Human Molecular Genetics, 2000, Vol. 9, No. 1 125-132
© 2000 Oxford University Press
Ser298 of MITF, a mutation site in Waardenburg syndrome type 2, is a phosphorylation site with functional significance
1Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai 980-8575, Japan, 2National Institute of Deafness and Other Communication Disorders, Bethesda, MD 20892, USA, 3Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA, 4National Institute of Mental Health, Bethesda, MD 20892, USA and 5Research Institute, Saitama Cancer Center, Ina, Saitama 362-0806, Japan
Received 29 September 1999; Revised and Accepted 27 October 1999.
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ABSTRACT |
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MITF (microphthalmia-associated transcription factor) is a basic-helix–loop–helix–leucine zipper (bHLHZip) factor which regulates expression of tyrosinase and other melanocytic genes via a CATGTG promoter sequence, and is involved in melanocyte differentiation. Mutations of MITF in mice or humans with Waardenburg syndrome type 2 (WS2) often severely disrupt the bHLHZip domain, suggesting the importance of this structure. Here, we show that Ser298, which locates downstream of the bHLHZip and was previously found to be mutated in individuals with WS2, plays an important role in MITF function. Glycogen synthase kinase 3 (GSK3) was found to phosphorylate Ser298 in vitro, thereby enhancing the binding of MITF to the tyrosinase promoter. The same serine was found to be phosphorylated in vivo, and expression of dominant-negative GSK3ß selectively suppressed the ability of MITF to transactivate the tyrosinase promoter. Moreover, mutation of Ser298, as found in a WS2 family, disabled phosphorylation of MITF by GSK3ß and impaired MITF function. These findings suggest that the Ser298 is important for MITF function and is phosphorylated probably by GSK3ß.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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MITF (microphthalmia-associated transcription factor) is a transcription factor with the basic-helix–loop–helix–leucine zipper (bHLHZip) structure (1,2). MITF is thought to regulate expression of tyrosinase and other melanocytic genes via a CATGTG promoter element (3–6), and is involved in melanocyte differentiation (7). Mutations of the MITF gene are associated with Waardenburg syndrome type 2 (WS2) (8,9) and albinism–deafness (Tietz) syndrome (10). Mutant MITF proteins predicted by these mutations often compromise the bHLHZip domain, suggesting the importance of this motif. HLH proteins play important roles in the development of a variety of tissue-specific lineages. The bHLHZip subfamily contains a C-terminal leucine zipper which essentially extends the helix 2
-helix of the HLH. These proteins bind DNA as homo- or heterodimers with the related proteins TFEB, TFEC or TFE3 (2), although it is unclear whether these other factors are co-expressed with MITF within melanocytes. The basic domain contacts DNA within the major groove of the E box element while the HLHZip represents the dimerization interface. For the MITF subfamily, the leucine zipper appears to be essential for dimerization since its deletion produces loss of function at the level of dimerization (and DNA binding) (2). Among naturally occurring MITF mutant mouse strains it was possible to correlate specific mutations using biochemical and in vivo structure–function analysis (2,11). In humans, haploinsufficiency appears to play a prominent role in the pigment and deafness phenotypes of affected WS2 patients, based on the locations of specific mutations (9). These structure–function analyses have been limited by the availability and recognition of distinct MITF mutations scattered throughout the MITF gene. One unexpected WS2 mutation was recognized (9): it predicts substitution of a serine (S298) located in a region C-terminal to the bHLHZip domain which has not previously been associated with any known function in the MITF protein. In this report, a series of deletion constructs of MITF were employed to map functionally important regions using transcriptional reporter and DNA-binding assays. One of the domains important for MITF transcriptional function was found to be this region downstream of the bHLHZip, which contains four putative phosphorylation sites. Mutation of only one of these sites, Ser298, profoundly affected MITF function. Glycogen synthase kinase 3 (GSK3) was found to phosphorylate Ser298 in vitro, significantly enhancing the ability of MITF to bind the tyrosinase promoter DNA element. The same serine was found to be phosphorylated within endogenous cellular MITF, and expression of dominant-negative GSK3ß selectively suppressed the ability of MITF to transactivate the tyrosinase promoter. Finally, replacement of Ser298 with either alanine or proline, a mutation found in a WS2 family, disabled phosphorylation of MITF by GSK3ß and impaired MITF function. These findings suggest that MITF may be regulated by GSK3ß and that perturbation of this regulation is a potential cause of WS2.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Functional regions of the MITF protein
Transactivation assays using MITF truncations on the tyrosinase promoter revealed several important MITF regions. Truncation of the first 74 amino acids (including a glutamine-rich region) or the last 95 amino acids (including threonine- and serine-rich regions) modestly decreased the transcriptional activity of MITF (Fig. 1A, MITF[75–419] and MITF[1–324]), although truncation of the serine-rich region alone did not (Fig. 1A, MITF[1–369]). A third region (MITF[294–324]) is adjacent to and downstream of the bHLHZip structure, and is critical, since deletion of this region drastically decreased transactivation activity (Fig. 1A, MITF[1–293]).
