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Transcriptional Activation Function of Zinc Finger Protein TIS11 and Its Negative Regulation by Phorbol Ester
Biochemical and Biophysical Research Communications 274, 526 –532 (2000) doi:10.1006/bbrc.2000.3182, available online at http://www.idealibrary.com on
Transcriptional Activation Function of Zinc Finger Protein TIS11 and Its Negative Regulation by Phorbol Ester Tomiyasu Murata, Kiyomi Hikita, and Norio Kaneda 1 Department of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan
Received June 28, 2000
TIS11, a CCCH zinc finger protein, is one of the typical growth factor-inducible nuclear proteins. We found that TIS11 possesses the potential to activate transcription when fused to the GAL4 DNA binding domain and transiently cotransfected into rat pheochromocytoma PC12 cells along with a GAL4responsive luciferase reporter gene. The study with deletion mutants of TIS11 revealed that the major transactivation region is located at the N-terminal 101 amino acid residues and that the remaining central and C-terminal region had a moderate transactivational activity. In addition, the transactivational activity of TIS11 was found to be significantly reduced by treating the transfectants with phorbol 12-myristate 13-acetate (PMA). PMA-induced inactivation of TIS11 was blocked by calphostin C, a protein kinase C inhibitor, and PD98059, a mitogen-activated protein (MAP) kinase kinase inhibitor. These results suggested that TIS11 functions as a positive transcriptional regulator and that the protein kinase C/MAP kinase signaling cascade is involved in negative regulation of TIS11 by PMA. © 2000 Academic Press Key Words: TIS11; zinc finger protein; phorbol ester; transcriptional activation; rat pheochromocytoma PC12 cells.
TIS11 was first isolated as a cDNA encoding an immediate early gene, which was induced by phorbol 12-myristate 13-acetate (PMA), serum and several growth factors in Swiss 3T3 fibroblasts (1–3). The mouse TIS11 cDNA was also cloned from 3T3 cells as an insulin-inducible gene, tristetraprolin (TTP) (4), and as a serum-inducible gene, Nup475 (5). We previously reported cDNA cloning of the rat TIS11 from nerve growth factor-treated pheochromocytoma PC12 cells (6). The human orthologue was reported as the human TTP cDNA (7) and as G0S24 cDNA (8). Mouse, To whom correspondence should be addressed. Fax: ⫹81-52-8348090. E-mail: [email protected]. 1
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
rat, and human TIS11 proteins all contain two putative zinc finger motifs, CX 8CX 5CX 3H, separated by 13 amino acid residues, and the recombinant protein has been shown to bind Zn 2⫹ with high affinity (5, 9). The three members of the CCCH class of zinc finger protein family in mammals have so far been identified: i.e., the first is TIS11/TTP/Nup475/G0S24; the second is mouse TIS11b (10), rat cMG1 (11), human ERF-1 (12), and human Berg36 (13); and the third is mouse TIS11d (10) and human ERF-2 (14). Among these members, two CX 8CX 5CX 3H zinc finger motifs and the spacing between the two repeats are completely conserved, although there is very little overall amino acid sequence homology. Other members of this family containing two or more CX 8CX 5CX 3H motifs have been identified in Xenopus (15), Drosophila (16 –22) and yeast (23–25). By immunocytochemistry, TTP/Nup475 protein was shown to be localized in the nucleus (5, 26). Considering the structural features of the zinc finger motif of TIS11 and its nuclear localization, TIS11 may function as an immediate early transcriptional regulator like Jun and Fos families and Erg-1. Recently, it was reported that TTP was translocated from the nucleus to the cytoplasm in NIH 3T3 cells in response to serum or other mitogens (26, 27). In addition, it was reported that, in the cytoplasm, TTP inhibits tumor necrosis factor-␣ production by destabilizing its mRNA as an RNA-binding protein (28), and that its binding to the AU-rich element of tumor necrosis factor-␣ mRNA depends on the integrity of the two zinc finger structures (29). These studies suggested that TIS11 possesses both nuclear and cytoplasmic functions. However, the physiological function of TIS11 in the nucleus has not been elucidated. A number of regulatory domains of transcription factors have been analyzed by fusing the region to the heterologous DNA binding domain such as the GAL4 DNA binding domain, and then by cotransfection with the corresponding reporter gene into cultured cells. To examine transcriptional activity of TIS11, we prepared several constructs composed of the full-length as well as partial fragments of TIS11 fused to GAL4 DNA
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binding domain, and cotransfected with the luciferase reporter gene into PC12 cells. With this approach, we characterized transcriptional activation function of TIS11. MATERIALS AND METHODS Materials. PMA, dithiothreitol, phenylmethylsulfonyl fluoride, leupeptin, and aprotinin were purchased from Sigma Chemical Co. (St. Louis, MO). Protein kinase C inhibitor, calphostin C, and mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor, PD98059, were obtained from Research Biochemicals International (Natick, MA). Restriction enzymes and Taq DNA polymerase were purchased from Takara Shuzo Co. (Ohtsu, Japan). pGEM-T Easy, pBIND, and pG5luc vectors, TransFast transfection reagent, and dual-luciferase reporter assay system were obtained from Promega (Madison, WI). Adenosine 5⬘-[␥- 32P] triphosphate ([␥- 32P] ATP; 110 TBq/mmol) and p42/p44 MAP kinase enzyme assay kit were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Fetal bovine serum (FBS), horse serum (HS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were obtained from Gibco BRL (Grand Island, NY). Working stocks of PMA (2 mM), calphostin C (1 mM), and PD98059 (50 mM) were prepared in dimethyl sulfoxide. Plasmid constructs. DNA for various TIS11 peptide fragments were prepared by PCR using the rat full-length TIS11 cDNA (6) as a template. The nucleotide sequences of primers used for PCR were as follows: primer I, 5⬘-ACTGTCGACTTATGGATCTCTCTGCCATCTAC3⬘; primer II, 5⬘-ACTGTCGACTTCCGGGCCCTGAGCTGTCACCC-3⬘; primer III, 5⬘-ACTGTCGACTTCAAAGCATCAGCTTCTCAGGC-3⬘; primer IV, 5⬘-ACTGATATCTCACTCAGAGACAGAGATGCG-3⬘; primer V, 5⬘-ACTGATATCTCAGGTTCTGCGGCCTGAAGGCAA-3⬘; and primer VI, 5⬘-ACTGATATCTCAGAGCTCAGTCTTGTATCGAGA3⬘. DNA Fragments 1–320 (which corresponds to TIS11 amino acids 1–320), 1–189, 1–101, 76 –320, 76 –189, and 176 –320 were prepared using the following primer pairs: primers I/IV for fragment 1–320; primers I/V for fragment 1–189; primers I/VI for fragment 1–101; primers II/IV for fragment 76 –320; primers II/V for fragment 76 –189; and primers III/IV for fragment 176 –320. PCR was performed for 30 cycles under the following conditions: 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Amplified DNA fragments were separated by electrophoresis on 1.5% low-melting agarose gels, cloned into the pGEM T-Easy vector, and sequenced using a fluorescence DNA sequencer (ABI PRISM 310-2 Genetic Analyzer, Perkin-Elmer Applied Biosystems, Foster City, CA). Targeted DNA fragments were prepared from each vector by SalI/ EcoRV digestion, and then inserted into the pBIND vector to generate fusion proteins with the GAL4 DNA binding domain. To generate the full-length TIS11 expression plasmid lacking the GAL4 DNA binding domain (TIS1-320), GAL-TIS1-320 construct was digested with NheI and SalI, and self-ligated. The pG5E1b-LUC containing five GAL4 binding sites upstream of a minimal TATA box derived from adenovius E1b that, in turn, was upstream of the firefly luciferase gene was used as a GAL4-responsive reporter plasmid. To generate E1b-LUC reporter plasmid lacking the GAL4 binding site, the pG5E1b-LUC plasmid was digested with KpnI and NheI, and self-ligated. Cell culture and transfection. Rat pheochromocytoma PC12 cells were maintained in DMEM containing 25 mM glucose, supplemented with 5% heat-inactivated FBS and 5% heat-inactivated HS in the presence of 50 U/ml penicillin and 50 g/ml streptomycin under a humidified atmosphere of 5% CO 2 and 95% air at 37°C. For the transfection experiments, cells were grown on 24-well tissue culture plates to approximately 70% confluence, and washed once with serum-free medium. Either pBIND plasmid (0.2 g/well) or an equivalent molar amount of test plasmid was cotransfected into the cells with the reporter plasmid (0.35 g/well) using TransFast reagent, a synthetic cationic lipid component, according to the manu-
