degradation (Fig. 4B). Consistent with these results and in contrast to that observed with the wild-type MITF (Fig. 3, lane 7), addition of MG132 did not increase MITF(S73A) levels (Fig. 4, lane 5). These results
139
FIG. 4. The serine 73 (S73) phosphorylation site is essential for MITF ubiquitination and hUBC9-dependent degradation. (A) In vitro translated wild-type (lane 1) and S73 mutant MITF (MITFS73A) (lane 2) were incubated with in vitro translated ubiquitin for 60 min at 30°C. The samples were then washed and separated by SDS–PAGE and visualized by autoradiography. (B) FLAGMITFS73A mutant was transfected in COS7 cells with (lanes 2– 4) or without (lane 1) increasing amounts of hUBC9, treated with (lane 5) or without (lanes 1– 4) MG132. Whole-cell extracts were made and Western blotted with an anti-FLAG antibody.
strongly suggest that S73 is critical for MITF ubiquitination in vivo. Identification of MITF Lysine 201 (K201) as a Potential Ubiquitination Site Protein modification by ubiquitination targets proteins for degradation by the 26S proteasome. Initially, ubiquitin is activated by an ATP-dependent pathway in a reaction catalyzed by the enzyme E1. Activated ubiquitin is then transferred to the E2 ubiquitin-conjugating enzyme, which catalyzes formation of an isopeptide bond between the C-terminal glycine of ubiquitin and the ⑀-amino group of a lysine residue on the target protein [15]. MITF contains 21 lysine residues that are distributed throughout the entire molecule. We attempted to identify the ubiquitination site(s) by constructing lysine to arginine substitutions spanning the MITF molecule. Wild-type and mutated MITF proteins were translated in vitro and incubated with ubiquitin. Ubiquitinated products were observed from wild-type MITF and from K182R and K205R mutant proteins (Fig. 5A). However, ubiquitination was almost completely abolished when the lysine residue at position 201 was replaced by arginine (Fig. 5A, lane 3), suggesting that this lysine residue is the target for ubiquitin conjugation. These results were confirmed in vivo by cotransfecting COS7 cells with the
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FIG. 5. Identification of MITF lysine 201 (K201) as a potential ubiquitination site. (A) In vitro translated wild-type (lane 1) and MITF mutant proteins (K182R, K201R, K205R) (lanes 2, 3, and 4) were incubated with in vitro translated ubiquitin for 60 min at 30°C. The samples were then separated by SDS–PAGE and visualized by autoradiography. Lane 5 shows in vitro translated wild-type MITF in the absence of ubiquitin. (B) FLAG-MITFK201R mutant was transfected in COS7 cells with (lanes 2– 4) or without (lane 1) increasing amounts of hUBC9, treated with (lane 5) or without (lanes 1– 4) MG132. Whole-cell extracts were made and Western blotted with an anti-FLAG antibody.
MITFK201R mutant alone or together with pFLAGCMV2– hUBC9. In contrast to that observed with the MITFS73A mutant (Fig. 4B), the anti-FLAG antibody detected two mutant MITFK201R bands which have an identical molecular weight as the wild-type MITF protein (Fig. 5B). Importantly, both MITFK201R isoforms were resistant to hUBC9-mediated degradation (Fig. 5B). Altogether, our results demonstrate that K201 is required for MITF ubiquitination. DISCUSSION
MITF regulates melanocyte differentiation and/or survival. Mutations at the mouse micropthalmia locus (mi) affect the development of melanocytes and several other cell types such as mast cells, osteoclasts, and retinal pigmented epithelial cells [40]. The mouse MITF protein interacts with the retinoblastoma tumor suppressor protein (pRb) [41] and with the coactivator p300 [42]. Using a cDNA library from human melanoma cells, we have identified hUBC9 as the only positive interacting protein for the human truncated micropthalmia protein MITF (MITF 168 –325). The yeast two-hybrid screen did not identify pRb or p300 as MITF 168 –325-interacting proteins. Because MITF and pRb have intrinsic Gal4 transactivation activities in the yeast system, we used the full-length
MITF coding region fused to the Gal4 transactivation domain and an N-terminal truncated pRb protein fused to the Gal4 DNA-binding domain to determine MITF–pRb interactions in yeast. No positive interaction was observed using these constructs, nor was interaction detected by coimmunoprecipitation (data not shown). These negative results may indicate either that MITF–pRb interact in a transient or weak manner or that such an interaction does not occur in human melanocytes. hUBC9 protein has been highly conserved throughout evolution. hUBC9 shares 75 and 82% amino acid similarity with the products of the yeast genes UBC9 (Saccharomyces cerevisiae) and hus5 (S. pombe), respectively [43, 44], and is 100% identical to the product of the mouse UBC9 gene [45]. A functional complementation assay showed that human UBC9 can substitute for the S. cerevisiae UBC9 gene, which is required for cell cycle progression [28]. In yeast, B-type-cyclin degradation is mediated by UBC9 [43]. Furthermore, repression of UBC9 prevents cell-cycle progression and causes yeast growth arrest in the G2 or early M phase [43]. The function of UBC9 in the G2/M phase could be to regulate levels of