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Activation domains of MITF
MITF[117–184], which contains an acidic region (Fig. 1A), showed transactivation function (Fig. 1B). In addition, a threonine-rich region (MITF[324–369]) also possesses modest transactivation propensity, whereas MITF[294–324] did not appear to contain an activation domain (Fig. 1C).
DNA-binding ability of MITF
DNA-binding assays revealed that deletion of MITF[294–324] decreases its ability to bind to the CATGTG core DNA sequence contained in the human tyrosinase promoter (Fig. 1D, MITF[1–293]), whereas MITF with deletion of the C-terminal 95 amino acids retained this ability (Fig. 1D, MITF[1–324]).
Mutation of Ser298 severely affects MITF function
Replacement of Ser298 by an alanine significantly diminished the transactivation of the tyrosinase promoter by MITF (Fig. 2A), whereas substitutions with alanine at Ser302, Thr303 or Ser307 did not. MITF[S298A] also displayed a decrease in binding to the CATGTG element, whereas binding of MITF[S302A], [T303A] and [S307A] was similar to that of wild-type (Fig. 2B).
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Phosphorylation of MITF at Ser298 by GSK3
Sequence inspection revealed that Ser298 matches the glycogen synthase kinase 3 phosphoacceptor consensus and might therefore be a phosphorylation target in vitro and in vivo. To test this, GSK3
and GSK3ß were examined for the ability to phosphorylate recombinant or mutant MITF proteins. Using in vitro kinase assays, wild-type His-MITF and MITF[S302A] were found to be phosphorylated by these kinases whereas His-MITF[S298A] was not phosphorylated (Fig. 3A).
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Phosphorylation sites of endogenous MITF
To determine whether Ser298 was phosphorylated in vivo, two-dimensional phosphotryptic peptide mapping was carried out using 32P-labeled endogenous MITF immunoprecipitated from melanoma cells. The position of the Ser298-dependent in vitro phosphorylated MITF phosphopeptide was compared with the pattern derived from cellular MITF, and a prominent spot displayed identical migration in two dimensions (Fig. 3B), suggesting that Ser298 is phosphorylated in vivo.
Dominant-negative GSK3ß suppresses MITF transactivation
Dominant-negative mutants of GSK3 and GSK3ß were employed in which consecutive lysine residues in the ATP-binding site were mutated to methionine and isoleucine (12). Transfection of CV1 cells, which express endogenous GSK3 and GSK3ß (data not shown), resulted in selective and dose-dependent inhibition of MITF transactivation by dominant-negative GSK3ß. This dominant-negative effect is selective for MITF transactivation. Neither dominant-negative GSK3 cDNA nor wild-type GSK3/ß cDNAs significantly altered MITF transactivation potential in this assay (Fig. 4).
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Phosphorylation of MITF by GSK3ß enhances the DNA-binding activity of MITF
We compared the abilities of unphoshorylated recombinant MITF and GSK3ß–phoshorylated MITF to bind the CATGTG-core sequence from the tyrosinase promoter, and found that phosphorylated MITF had stronger DNA-binding ability than unphoshorylated MITF (Fig. 5).