facturer’s instructions. For analysis of the transcriptional regulatory region of TIS11 protein, transfected PC12 cells were cultured for 24 h in 5% FBS and 5% HS-supplemented medium before harvesting. For analysis of regulation of the TIS11 transcriptional activity by PMA, transfected PC12 cells were cultured for 20 h in 5% FBS and 5% HS-supplemented medium, followed by culture for 4 h in 0.1% FBSsupplemented medium, and then for 4 or 8 h in the same medium with or without PMA (1 M) before harvesting. To study the effects of calphostin C and PD98059 on transcriptional inhibition by PMA, cells were preincubated with the inhibitor 30 min prior to treatment with PMA. In control experiments, cells were treated with the inhibitor alone. At the end of the culture period, transfectants were lysed, and the luciferase activity in each cell lysate was measured by the dual-luciferase assay system according to the manufacturer’s instructions. The pBIND plasmid also contains the Renilla luciferase gene under the control of the promoter derived from SV40. To correct for transfection efficiency and differences in cell number, luciferase activity was expressed as the ratio of firefly luciferase activity of reporter plasmid to the Renilla luciferase activity of the pBIND or test expression plasmid. Measurement of p42/p44 MAP kinase activity. The BIOTRAK MAP kinase enzyme assay kit was used to measure p42/p44 MAP kinase activity. After treatment for indicated time, the cells were washed with ice-cold PBS and lysed with 500 l of lysis buffer containing 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin. Cell lysates were then centrifuged for 20 min at 25,000g at 4°C (TL-100; Beckman, Palo Alto, CA). To determine p42/p44 MAP kinase activity, 15 l of the supernatant and 10 l of peptide buffer were mixed, and the reaction was started by the addition of [␥- 32P] ATP. After incubating at 37°C for 30 min, the reaction was stopped by the addition of 10 l of stop buffer. Aliquot of the sample (30 l) was pipetted onto the center of a filter disc. To remove nonspecifically bound [␥- 32P]ATP, discs were washed twice (2 min/wash) with 75 mM orthophosphoric acid, followed by an additional wash with distilled H 2O. Phosphorylation was quantitated by measuring radioactivity of the disc with a scintillation counter. Protein concentration was determined by the Bradford method with a kit from Bio-Rad (Hercules, CA) and bovine plasma ␥-globulin as a standard. Statistical analysis. The significance of differences was estimated by Student’s t test. A P value less than 0.05 was considered significant.
RESULTS Transactivational Activity of TIS11 and Mapping of Its Regulatory Regions To elucidate the transcriptional properties of TIS11, we constructed a fusion protein of the GAL4 DNA binding domain with TIS11 as a test plasmid. We used the adenoviral E1b promoter bearing five GAL4 binding sites that was linked to the firefly luciferase gene as the target promoter (Fig. 1A). Cotransfection of GAL4 full-length TIS11 test plasmid with the pG5E1b-LUC reporter plasmid into PC12 cells demonstrated that the GAL4 full-length TIS11 fusion protein (GAL-TIS1-320) activated transcription of the luciferase reporter gene by 4.7-fold compared with the control plasmid containing only the GAL4 DNA binding domain (GAL4DBD) (Fig. 1A). In addition, a construct of TIS11 protein without the DNA binding domain (TIS1-320) showed no transcriptional activity toward the pG5E1b-LUC
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FIG. 1. Identification of transcriptional activation region of TIS11. (A) Transactivational activity of TIS11. The pG5E1b-LUC and E1b-LUC were used as the reporter plasmids. A cDNA fragment encoding the full-length TIS11 was cloned into the pBIND expression test plasmid to generate a fusion protein with the GAL4 DNA binding domain at the N-terminus. The full-length TIS11 test plasmid lacking the GAL4 DNA binding domain was also constructed. The test plasmids were transiently cotransfected with reporter plasmid into PC12 cells. The relative luciferase activity was determined as described under Materials and Methods. The values are expressed as -fold increase, taking the relative luciferase activity of GAL4DBD as 1.0. All values represent the means ⫾ SD of four independent experiments, in which measurements were made in triplicate. *P ⬍ 0.05 compared with GAL4DBD and pG5E1b-LUC cotransfectants. (B) Mapping of the transcriptional activation regions of TIS11. Test plasmids with either full-length or variously deleted forms of TIS11 fused to the GAL4 DNA-binding domain were constructed. Each plasmid expressing GAL4-TIS11 chimera protein was tested for its ability to transactivate the pG5E1b-LUC reporter gene in PC12 cells. All values represent the means ⫾ SD of four independent experiments, in which measurements were made in triplicate. *P ⬍ 0.05 compared with GAL4DBD transfectants. **P ⬍ 0.05 compared with GAL-TIS1-189 transfectants.
reporter plasmid (Fig. 1A). When E1b-LUC, a reporter plasmid lacking the five GAL4 binding sites, was used, neither GAL-TIS1-320 nor TIS1-320 affected transcription of the reporter plasmid (Fig. 1A). These results indicated that transcriptional activation with TIS11 was brought about through binding of the chimeric molecule to the GAL4 binding site of the reporter plasmid. To define which region of TIS11 was responsible for transactivational activity, we constructed a series of mutants in which variously deleted TIS11 proteins were fused to the GAL4 DNA binding domain. Test plasmids for these mutant constructs were cotransfected into PC12 cells together with the luciferase reporter plasmid pG5E1b-LUC. The transactivational activities of these partial fragments of TIS11 are
shown in Fig. 1B. The N-terminal fragment of TIS11, GAL-TIS1-101, activated transcription of the reporter gene to a level similar to that of the full-length TIS11, GAL-TIS1-320, indicating that the N-terminal region consisting of 101 amino acid residues functions as a potent transcriptional activator domain. However, a longer N-terminal fragment, GAL-TIS1-189, showed reduced transactivation of the reporter gene relative to GAL-TIS1-101, indicating that amino acids 102–189 which cover two zinc finger motifs impede the transactivational activity of the N-terminal fragment as a transcriptional inhibitory region. On the other hand, fragment 76 –320 lacking the N-terminal region moderately activated transcription with half the potency of the full-length protein. Further deletions of the C-terminal amino acids from 190 to 320 of GAL-TIS76-
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Effects of Protein Kinase C and MEK Inhibitors on PMA Repression of the Transactivational Activity of TIS11
FIG. 2. Effects of PMA on the transactivational activity of TIS11. Test plasmids used were the same as in Fig. 1B. They were transiently cotransfected with pG5E1b-LUC reporter plasmid into PC12 cells. The transfectants were cultured in DMEM containing 5% FBS and 5% HS for 20 h, followed by DMEM containing 0.1% FBS for 4 h, and then for an additional 8 h in the same medium with or without 1 M PMA. The relative luciferase activity was determined as described under Materials and Methods. All values represent the means ⫾ SD of four independent experiments, in which measurements were made in triplicate. *P ⬍ 0.05 compared with untreated transfectants in each test plasmid.