centromere proteins that direct chromosome segregation and mitosis through interaction with the centromere DNA-binding core complex CBF3 [27]. The crystal structure of the mammalian mUBC9 protein [46] revealed a number of unique features, including a protruding surface loop and a strong overall electrostatic dipole. Such features may have a role in allowing mUBC9 to interact with an exceptionally large variety of proteins. Nuclear magnetic resonance measurements show that two regions in murine/human UBC9 are likely to be important in protein–protein recognition in the ubiquitin pathway. The region near the C-terminus, which undergoes conformational change, may be important for UBC9 –substrate binding and catalytic activity [47]. Proteins that have been shown to interact with mUBC9 include the human papillomavirus type 16 E1 replication protein [28], the glucocorticoid receptor, c-Jun [30], the human Fas (CD95) protein [26], the Rad 51 recombination protein [31], the adenovirus E1A protein [45], the transcription factor ETS-1 [32], the helix-loop-helix protein E2A [25], and the transcription factor MITF (this communication). hUBC9-mediated MITF, E2A, and ATF2 [14, 25, 48] degradation stand in contrast to recent work showing that hUBC9 probably forms conjugates with ubiquitin-like proteins such as SUMO [33–37]. Modification of Ran GTPase protein by sentrin/SUMO requires UBC9 for nuclear transport [49]. However, using an in vitro ubiquitination assay, we demonstrated that MITF is preferentially ubiquitinized rather than sentrinized. Thereby, hUBC9 association with MITF ap-
141
UBIQUITINATION OF MITF PROTEIN
pears to be necessary for MITF recognition by the ubiquitin pathway. Phosphorylation of MITF does not alter its nuclear localization, DNA binding, or dimerization; however, phosphorylation facilitates association with the transcriptional coactivator p300 [19]. We have demonstrated that S73 phosphorylation favors MITF ubiquitination in vitro. Moreover, in vivo studies demonstrated that hUBC9 preferentially targets the 59-kDa MITF protein, previously demonstrated to be transcriptionally active, S73-phosphorylated MITF [19]. Recently, Ser 298, which locates downstream of the bHLH/ZIP, was found to be phosphorylated in melanoma cells [50]. However, S298 does not appear to be critical for MITF degradation since S73A mutation was sufficient to create a stable, 55-kDa protein. We propose that whereas S73 phosphorylation may trigger MITF activation, it may also provide a mechanism for degradation by active proteolysis. Phosphorylation and dephosphorylation have frequently been coupled with the ubiquitin-dependent proteasome pathway, since many substrates contain the acidic PEST sequences [39]. Activation of NFB by IB␣ ubiquitination and consequent degradation requires phosphorylation of S32 and S36 in the IB␣ molecule [51, 52]. Specific inhibition of the proteolytic activity results in accumulation of ubiquitinated forms of IB␣, indicating that IB␣ is targeted for degradation by a phosphorylation-dependent ubiquitination process [53]. The precise mechanism for how a PEST sequence is recognized by the ubiquitin-dependent proteasome pathway is not known. Protein phosphorylation may induce a conformational change that unmasks regions required for proteolysis [54]. Within this hypothesis, it is then likely that phosphorylation on S73 favors hUBC9 association and targets MITF protein for ubiquitination on K201. Importantly, K201 resides within the DNA-binding domain of the MITF protein. Thus, K201 ubiquitination may inhibit MITF transcriptional activity before it is recognized by the proteasome complex. Targeting phosphorylated MITF for proteasome degradation may not require its nuclear export since we have found that hUBC9 localizes in both the nuclear and cytoplasmic compartments (data not shown). This feature, in addition to the molecular structure [46], should explain hUBC9 multifunctionality and accessibility to a variety of cytoplasmic and nuclear partners. MITF overexpression results in apoptosis of mast cells [55]. Importantly, MITF protein levels increase during the commitment to differentiation but are almost undetectable in senescent human normal melanocytes in culture [56]. In these senescent melanocytes, absence of MITF protein was not accompanied by reduction in its mRNA levels, suggesting active degra-
dation of the MITF protein (unpublished results). It is thus likely that MITF levels are also critically regulated during development and differentiation of the melanocyte lineage. In addition, downregulation of melanin production and expression of a poorly or nondifferentiated phenotype is a common feature of metastatic human melanomas, both in vivo and in tissue culture. Our results raise the possibility that MITF– hUBC9 interaction and consequent MITF degradation may contribute to the poorly differentiated, nonpigmented phenotype of human metastatic melanoma tumors. We are grateful to Amey Herrin for assistance with tissue culture. This work was supported by Grants AG00594 and AG13663 (E.E.M) and AG09909 and AG11658 (J.C). M.M.H. was supported by a Training Grant (AG00183) from the National Institute on Aging.
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