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Ser298Pro mutation found in WS2 also affects the function of MITF
Substitution of a proline for Ser298 strongly inhibited MITF transactivation and was comparable with S298A substitution (Fig. 6A). DNA binding was also inhibited by S298P mutation (Fig. 6B), and this mutant was not phosphorylated by purified GSK3ß in vitro (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Although the bHLHZip structure of MITF seems to be essential for its function, we found in this study that regions flanking this structure are also important. MITF contains glutamine-rich, serine-rich, threonine-rich and acidic domains, homologs of which are often important in transcription factor function. Glutamine-rich regions of Sp1, CREB, Oct1, Oct2 and AP-2 have been shown to possess transcriptional activities (13–15). Likewise, serine/threonine-rich regions of ITF1, ITF2, TFE3 and TREB5 also function as activation domains (14,16). We examined whether these regions of MITF function as activation domains in Gal4 chimeras. Consistent with previous findings in murine MITF (17), we found that MITF[117–184], which contains an acidic region, functions as an activation domain. In addition, we found that a threonine-rich region (MITF[324–369]) also possesses modest activation propensity. However, a region immediately downstream of the bHLHZip structure of MITF (MITF[294–324]), which is important for tyrosinase gene activation, did not prove to contain an activation domain. In addition, deletion of this region did not change the nuclear localization of MITF as judged by immunoblot analysis using antibody to recombinant MITF (data not shown). However, this deletion dramatically decreased DNA-binding activity, whereas MITF with deletion of the C-terminal 95 amino acids retained DNA binding. Therefore, MITF[294–324] may regulate MITF function through modulation of its DNA-binding activity. The modulation of DNA-binding activity by region MITF[294–324] may be specific to MITF, since fusion of this region to a GAL4-binding domain did not significantly increase the transactivational activity of the domain (Fig. 1C).
Phosphorylation is one of the major post-translational mechanisms by which the activity of transcription factors is regulated. Phosphorylation of certain transcription factors may occur within the DNA-binding domain and positively or negatively affect sequence-specific interactions of transcription factors to DNA (18). Myogenin, which activates muscle-specific genes through binding to an E box, provides an example wherein phosphorylation by fibroblast growth factor and protein kinase C occurs at Thr87, within the DNA-binding domain, and this phosphorylation inhibits DNA binding (19). On the other hand, phosphorylation of the Pit-1 DNA recognition domain enhances DNA-binding activity (20). Phosphorylation of transcription factors outside the DNA-binding motif may also alter DNA-binding activity. For example, phosphorylation of serum response factor (SRF) by casein kinase 2 at the N-terminus strongly enhances DNA-binding activity of SRF through a conformational change of the DNA-binding motif (21,22). MITF is known to be phosphorylated and has been shown to be phosphorylated in response to c-Kit stimulation by MAP kinase at Ser73 (23).
These findings suggested the possibility that phosphorylation of MITF at serine or threonine residues in MITF[294–324] is involved in regulating the function of MITF. To test this, we carried out alanine scanning mutagenesis of these residues and examined transactivation and DNA binding. Substitution of alanine for Ser298 significantly diminished the transactivation of the tyrosinase promoter by MITF, a result not found with substitution for Ser302, Thr303 or Ser307. MITF[S298A] also displayed a dramatic decrease in binding to the CATGTG element, whereas the binding of MITF[S302A], [T303A] and [S307A] was similar to that of wild-type. MITF[S298A] displayed expression and stability comparable with wild-type MITF (data not shown). Since the sequence context of S298 (Ser-Lys-Ile-Pro-Ser) matches the consensus substrate sequence for GSK3 (Ser*-X-X-X-Ser, asterisk indicates GSK3 phosphoacceptor site) (24–26), we tested whether GSK3 could phosphorylate recombinant histidine-tagged MITF (His-MITF) in vitro. Both GSK3 and GSK3ß phosphorylated wild-type His-MITF, but not His-MITF[S298A]. Consistent with the transactivation and DNA-binding assays, MITF[S302A] was phosphorylated by GSK3. These data imply that Ser302 of MITF (the second serine in the GSK3 consensus) is not essential for GSK3-mediated phosphorylation of MITF as is the case for other transcription factor substrates such as c-Jun (27) and c-Myb (28). Ser298, however, appears to be required for phosphorylation of recombinant MITF by GSK3.
To determine whether endogenous MITF is phosphorylated at Ser298 within cells, we carried out two-dimensional phosphopeptide mapping of trypsin-digested 32P-labeled endogenous MITF immunoprecipitated from melanoma cells. A unique phosphotryptic fragment was identified using recombinant MITF phosphorylated by GSK3ß in vitro. Comparison of this spot with the pattern generated from endogenous MITF from 32P-labeled melanoma cells resulted in identification of a perfectly co-migrating species, suggesting that Ser298 is phosphorylated within cells. To examine whether GSK3 is involved in MITF function in vivo and, if so, to determine which isoform of GSK3 is involved, we examined the effects of overexpressing wild-type or dominant-negative forms of either GSK3 or GSK3ß on the ability of MITF to transactivate the tyrosinase promoter (Fig. 4). Transfection of CV1 cells with dominant-negative GSK3ß cDNA resulted in selective and dose-dependent inhibition of MITF transactivation. Expression of wild-type GSK3ß cDNA, however, did not superactivate the ability of MITF to transactivate the tyrosinase promoter. Thus, it is likely that endogenous GSK3ß levels are not rate limiting in the ability of MITF to transactivate. To determine whether GSK3ß-phosphorylation influences the DNA-binding activity of MITF, DNA-binding studies were undertaken, and it was found that phosphorylation of recombinant MITF by GSK3ß markedly enhanced the ability of MITF to bind the CATGTG core sequence from the tyrosinase promoter (Fig. 5). Although there may be other underlying mechanisms, the increment DNA-binding activity by phosphorylation of MITF at Ser298 accounts for the importance of this amino acid for transcriptional activity of MITF at least in part.