320 resulted in complete loss of transactivational activity, as demonstrated with GAL-TIS76-189. Although this result indicated the importance of amino acids 190 –320 for transcriptional activation, GALTIS176-320, which covered amino acids 190 –320, showed no activity by itself. These results suggested that two subregions (76 –189 and 190 –320) may synergistically function in the transactivational activity of the fragment 76 –320.
Since PMA activates protein kinase C and stimulates a signaling cascade involving Raf-MEK-p42/p44 MAP kinase, we postulated that this pathway may play an important role for the inhibitory effect of PMA on the transcriptional activation of TIS11. As shown in Fig. 3, PMA treatment of PC12 cells resulted in 6.4-fold increase in p42/p44 MAP kinase activity within 15 min, which was followed by a reduced, but sustained activation lasting until 60 min. Treatment of the cells with protein kinase C inhibitor calphostin C (0.25 M) and MEK inhibitor PD98059 (50 M) completely inhibited the PMA-induced activation of MAP kinase activity. Both of these inhibitors had no effects on basal MAP kinase activity (data not shown). Therefore, these inhibitor concentrations were used to test whether the inhibition of protein kinase C/MAP kinase pathway affected PMA repression of TIS11 transcriptional activation. As shown in Table 1, treatment with calphostin C significantly reduced the inhibitory effect of PMA in each test plasmid examined (GAL-TIS1-320, GALTIS1-101, and GAL-TIS76-320). Similarly, treatment with PD98059, significantly reduced the inhibitory effect of PMA in each test plasmid examined (Table 2). These results clearly showed that PMA inhibited the transactivational activity of TIS11 through a mechanism dependent on activation of the protein kinase C/MAP kinase pathway.
Negative Regulation of TIS11 Transcriptional Activation by PMA To further investigate the nature of transactivation, we examined the effect of PMA on the transactivational activity of TIS11. Full-length and variously deleted TIS11 test plasmids were cotransfected into PC12 cells with the luciferase reporter plasmid pG5E1bLUC, and then the transfected cells were treated with PMA (1 M). PMA treatment for 8 h of PC12 cells transfected with GAL-TIS1-320, GAL-TIS1-189, GALTIS1-101, and GAL-TIS76-320 caused significant decreases in their relative luciferase activities, whereas cells transfected with GAL4DBD, GAL-TIS76-189, and GAL-TIS176-320 showed no significant responses following PMA treatment (Fig. 2). These results suggested that PMA inhibition of TIS11 transactivational activity resulted from inactivation of functions of both fragments 1–101 and 76 –320.
FIG. 3. Inhibitory effects of calphostin C and PD98059 on activation of p42/p44 MAP kinase by PMA in PC12 cells. PC12 cells were cultured in DMEM containing 5% FBS and 5% HS for 20 h, followed by DMEM containing 0.1% FBS for 4 h, and then for the indicated time in the same medium with or without 1 M PMA. The protein kinase C inhibitor calphostin C (0.25 M) and MEK inhibitor PD98059 (50 M) were added 30 min prior to adding PMA. MAP kinase activity was determined as described under Materials and Methods. All values represent the means ⫾ SD of three independent experiments, in which measurements were made in triplicate. *P ⬍ 0.05 compared with untreated cells.
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Effects of a Protein Kinase C Inhibitor, Calphostin C, on PMAInduced Inhibition of TIS11 Transactivational Activity
Test plasmid GAL-TIS1-320
GAL-TIS1-101
GAL-TIS76-320
Treatment None PMA Calphostin Calphostin None PMA Calphostin Calphostin None PMA Calphostin Calphostin
C C ⫹ PMA C C ⫹ PMA C C ⫹ PMA
Relative luciferase activity 581 ⫾ 27 291 ⫾ 18* 586 ⫾ 25 511 ⫾ 22 530 ⫾ 28 345 ⫾ 18* 524 ⫾ 20 492 ⫾ 25 201 ⫾ 18 80 ⫾ 7* 198 ⫾ 12 170 ⫾ 20
PMA inhibition (%)
] ] ] ] ] ]
50 13 35 6 60 14
Note. PC12 cells were transiently cotransfected with test plasmid (GAL4DBD, GAL-TIS1-320, GAL-TIS1-101, or GAL-TIS76-320) and pG5E1b-LUC reporter plasmid. The transfectants were stimulated with 1 M PMA for 4 h. The protein kinase C inhibitor calphostin C (final concentration, 0.25 M) was added 30 min prior to adding PMA. To demonstrate the effects of the reagents, relative luciferase activity is shown after subtracting the activity of the control GAL4DBD transfectant (untreated, 181 ⫾ 12; PMA-treated, 170 ⫾ 10; calphostin C-treated, 186 ⫾ 8; and calphostin C ⫹ PMA-treated, 176 ⫾ 11) from the value of each test plasmid. Other experimental conditions were the same as those described in the legend to Fig. 2. Results are expressed as means ⫾ SD from four independent experiments, in which measurements were made in triplicate. * P ⬍ 0.05 compared with untreated transfectants.