It was previously reported that affected members of a WS2 family are associated with an MITF mutation which substitutes a proline for Ser298 (9). It is reasonable to expect that this S298P mutation would cause effects similar to those caused by the S298A mutation shown above, since both mutations would make the phoshorylation of MITF at amino acid 298 impossible. Therefore, we examined the effect of this mutation on MITF function by transactivation and DNA-binding assays. As expected, substitution of a proline for Ser298 strongly inhibited the transactivation and DNA-binding activities of MITF to levels comparable with alanine substitution at S298. Thus, phosphorylation of MITF at Ser298 by GSK3ß appears to be crucially important for melanocyte differentiation. A melanocyte signal transduction pathway for MITF activation by MAP kinase was recently shown to involve Steel factor/c-Kit (23). Although WNT and HGF/SF have been implicated in melanocyte development (29,30) and shown to stimulate pathways known to regulate GSK3ß in other cell types (31), upstream regulators of GSK3ß activity in melanocytes remain to be elucidated.
In summary, our results suggest that phosphorylation of MITF at Ser298, which is probably mediated by GSK3ß, is important for the ability of MITF to bind and transactivate the tyrosinase promoter, implying that this phosphorylation event may be pivotal for the role of MITF in melanocyte differentiation. Furthermore, a WS2-associated mutation of MITF at Ser298 disables the transcriptional activity of MITF, probably through failure of phosphorylation by GSK3ß. Thus, perturbation of MITF phosphorylation by GSK3ß is a potential cause of WS2. Further study is necessary to determine whether this type of positive regulation of MITF by endogenous GSK3 occurs in melanocytes. Also it is necessary to elucidate how phoshorylation of MITF at Ser298 increases its DNA-binding activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmid constructs
All plasmids were constructed by standard methods using restriction enzymes and/or PCR. The MITF cDNA used in this study was reported previously (1). When PCR was employed, the validity of nucleotide sequences was confirmed by the dideoxy chain termination method.
pRc/CMV-MITF series.
An MITF cDNA encoding wild-type MITF (MITF[1–419]) was inserted into the mammalian expression vector pRc/CMV (Invitrogen, Carlsbad, CA), and termed pRc/CMV-MITF[1–419]. Likewise, MITF cDNAs encoding the truncated MITF proteins (MITF[1–369], MITF[1–324], MITF[1–293] and MITF[75–419]) were inserted into pRc/CMV and termed pRc/CMV-MITF[1–369], -MITF[1–324], -MITF[1–293] and -MITF[75–419], respectively. For the construct encoding the N-terminal truncated MITF protein, nine nucleotides ATGCTGGAA (encoding Met-Leu-Glu) were added at the 5' end of the truncated cDNA sequence for the initiation of protein synthesis. Constructs encoding MITF mutants predicting the amino acid substitutions indicated in the brackets, i.e. pRc/CMV-MITF[S298A], -MITF[S302A], -MITF[S307A], -MITF[T303A] and -MITF[S298P], were made by PCR-mediated mutagenesis (32).
pET-MITF series.
To express proteins in Escherichia coli, wild-type and point-mutated MITF cDNAs were inserted into pET-28a(+) (Novagen, Madison, WI), and termed pET-MITF[1–419], -MITF[S298A], -MITF[S298P] and -MITF[S302A].
pM-MITF series.
Wild-type and truncated MITF cDNAs were inserted downstream of the sequence encoding GAL4 DNA-binding domain in the mammalian expression vector pM (Clontech, Palo Alto, CA). These constructs (pM-MITF[1–293], -MITF[117–184], -MITF[14–47], -MITF[293–419], -MITF[293–369], -MITF[324–369] and -MITF[293–324]) are predicted to encode GAL4/MITF fusion proteins.
GSK3 expression vectors.
To express a wild-type or dominant-negative type of GSK3 or GSK3ß (12), each cDNA was inserted at the EcoRI site in the mammalian expression vector pcDNA3.1 (Invitrogen).
pHTL4.
The fusion gene construct containing the promoter region and a part of exon 1 (–7400/+56) of the human tyrosinase gene upstream of the firefly luciferase gene (33) was used to assay the ability of MITFs to transactivate the tyrosinase gene.
pG5-Luc.
This construct contains five copies of a GAL4-binding site upstream of the E1b-TATA promoter, along with the luciferase gene. To make this construct, pG5CAT (Clontech) was cleaved with BamHI/EcoRI, filled in at the 3' end and ligated with HindIII linker. This fragment was inserted into a luciferase reporter vector pGL2-Basic (Promega, Madison, WI) cleaved with BglII and HindIII.
Cell culture
NIH/3T3 and CV1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum and 50 µg/ml of gentamicin. Human melanoma cells were maintained in RPMI1640 supplemented with 10% fetal calf serum and 50 µg/ml of gentamicin. Primary culture cells of melanocytes were obtained from Clonetics (Walkersville, MD) and maintained following the manufacturer’s recommendations.
Tyrosinase gene transactivation assay
NIH/3T3 or CV1 cells were split 24 h prior to transfection into six-well plates (Costar, Acton, MA), transfected for 16 h, re-fed with fresh medium and harvested 24–36 h later. These two cell lines were interchangeable and yielded essentially the same results in reporter assays. Transfections were carried out by calcium phosphate/DNA coprecipitation (5). In brief, ~60% confluent cells were cotransfected with 6 µg of DNA consisting of a mammalian expression vector plasmid containing wild-type, truncated or point-mutated MITF cDNAs (pRc/CMV-MITF series, 1 µg), a reporter plasmid (pHTL4, 1 µg), pCH110 (1 µg; Amersham Pharmacia Biotech, Piscataway, NJ) for internal control, and pUC19 (varying amounts) as a carrier. At harvest, cells were washed once with phosphate-buffered saline, lysed and assayed using the Luciferase Assay System kit (Promega) and a luminometer. Luciferase data were normalized using ß-galactosidase activity in the cell lysate as a measure of relative transfection efficiencies (6).
To examine the effects of GSK3 cDNAs on the ability of MITF to transactivate the tyrosinase gene, wild-type or dominant-negative GSK3 or GSK3ß cDNAs (12) were included in the assay system. Cells were cotransfected with 6.5 µg of DNA consisting of pRc/CMV-MITF[1–419] (1 µg), pHTL4 (1 µg), a GSK3 or GSK3ß expression vector (0.5–4 µg), pcDNA3.1/His/lacZ (0.5 µg) and pUC19 (varying amounts) as a filler.
Activation domain assay
The effector plasmid encodes a test protein fused to the GAL4 DNA-binding domain. pG5-Luc reporter plasmid is a fusion construct containing five copies of the GAL4-binding sequence, E1b-TATA minimal promoter and the luciferase gene using an effector plasmid containing a sequence encoding the DNA-binding domain of GAL4 (amino acids 1–147) (34), fused to wild-type or mutant MITF cDNA (pM-MITF series) and a reporter pG5-Luc plasmid containing five copies of a GAL4-binding DNA element upstream of the luciferase gene (34). Activation domains of MITF were assayed as described (35). In brief, the ability of GAL4/MITF fusion proteins to activate transcription from GAL4 target sequences was assessed by transfecting cultured cells with 1 µg each of effector (pM-MITF), reporter (pG5-Luc) and internal control (pCH110) vectors, and 3 µg of carrier plasmid pUC19. After 24–36 h, cells were harvested and luciferase activity was determined as described above.