common (30, 31). Although TIS11 does not show any homology to activation domains of known transcription factors, rat TIS11 is generally rich in proline residues (amino acids 1–101, 13.9%; amino acids 76 –320, 16.5%) including unique clusters of proline residues, PPPPG (amino acids 64 – 68, 191–195, and 212–216), suggesting that the proline-rich may be involved in the transactivational activity of TIS11. Interestingly, the transactivational activity of N-terminal region was reduced by the presence of an internal region (residues 102 to 189) which includes two zinc finger motifs. Recently, Batchelder et al. (32) reported that zinc finger region of mouse TTP (amino acids 91–165 relative to amino acids 92–166 of rat TIS11) significantly inhibited gene transcription from heterologous herpes simplex virus thymidine kinase promoter having GAL4 DNA binding sites. Thus, it is likely that the internal amino acids 102–189 of TIS11 acts as the intramolecular repressor region. Our data showed that central amino acids 76 –189 containing two zinc finger domains functions as a subregion for the moderate transactivational activity of the fragment 76 –320. In the present study, however, possible inhibitory effect of the amino acids 76 –189 against the fragment 76 –320 activity could not be estimated because deletion of amino acids from 76 to 176 in the fragment TABLE 2
Effects of a MAP Kinase Kinase Inhibitor, PD98059, on PMAInduced Inhibition of TIS11 Transactivational Activity
Test plasmid
Treatment
Relative luciferase activity
GAL-TIS1-320
None PMA PD98059 PD98059 ⫹ PMA None PMA PD98059 PD98059 ⫹ PMA None PMA PD98059 PD98059 ⫹ PMA
523 ⫾ 26 251 ⫾ 13* 501 ⫾ 22 412 ⫾ 21 476 ⫾ 21 299 ⫾ 15* 452 ⫾ 20 407 ⫾ 16 174 ⫾ 16 64 ⫾ 8* 162 ⫾ 15 121 ⫾ 20
DISCUSSION In the present study, we demonstrated that CCCHtype zinc finger protein TIS11, a typical growth factorinducible nuclear protein, possesses transcriptional activation potential when fused downstream of a heterologous DNA-binding domain, and measured its ability to activate transcription of a reporter gene in mammalian cells. This observation suggests that TIS11 can act as a positive regulator of transcription. Analyses using fusion proteins of GAL4 DNA-binding domain and variously deleted TIS11 proteins revealed that the major transactivation region of TIS11 was located at the N-terminal 101 amino acid residues, though the central and C-terminal region (residues 76 to 320) also exhibited moderate transactivational activity. The essential transactivational regions of TIS11 may function through a direct interaction with components of the basal transcription initiation complex or, alternatively, through the interaction with an intermediary coactivator/associated protein(s) that in turn recognizes the initiation complex. Transcriptional activation domains are grouped according to the content of certain amino acid residues with proline-rich, glutamine-rich, and acidic domains being the most
GAL-TIS1-101
GAL-TIS76-320
PMA inhibition (%)
] ] ] ] ] ]
52 18 37 10 63 25
Note. PC12 cells were transiently cotransfected with test plasmid (GAL4DBD, GAL-TIS1-320, GAL-TIS1-101, or GAL-TIS76-320) and pG5E1b-LUC reporter plasmid. The transfectants were stimulated with 1 M PMA for 4 h. The MAP kinase kinase inhibitor PD98059 (final concentration, 50 M) was added 30 min prior to adding PMA. To demonstrate the effects of the reagents, relative luciferase activity is shown after subtracting the activity of the control GAL4DBD transfectant (untreated, 163 ⫾ 8; PMA-treated, 153 ⫾ 12; PD98059treated, 155 ⫾ 11; and PD98059 ⫹ PMA-treated, 151 ⫾ 13) from the value of each test plasmid. Other experimental conditions were the same as those described in the legend to Fig. 2. Results are expressed as means ⫾ SD from four independent experiments, in which measurements were made in triplicate. * P ⬍ 0.05 compared with untreated transfectants.
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76 –320 resulted in complete loss of transactivational activity, as demonstrated with GAL-TIS76-320 and GAL-TIS176-320 (Fig. 1B). The intramolecular repression region probably acts as a buffer for the transactivation function of TIS11. Activation of intracellular signal transduction pathways often results in modulation of function of transcription factors. In the present study, we observed that PMA significantly reduces TIS11 transactivational activity. Activation of protein kinase C by phorbol esters stimulates the MAP kinase signaling cascade, and this appeared to occur through activation of Raf, which lies immediately upstream of MEK (33, 34). Therefore, the effects of protein kinase C and MEK inhibitors on PMA signaling were examined. The specific protein kinase C inhibitor, calphostin C, and the MEK inhibitor, PD98059, each blocked the PMAinduced decrease in transactivational activity of TIS11. These results suggested that activations of protein kinase C and MAP kinase signaling pathway are involved in the PMA-induced repression of transactivational activity of TIS11. Phosphorylation of MAP kinase has been shown to regulate transactivational activities of several transcription factors (35–37). It has been reported that TTP is phosphorylated by several mitogens including PMA, serum, fibroblast growth factor and platelet-derived growth factor, and that Ser220 is the primary site phosphorylated by p42 MAP kinase both in vitro and in intact cells (27). When GAL-mtTIS1-320 (S221A) and GAL-mtTIS76-320 (S221A), in which Ser221 (equivalent to Ser220 in mouse TTP) was replaced with an alanine residue, were used as test plasmids, they showed no effect on the action of PMA, which was indistinguishable from that of wild-type fusion protein (data not shown). This suggested that the phosphorylation of Ser221 is not essential for down-regulation of transactivational activity of TIS11 by PMA, though we cannot rule out the possibility of other minor phosphorylation sites (27). It is also likely that PMA inhibition of TIS11 function results from the modulation of a putative MAP kinase pathway-sensitive transcriptional coactivator protein(s) which associates with TIS11. Recently, PMA has been shown to stimulate translocation of TTP from the nucleus to the cytoplasm in serum-deprived NIH 3T3 cells (26). Thus, this nuclear export may lead to the dissociation of TIS11 from functional transcription complex in PMA-treated PC12 cells, and decrease in level of TIS11 molecule in the transactivational signal. It was reported that YTIS11/CTH2, which is a member of the CCCH zinc finger family in yeast, transactivated the transcription of a reporter gene through its N-terminal activation region (24). Although amino acid sequences of the two CCCH motifs are highly conserved between TIS11 and YTIS11/CTH2, there observed no overall homology in amino acid sequence including the N-terminal region. It was reported that
overexpression of TIS11/TTP markedly inhibited the cell proliferation of yeast as well as TIS11b, CTH1, and YTIS11/CTH2 (24, 25), suggesting that CCCH zinc finger structure is critical for the growth inhibition. Therefore, TIS11 may function as a transactivator that promotes gene expression, which leads to inhibition of the cell growth. It is possible that TIS11 functions as a positive transcription factor that binds to a target DNA sequence or as a coactivator protein that interacts with other transcription factor(s). Identification of downstream genes that associate with TIS11 will provide more insight into molecular mechanisms underlying the function of this protein. ACKNOWLEDGMENTS This work was supported in part by Scientific Research Grants (11672196, 11771456, and High-Tech Research Center Project) from the Ministry of Education, Science, Sports, and Culture of Japan.
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21. Guedes, S., and Priess, J. R. (1997) Development 124, 731–739. 22. Tabara, H., Hill, R. J., Mello, C. C., Priess, J. R., and Kohara, Y. (1999) Development 126, 1–11. 23. Kanoh, J., Sugimoto, A., and Yamamoto, M. (1995) Mol. Biol. Cell 6, 1185–1195. 24. Ma, Q., and Herschman, H. R. (1995) Oncogene 10, 487– 494. 25. Thompson, M. J., Lai, W. S., Taylor, G. A., and Blackshear, P. J. (1996) Gene 174, 225–233. 26. Taylor, G. A., Thompson, M. J., Lai, W. S., and Blackshear, P. J. (1996) Mol. Endocrinol. 10, 140 –146. 27. Taylor, G. A., Thompson, M. J., Lai, W. S., and Blackshear, P. J. (1995) J. Biol. Chem. 270, 13341–13347. 28. Carballo, E., Lai, W. S., and Blackshear, P. J. (1998) Science 281, 1001–1005. 29. Lai, W. S., Carballo, E., Strum, J. R., Kennington, E. A., Phillips, R. S., and Blackshear, P. J. (1999) Mol. Cell. Biol. 19, 4311– 4323.