DNA-binding assay
Wild-type and mutant MITF proteins were synthesized using an in vitro transcription/translation kit (TNT T7 Coupled Reticulocyte Lysate System; Promega) with the pRc/CMV series and T7 RNA polymerase. Equivalent amounts of wild-type and mutant proteins, as determined by autoradiography following separation of [35S]Met-labeled translation products on a 10–20% gradient polyacrylamide gel (Novex, Encinitas, CA), were used for electrophoretic mobility shift assays as previously described (6). In brief, double-stranded oligonucleotides, encompassing the CATGTG core sequence of the human tyrosinase promoter (5'-GATCTGTGGAGATCATGTGATGACTTCCTGGATC-3') (33) were filled in with [-32P]dCTP and used as probe. After preincubation without the probe for 30 min, the translated proteins were incubated with probe DNA for 30 min at room temperature in 20 µl of solution containing 1 µg of poly(dI·dC), 5% glycerol, 100 mM KCl, 10 mM Tris–HCl pH 7.4, 1 mM dithiothreitol (DTT). As a negative control the plasmid was omitted from the reaction mixture for in vitro transcription/translation. After incubation, the reaction mixture was loaded on 6% polyacrylamide gel, and run in 0.25x TBE buffer. Gels were dried and autoradiographed to detect mobility shift. To examine the effect of phosphorylation of MITF on DNA-binding activity, His-MITF protein was expressed in E.coli BL21(DE3) (Novagen), and purified using an Ni2+-NTA-agarose column (Qiagen, Chatsworth, CA). Protein concentration of purified protein was then determined by Bradford protein assay kit (BioRad, Hercules, CA). His-MITF (1 µg) was incubated at 30°C for 30 min in a reaction mixture (10 µl) with or without GSK3ß (see below). Using these phosphorylated or unphosphorylated recombinant MITFs, the DNA-binding assay was performed as above.
In vitro phosphorylation of MITF by GSK3
Wild-type His-MITF and His-MITF[S298A] were expressed in E.coli BL21(DE3) and purified using an Ni2+-NTA-agarose column. One microgram of these proteins was incubated at 30°C for 30 min in a reaction mixture containing 5 µCi of [
-32P]ATP, GSK3 or GSK3ß (5 x 10–3 U/ml; Upstate Biotechnology, Lake Placid, NY), 20 mM Tris–HCl pH 7.5, 10 mM MgCl2, 5 mM DTT and 25 µM of ATP. The reaction was terminated by addition of loading buffer, and the samples were boiled and applied to 10–20% gradient denaturing polyacrylamide gels (Novex). After separation by polyacrylamide gel electrophoresis (PAGE), phosphorylated products were visualized by autoradiography.
Phosphotryptic mapping
For in vivo labeling of MITF, human melanoma cells were washed in phosphate-free, serum-free DMEM (Gibco, Rockville, MD) and labeled in vivo with 1 mCi/ml of inorganic 32P for 8 h. Washed cells were lysed in 1% Triton X-100, 170 mM NaCl, 20 mM Tris–HCl pH 7.4 plus protease and phosphatase inhibitors, immunoprecipitated with anti-MITF monoclonal antibody C5 and protein G agarose beads (Gibco) at 4°C overnight, washed, and boiled in 3% SDS, 10% glycerol, 120 mM Tris and 0.1 M DTT. Proteins were resolved on PAGE, transferred to nitrocellulose, cut out, and digested with 20 mg of TPCK-treated trypsin (Sigma, St Louis, MO) overnight at 37°C. Two-dimensional tryptic analysis employed 1% ammonium carbonate (pH 8.9) for electrophoresis and n-butanol:pyridine:acetic acid:water (37.5:25:7.5:30%) for thin-layer chromatography (36). One hundred nanograms of a histidine-tagged recombinant fragment of human MITF (amino acids 1–325) was phosphorylated with 0.02 U of purified GSK3ß (Upstate Biotechnology) in 20 µl of 50 mM HEPES pH 7.6, 10 mM MgCl2, 2 mM NaVO4, 1 mg/ml of Pefabloc, 2 mM DTT and 50 µM ATP plus 10 µCi of [-32P]ATP at 32°C for 30 min, followed by PAGE and tryptic digestion and two-dimensional analysis as above.
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ACKNOWLEDGEMENTS |
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We thank Drs H. Varmus and X. He for wild-type and dominant-negative GSK3 cDNAs, Dr S. Shibahara for pHTL4 and Dr Y. Kawakami for human melanoma cells. Thanks are also due to Ms B. Ploplis and A. Wells for technical assistance. C.T. is supported by NIH Fellowship KO8 HL3700; D.E.F. is supported by NIH grant AR43369 and is a Pew Foundation Scholar.
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FOOTNOTES |
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+ Present address: Department of Pediatrics, Hokkaido University School of Medicine, Sapporo 060-8638, Japan
§ Present address: Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo 113-8602, Japan
¶ Present address: Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
To whom correspondence should be addressed at: Research Institute, Saitama Cancer Center, 818 Komuro, Ina, Saitama 362-0806, Japan. Tel: +81 48 722 111 ext. 4693; Fax +81 48 722 1739; Email: mtachiba@cancer-c.pref.saitama.jp
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REFERENCES |
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