30. Mitchell, P. J., and Tjian, R. (1989) Science 245, 371–378. 31. Triezenberg, S. J. (1995) Curr. Opin. Genet. Dev. 5, 190 –196. 32. Batchelder, C., Dunn, M. A., Choy, B., Suh, Y., Cassie, C., Shim, E. Y., Shin, T. H., Mello, C., Seydoux, G., and Blackwell, T. K. (1999) Genes Dev. 13, 202–212. 33. Sozeri, O., Vollmer, K., Liyanage, M., Frith, D., Kour, G., Mark, G. E., III, and Stabel, S. (1992) Oncogene 7, 2259 –2262. 34. Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D., and Rapp, U. R. (1993) Nature 364, 249 –252. 35. Nakajima, T., Kinoshita, S., Sasagawa, T., Sasaki, K., Naruto, M., Kishimoto, T., and Akira, S. (1993) Proc. Natl. Acad. Sci. USA 90, 2207–2211. 36. Hill, C. S., and Treisman, R. (1995) Cell 80, 199 –211. 37. Chu, B., Soncin, F., Price, B. D., Stevenson, M. A., and Calderwood, S. K. (1996) J. Biol. Chem. 271, 30847–30857.
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Transcriptional Activation Function of Zinc Finger Protein TIS11 and Its Negative Regulation by Phorbol Ester Tomiyasu Murata, Kiyomi Hikita, and Norio Kaneda 1 Department of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan
Received June 28, 2000
TIS11, a CCCH zinc finger protein, is one of the typical growth factor-inducible nuclear proteins. We found that TIS11 possesses the potential to activate transcription when fused to the GAL4 DNA binding domain and transiently cotransfected into rat pheochromocytoma PC12 cells along with a GAL4responsive luciferase reporter gene. The study with deletion mutants of TIS11 revealed that the major transactivation region is located at the N-terminal 101 amino acid residues and that the remaining central and C-terminal region had a moderate transactivational activity. In addition, the transactivational activity of TIS11 was found to be significantly reduced by treating the transfectants with phorbol 12-myristate 13-acetate (PMA). PMA-induced inactivation of TIS11 was blocked by calphostin C, a protein kinase C inhibitor, and PD98059, a mitogen-activated protein (MAP) kinase kinase inhibitor. These results suggested that TIS11 functions as a positive transcriptional regulator and that the protein kinase C/MAP kinase signaling cascade is involved in negative regulation of TIS11 by PMA. © 2000 Academic Press Key Words: TIS11; zinc finger protein; phorbol ester; transcriptional activation; rat pheochromocytoma PC12 cells.
TIS11 was first isolated as a cDNA encoding an immediate early gene, which was induced by phorbol 12-myristate 13-acetate (PMA), serum and several growth factors in Swiss 3T3 fibroblasts (1–3). The mouse TIS11 cDNA was also cloned from 3T3 cells as an insulin-inducible gene, tristetraprolin (TTP) (4), and as a serum-inducible gene, Nup475 (5). We previously reported cDNA cloning of the rat TIS11 from nerve growth factor-treated pheochromocytoma PC12 cells (6). The human orthologue was reported as the human TTP cDNA (7) and as G0S24 cDNA (8). Mouse, To whom correspondence should be addressed. Fax: ⫹81-52-8348090. E-mail: [email protected]. 1
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
rat, and human TIS11 proteins all contain two putative zinc finger motifs, CX 8CX 5CX 3H, separated by 13 amino acid residues, and the recombinant protein has been shown to bind Zn 2⫹ with high affinity (5, 9). The three members of the CCCH class of zinc finger protein family in mammals have so far been identified: i.e., the first is TIS11/TTP/Nup475/G0S24; the second is mouse TIS11b (10), rat cMG1 (11), human ERF-1 (12), and human Berg36 (13); and the third is mouse TIS11d (10) and human ERF-2 (14). Among these members, two CX 8CX 5CX 3H zinc finger motifs and the spacing between the two repeats are completely conserved, although there is very little overall amino acid sequence homology. Other members of this family containing two or more CX 8CX 5CX 3H motifs have been identified in Xenopus (15), Drosophila (16 –22) and yeast (23–25). By immunocytochemistry, TTP/Nup475 protein was shown to be localized in the nucleus (5, 26). Considering the structural features of the zinc finger motif of TIS11 and its nuclear localization, TIS11 may function as an immediate early transcriptional regulator like Jun and Fos families and Erg-1. Recently, it was reported that TTP was translocated from the nucleus to the cytoplasm in NIH 3T3 cells in response to serum or other mitogens (26, 27). In addition, it was reported that, in the cytoplasm, TTP inhibits tumor necrosis factor-␣ production by destabilizing its mRNA as an RNA-binding protein (28), and that its binding to the AU-rich element of tumor necrosis factor-␣ mRNA depends on the integrity of the two zinc finger structures (29). These studies suggested that TIS11 possesses both nuclear and cytoplasmic functions. However, the physiological function of TIS11 in the nucleus has not been elucidated. A number of regulatory domains of transcription factors have been analyzed by fusing the region to the heterologous DNA binding domain such as the GAL4 DNA binding domain, and then by cotransfection with the corresponding reporter gene into cultured cells. To examine transcriptional activity of TIS11, we prepared several constructs composed of the full-length as well as partial fragments of TIS11 fused to GAL4 DNA
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binding domain, and cotransfected with the luciferase reporter gene into PC12 cells. With this approach, we characterized transcriptional activation function of TIS11. MATERIALS AND METHODS Materials. PMA, dithiothreitol, phenylmethylsulfonyl fluoride, leupeptin, and aprotinin were purchased from Sigma Chemical Co. (St. Louis, MO). Protein kinase C inhibitor, calphostin C, and mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor, PD98059, were obtained from Research Biochemicals International (Natick, MA). Restriction enzymes and Taq DNA polymerase were purchased from Takara Shuzo Co. (Ohtsu, Japan). pGEM-T Easy, pBIND, and pG5luc vectors, TransFast transfection reagent, and dual-luciferase reporter assay system were obtained from Promega (Madison, WI). Adenosine 5⬘-[␥- 32P] triphosphate ([␥- 32P] ATP; 110 TBq/mmol) and p42/p44 MAP kinase enzyme assay kit were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Fetal bovine serum (FBS), horse serum (HS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were obtained from Gibco BRL (Grand Island, NY). Working stocks of PMA (2 mM), calphostin C (1 mM), and PD98059 (50 mM) were prepared in dimethyl sulfoxide. Plasmid constructs. DNA for various TIS11 peptide fragments were prepared by PCR using the rat full-length TIS11 cDNA (6) as a template. The nucleotide sequences of primers used for PCR were as follows: primer I, 5⬘-ACTGTCGACTTATGGATCTCTCTGCCATCTAC3⬘; primer II, 5⬘-ACTGTCGACTTCCGGGCCCTGAGCTGTCACCC-3⬘; primer III, 5⬘-ACTGTCGACTTCAAAGCATCAGCTTCTCAGGC-3⬘; primer IV, 5⬘-ACTGATATCTCACTCAGAGACAGAGATGCG-3⬘; primer V, 5⬘-ACTGATATCTCAGGTTCTGCGGCCTGAAGGCAA-3⬘; and primer VI, 5⬘-ACTGATATCTCAGAGCTCAGTCTTGTATCGAGA3⬘. DNA Fragments 1–320 (which corresponds to TIS11 amino acids 1–320), 1–189, 1–101, 76 –320, 76 –189, and 176 –320 were prepared using the following primer pairs: primers I/IV for fragment 1–320; primers I/V for fragment 1–189; primers I/VI for fragment 1–101; primers II/IV for fragment 76 –320; primers II/V for fragment 76 –189; and primers III/IV for fragment 176 –320. PCR was performed for 30 cycles under the following conditions: 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Amplified DNA fragments were separated by electrophoresis on 1.5% low-melting agarose gels, cloned into the pGEM T-Easy vector, and sequenced using a fluorescence DNA sequencer (ABI PRISM 310-2 Genetic Analyzer, Perkin-Elmer Applied Biosystems, Foster City, CA). Targeted DNA fragments were prepared from each vector by SalI/ EcoRV digestion, and then inserted into the pBIND vector to generate fusion proteins with the GAL4 DNA binding domain. To generate the full-length TIS11 expression plasmid lacking the GAL4 DNA binding domain (TIS1-320), GAL-TIS1-320 construct was digested with NheI and SalI, and self-ligated. The pG5E1b-LUC containing five GAL4 binding sites upstream of a minimal TATA box derived from adenovius E1b that, in turn, was upstream of the firefly luciferase gene was used as a GAL4-responsive reporter plasmid. To generate E1b-LUC reporter plasmid lacking the GAL4 binding site, the pG5E1b-LUC plasmid was digested with KpnI and NheI, and self-ligated. Cell culture and transfection. Rat pheochromocytoma PC12 cells were maintained in DMEM containing 25 mM glucose, supplemented with 5% heat-inactivated FBS and 5% heat-inactivated HS in the presence of 50 U/ml penicillin and 50 g/ml streptomycin under a humidified atmosphere of 5% CO 2 and 95% air at 37°C. For the transfection experiments, cells were grown on 24-well tissue culture plates to approximately 70% confluence, and washed once with serum-free medium. Either pBIND plasmid (0.2 g/well) or an equivalent molar amount of test plasmid was cotransfected into the cells with the reporter plasmid (0.35 g/well) using TransFast reagent, a synthetic cationic lipid component, according to the manu-
facturer’s instructions. For analysis of the transcriptional regulatory region of TIS11 protein, transfected PC12 cells were cultured for 24 h in 5% FBS and 5% HS-supplemented medium before harvesting. For analysis of regulation of the TIS11 transcriptional activity by PMA, transfected PC12 cells were cultured for 20 h in 5% FBS and 5% HS-supplemented medium, followed by culture for 4 h in 0.1% FBSsupplemented medium, and then for 4 or 8 h in the same medium with or without PMA (1 M) before harvesting. To study the effects of calphostin C and PD98059 on transcriptional inhibition by PMA, cells were preincubated with the inhibitor 30 min prior to treatment with PMA. In control experiments, cells were treated with the inhibitor alone. At the end of the culture period, transfectants were lysed, and the luciferase activity in each cell lysate was measured by the dual-luciferase assay system according to the manufacturer’s instructions. The pBIND plasmid also contains the Renilla luciferase gene under the control of the promoter derived from SV40. To correct for transfection efficiency and differences in cell number, luciferase activity was expressed as the ratio of firefly luciferase activity of reporter plasmid to the Renilla luciferase activity of the pBIND or test expression plasmid. Measurement of p42/p44 MAP kinase activity. The BIOTRAK MAP kinase enzyme assay kit was used to measure p42/p44 MAP kinase activity. After treatment for indicated time, the cells were washed with ice-cold PBS and lysed with 500 l of lysis buffer containing 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin. Cell lysates were then centrifuged for 20 min at 25,000g at 4°C (TL-100; Beckman, Palo Alto, CA). To determine p42/p44 MAP kinase activity, 15 l of the supernatant and 10 l of peptide buffer were mixed, and the reaction was started by the addition of [␥- 32P] ATP. After incubating at 37°C for 30 min, the reaction was stopped by the addition of 10 l of stop buffer. Aliquot of the sample (30 l) was pipetted onto the center of a filter disc. To remove nonspecifically bound [␥- 32P]ATP, discs were washed twice (2 min/wash) with 75 mM orthophosphoric acid, followed by an additional wash with distilled H 2O. Phosphorylation was quantitated by measuring radioactivity of the disc with a scintillation counter. Protein concentration was determined by the Bradford method with a kit from Bio-Rad (Hercules, CA) and bovine plasma ␥-globulin as a standard. Statistical analysis. The significance of differences was estimated by Student’s t test. A P value less than 0.05 was considered significant.
RESULTS Transactivational Activity of TIS11 and Mapping of Its Regulatory Regions To elucidate the transcriptional properties of TIS11, we constructed a fusion protein of the GAL4 DNA binding domain with TIS11 as a test plasmid. We used the adenoviral E1b promoter bearing five GAL4 binding sites that was linked to the firefly luciferase gene as the target promoter (Fig. 1A). Cotransfection of GAL4 full-length TIS11 test plasmid with the pG5E1b-LUC reporter plasmid into PC12 cells demonstrated that the GAL4 full-length TIS11 fusion protein (GAL-TIS1-320) activated transcription of the luciferase reporter gene by 4.7-fold compared with the control plasmid containing only the GAL4 DNA binding domain (GAL4DBD) (Fig. 1A). In addition, a construct of TIS11 protein without the DNA binding domain (TIS1-320) showed no transcriptional activity toward the pG5E1b-LUC
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FIG. 1. Identification of transcriptional activation region of TIS11. (A) Transactivational activity of TIS11. The pG5E1b-LUC and E1b-LUC were used as the reporter plasmids. A cDNA fragment encoding the full-length TIS11 was cloned into the pBIND expression test plasmid to generate a fusion protein with the GAL4 DNA binding domain at the N-terminus. The full-length TIS11 test plasmid lacking the GAL4 DNA binding domain was also constructed. The test plasmids were transiently cotransfected with reporter plasmid into PC12 cells. The relative luciferase activity was determined as described under Materials and Methods. The values are expressed as -fold increase, taking the relative luciferase activity of GAL4DBD as 1.0. All values represent the means ⫾ SD of four independent experiments, in which measurements were made in triplicate. *P ⬍ 0.05 compared with GAL4DBD and pG5E1b-LUC cotransfectants. (B) Mapping of the transcriptional activation regions of TIS11. Test plasmids with either full-length or variously deleted forms of TIS11 fused to the GAL4 DNA-binding domain were constructed. Each plasmid expressing GAL4-TIS11 chimera protein was tested for its ability to transactivate the pG5E1b-LUC reporter gene in PC12 cells. All values represent the means ⫾ SD of four independent experiments, in which measurements were made in triplicate. *P ⬍ 0.05 compared with GAL4DBD transfectants. **P ⬍ 0.05 compared with GAL-TIS1-189 transfectants.
reporter plasmid (Fig. 1A). When E1b-LUC, a reporter plasmid lacking the five GAL4 binding sites, was used, neither GAL-TIS1-320 nor TIS1-320 affected transcription of the reporter plasmid (Fig. 1A). These results indicated that transcriptional activation with TIS11 was brought about through binding of the chimeric molecule to the GAL4 binding site of the reporter plasmid. To define which region of TIS11 was responsible for transactivational activity, we constructed a series of mutants in which variously deleted TIS11 proteins were fused to the GAL4 DNA binding domain. Test plasmids for these mutant constructs were cotransfected into PC12 cells together with the luciferase reporter plasmid pG5E1b-LUC. The transactivational activities of these partial fragments of TIS11 are
shown in Fig. 1B. The N-terminal fragment of TIS11, GAL-TIS1-101, activated transcription of the reporter gene to a level similar to that of the full-length TIS11, GAL-TIS1-320, indicating that the N-terminal region consisting of 101 amino acid residues functions as a potent transcriptional activator domain. However, a longer N-terminal fragment, GAL-TIS1-189, showed reduced transactivation of the reporter gene relative to GAL-TIS1-101, indicating that amino acids 102–189 which cover two zinc finger motifs impede the transactivational activity of the N-terminal fragment as a transcriptional inhibitory region. On the other hand, fragment 76 –320 lacking the N-terminal region moderately activated transcription with half the potency of the full-length protein. Further deletions of the C-terminal amino acids from 190 to 320 of GAL-TIS76-
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Effects of Protein Kinase C and MEK Inhibitors on PMA Repression of the Transactivational Activity of TIS11
FIG. 2. Effects of PMA on the transactivational activity of TIS11. Test plasmids used were the same as in Fig. 1B. They were transiently cotransfected with pG5E1b-LUC reporter plasmid into PC12 cells. The transfectants were cultured in DMEM containing 5% FBS and 5% HS for 20 h, followed by DMEM containing 0.1% FBS for 4 h, and then for an additional 8 h in the same medium with or without 1 M PMA. The relative luciferase activity was determined as described under Materials and Methods. All values represent the means ⫾ SD of four independent experiments, in which measurements were made in triplicate. *P ⬍ 0.05 compared with untreated transfectants in each test plasmid.
320 resulted in complete loss of transactivational activity, as demonstrated with GAL-TIS76-189. Although this result indicated the importance of amino acids 190 –320 for transcriptional activation, GALTIS176-320, which covered amino acids 190 –320, showed no activity by itself. These results suggested that two subregions (76 –189 and 190 –320) may synergistically function in the transactivational activity of the fragment 76 –320.
Since PMA activates protein kinase C and stimulates a signaling cascade involving Raf-MEK-p42/p44 MAP kinase, we postulated that this pathway may play an important role for the inhibitory effect of PMA on the transcriptional activation of TIS11. As shown in Fig. 3, PMA treatment of PC12 cells resulted in 6.4-fold increase in p42/p44 MAP kinase activity within 15 min, which was followed by a reduced, but sustained activation lasting until 60 min. Treatment of the cells with protein kinase C inhibitor calphostin C (0.25 M) and MEK inhibitor PD98059 (50 M) completely inhibited the PMA-induced activation of MAP kinase activity. Both of these inhibitors had no effects on basal MAP kinase activity (data not shown). Therefore, these inhibitor concentrations were used to test whether the inhibition of protein kinase C/MAP kinase pathway affected PMA repression of TIS11 transcriptional activation. As shown in Table 1, treatment with calphostin C significantly reduced the inhibitory effect of PMA in each test plasmid examined (GAL-TIS1-320, GALTIS1-101, and GAL-TIS76-320). Similarly, treatment with PD98059, significantly reduced the inhibitory effect of PMA in each test plasmid examined (Table 2). These results clearly showed that PMA inhibited the transactivational activity of TIS11 through a mechanism dependent on activation of the protein kinase C/MAP kinase pathway.
Negative Regulation of TIS11 Transcriptional Activation by PMA To further investigate the nature of transactivation, we examined the effect of PMA on the transactivational activity of TIS11. Full-length and variously deleted TIS11 test plasmids were cotransfected into PC12 cells with the luciferase reporter plasmid pG5E1bLUC, and then the transfected cells were treated with PMA (1 M). PMA treatment for 8 h of PC12 cells transfected with GAL-TIS1-320, GAL-TIS1-189, GALTIS1-101, and GAL-TIS76-320 caused significant decreases in their relative luciferase activities, whereas cells transfected with GAL4DBD, GAL-TIS76-189, and GAL-TIS176-320 showed no significant responses following PMA treatment (Fig. 2). These results suggested that PMA inhibition of TIS11 transactivational activity resulted from inactivation of functions of both fragments 1–101 and 76 –320.
FIG. 3. Inhibitory effects of calphostin C and PD98059 on activation of p42/p44 MAP kinase by PMA in PC12 cells. PC12 cells were cultured in DMEM containing 5% FBS and 5% HS for 20 h, followed by DMEM containing 0.1% FBS for 4 h, and then for the indicated time in the same medium with or without 1 M PMA. The protein kinase C inhibitor calphostin C (0.25 M) and MEK inhibitor PD98059 (50 M) were added 30 min prior to adding PMA. MAP kinase activity was determined as described under Materials and Methods. All values represent the means ⫾ SD of three independent experiments, in which measurements were made in triplicate. *P ⬍ 0.05 compared with untreated cells.
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Effects of a Protein Kinase C Inhibitor, Calphostin C, on PMAInduced Inhibition of TIS11 Transactivational Activity
Test plasmid GAL-TIS1-320
GAL-TIS1-101
GAL-TIS76-320
Treatment None PMA Calphostin Calphostin None PMA Calphostin Calphostin None PMA Calphostin Calphostin
C C ⫹ PMA C C ⫹ PMA C C ⫹ PMA
Relative luciferase activity 581 ⫾ 27 291 ⫾ 18* 586 ⫾ 25 511 ⫾ 22 530 ⫾ 28 345 ⫾ 18* 524 ⫾ 20 492 ⫾ 25 201 ⫾ 18 80 ⫾ 7* 198 ⫾ 12 170 ⫾ 20
PMA inhibition (%)
] ] ] ] ] ]
50 13 35 6 60 14
Note. PC12 cells were transiently cotransfected with test plasmid (GAL4DBD, GAL-TIS1-320, GAL-TIS1-101, or GAL-TIS76-320) and pG5E1b-LUC reporter plasmid. The transfectants were stimulated with 1 M PMA for 4 h. The protein kinase C inhibitor calphostin C (final concentration, 0.25 M) was added 30 min prior to adding PMA. To demonstrate the effects of the reagents, relative luciferase activity is shown after subtracting the activity of the control GAL4DBD transfectant (untreated, 181 ⫾ 12; PMA-treated, 170 ⫾ 10; calphostin C-treated, 186 ⫾ 8; and calphostin C ⫹ PMA-treated, 176 ⫾ 11) from the value of each test plasmid. Other experimental conditions were the same as those described in the legend to Fig. 2. Results are expressed as means ⫾ SD from four independent experiments, in which measurements were made in triplicate. * P ⬍ 0.05 compared with untreated transfectants.
common (30, 31). Although TIS11 does not show any homology to activation domains of known transcription factors, rat TIS11 is generally rich in proline residues (amino acids 1–101, 13.9%; amino acids 76 –320, 16.5%) including unique clusters of proline residues, PPPPG (amino acids 64 – 68, 191–195, and 212–216), suggesting that the proline-rich may be involved in the transactivational activity of TIS11. Interestingly, the transactivational activity of N-terminal region was reduced by the presence of an internal region (residues 102 to 189) which includes two zinc finger motifs. Recently, Batchelder et al. (32) reported that zinc finger region of mouse TTP (amino acids 91–165 relative to amino acids 92–166 of rat TIS11) significantly inhibited gene transcription from heterologous herpes simplex virus thymidine kinase promoter having GAL4 DNA binding sites. Thus, it is likely that the internal amino acids 102–189 of TIS11 acts as the intramolecular repressor region. Our data showed that central amino acids 76 –189 containing two zinc finger domains functions as a subregion for the moderate transactivational activity of the fragment 76 –320. In the present study, however, possible inhibitory effect of the amino acids 76 –189 against the fragment 76 –320 activity could not be estimated because deletion of amino acids from 76 to 176 in the fragment TABLE 2
Effects of a MAP Kinase Kinase Inhibitor, PD98059, on PMAInduced Inhibition of TIS11 Transactivational Activity
Test plasmid
Treatment
Relative luciferase activity
GAL-TIS1-320
None PMA PD98059 PD98059 ⫹ PMA None PMA PD98059 PD98059 ⫹ PMA None PMA PD98059 PD98059 ⫹ PMA
523 ⫾ 26 251 ⫾ 13* 501 ⫾ 22 412 ⫾ 21 476 ⫾ 21 299 ⫾ 15* 452 ⫾ 20 407 ⫾ 16 174 ⫾ 16 64 ⫾ 8* 162 ⫾ 15 121 ⫾ 20
DISCUSSION In the present study, we demonstrated that CCCHtype zinc finger protein TIS11, a typical growth factorinducible nuclear protein, possesses transcriptional activation potential when fused downstream of a heterologous DNA-binding domain, and measured its ability to activate transcription of a reporter gene in mammalian cells. This observation suggests that TIS11 can act as a positive regulator of transcription. Analyses using fusion proteins of GAL4 DNA-binding domain and variously deleted TIS11 proteins revealed that the major transactivation region of TIS11 was located at the N-terminal 101 amino acid residues, though the central and C-terminal region (residues 76 to 320) also exhibited moderate transactivational activity. The essential transactivational regions of TIS11 may function through a direct interaction with components of the basal transcription initiation complex or, alternatively, through the interaction with an intermediary coactivator/associated protein(s) that in turn recognizes the initiation complex. Transcriptional activation domains are grouped according to the content of certain amino acid residues with proline-rich, glutamine-rich, and acidic domains being the most
GAL-TIS1-101
GAL-TIS76-320
PMA inhibition (%)
] ] ] ] ] ]
52 18 37 10 63 25
Note. PC12 cells were transiently cotransfected with test plasmid (GAL4DBD, GAL-TIS1-320, GAL-TIS1-101, or GAL-TIS76-320) and pG5E1b-LUC reporter plasmid. The transfectants were stimulated with 1 M PMA for 4 h. The MAP kinase kinase inhibitor PD98059 (final concentration, 50 M) was added 30 min prior to adding PMA. To demonstrate the effects of the reagents, relative luciferase activity is shown after subtracting the activity of the control GAL4DBD transfectant (untreated, 163 ⫾ 8; PMA-treated, 153 ⫾ 12; PD98059treated, 155 ⫾ 11; and PD98059 ⫹ PMA-treated, 151 ⫾ 13) from the value of each test plasmid. Other experimental conditions were the same as those described in the legend to Fig. 2. Results are expressed as means ⫾ SD from four independent experiments, in which measurements were made in triplicate. * P ⬍ 0.05 compared with untreated transfectants.
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76 –320 resulted in complete loss of transactivational activity, as demonstrated with GAL-TIS76-320 and GAL-TIS176-320 (Fig. 1B). The intramolecular repression region probably acts as a buffer for the transactivation function of TIS11. Activation of intracellular signal transduction pathways often results in modulation of function of transcription factors. In the present study, we observed that PMA significantly reduces TIS11 transactivational activity. Activation of protein kinase C by phorbol esters stimulates the MAP kinase signaling cascade, and this appeared to occur through activation of Raf, which lies immediately upstream of MEK (33, 34). Therefore, the effects of protein kinase C and MEK inhibitors on PMA signaling were examined. The specific protein kinase C inhibitor, calphostin C, and the MEK inhibitor, PD98059, each blocked the PMAinduced decrease in transactivational activity of TIS11. These results suggested that activations of protein kinase C and MAP kinase signaling pathway are involved in the PMA-induced repression of transactivational activity of TIS11. Phosphorylation of MAP kinase has been shown to regulate transactivational activities of several transcription factors (35–37). It has been reported that TTP is phosphorylated by several mitogens including PMA, serum, fibroblast growth factor and platelet-derived growth factor, and that Ser220 is the primary site phosphorylated by p42 MAP kinase both in vitro and in intact cells (27). When GAL-mtTIS1-320 (S221A) and GAL-mtTIS76-320 (S221A), in which Ser221 (equivalent to Ser220 in mouse TTP) was replaced with an alanine residue, were used as test plasmids, they showed no effect on the action of PMA, which was indistinguishable from that of wild-type fusion protein (data not shown). This suggested that the phosphorylation of Ser221 is not essential for down-regulation of transactivational activity of TIS11 by PMA, though we cannot rule out the possibility of other minor phosphorylation sites (27). It is also likely that PMA inhibition of TIS11 function results from the modulation of a putative MAP kinase pathway-sensitive transcriptional coactivator protein(s) which associates with TIS11. Recently, PMA has been shown to stimulate translocation of TTP from the nucleus to the cytoplasm in serum-deprived NIH 3T3 cells (26). Thus, this nuclear export may lead to the dissociation of TIS11 from functional transcription complex in PMA-treated PC12 cells, and decrease in level of TIS11 molecule in the transactivational signal. It was reported that YTIS11/CTH2, which is a member of the CCCH zinc finger family in yeast, transactivated the transcription of a reporter gene through its N-terminal activation region (24). Although amino acid sequences of the two CCCH motifs are highly conserved between TIS11 and YTIS11/CTH2, there observed no overall homology in amino acid sequence including the N-terminal region. It was reported that
overexpression of TIS11/TTP markedly inhibited the cell proliferation of yeast as well as TIS11b, CTH1, and YTIS11/CTH2 (24, 25), suggesting that CCCH zinc finger structure is critical for the growth inhibition. Therefore, TIS11 may function as a transactivator that promotes gene expression, which leads to inhibition of the cell growth. It is possible that TIS11 functions as a positive transcription factor that binds to a target DNA sequence or as a coactivator protein that interacts with other transcription factor(s). Identification of downstream genes that associate with TIS11 will provide more insight into molecular mechanisms underlying the function of this protein. ACKNOWLEDGMENTS This work was supported in part by Scientific Research Grants (11672196, 11771456, and High-Tech Research Center Project) from the Ministry of Education, Science, Sports, and Culture of Japan.
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