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G0/G1 Growth Arrest Mediated by a Region Encompassing the Basic Leucine Zipper (bZIP) Domain of the Epstein-Barr Virus Transactivator Zta

1996; Elsevier BV; Volume: 271; Issue: 50 Linguagem: Inglês

10.1074/jbc.271.50.31799

1083-351X

Corinne Cayrol, Erik K. Flemington,

Cancer-related Molecular Pathways

The Epstein-Barr virus (EBV) immediate early transactivator Zta is a basic leucine zipper (bZIP) transcription factor that causes G0/G1 cell cycle arrest through induction of the tumor suppressor protein, p53, and the cyclin-dependent kinase inhibitors, p21 and p27 (Cayrol, C., and Flemington, E. K. (1996) EMBO J. 15, 2748-2759). Here, we report a genetic analysis of Zta-mediated G0/G1 growth arrest and p21 induction. The majority of the Zta transactivation domain can be deleted (ZΔ1-128) without significantly affecting the ability of Zta to elicit growth arrest. A larger amino-terminal deletion (ZΔ1-167) abrogates the ability of Zta to inhibit proliferation, mapping the growth-inhibitory domain to a carboxyl-terminal region encompassing the bZIP domain (amino acids 128-245). The integrity of the bZIP domain is required for growth suppression since a two-amino acid mutant which is defective for homodimerization, fails to induce cell cycle arrest. Western blot analysis of p21 expression in cells expressing Zta mutants reveals that the ability of Zta mutants to cause G0/G1 growth arrest is intimately related to their capacity to induce p21 expression. Together, these data demonstrate that a carboxyl-terminal region of Zta that includes the bZIP domain is sufficient to mediate G0/G1 growth arrest and p21 induction. The Epstein-Barr virus (EBV) immediate early transactivator Zta is a basic leucine zipper (bZIP) transcription factor that causes G0/G1 cell cycle arrest through induction of the tumor suppressor protein, p53, and the cyclin-dependent kinase inhibitors, p21 and p27 (Cayrol, C., and Flemington, E. K. (1996) EMBO J. 15, 2748-2759). Here, we report a genetic analysis of Zta-mediated G0/G1 growth arrest and p21 induction. The majority of the Zta transactivation domain can be deleted (ZΔ1-128) without significantly affecting the ability of Zta to elicit growth arrest. A larger amino-terminal deletion (ZΔ1-167) abrogates the ability of Zta to inhibit proliferation, mapping the growth-inhibitory domain to a carboxyl-terminal region encompassing the bZIP domain (amino acids 128-245). The integrity of the bZIP domain is required for growth suppression since a two-amino acid mutant which is defective for homodimerization, fails to induce cell cycle arrest. Western blot analysis of p21 expression in cells expressing Zta mutants reveals that the ability of Zta mutants to cause G0/G1 growth arrest is intimately related to their capacity to induce p21 expression. Together, these data demonstrate that a carboxyl-terminal region of Zta that includes the bZIP domain is sufficient to mediate G0/G1 growth arrest and p21 induction. INTRODUCTIONThe Epstein-Barr virus (EBV) 1The abbreviations used are: EBVEpstein-Barr virusbZIPbasic leucine zipperTBPTATA box-binding proteinCDKcyclin-dependent kinaseRbretinoblastoma proteinZREZta-responsive elementsPBSphosphate-buffered salineFACSfluorescence-activated cell sorting. lytic switch transactivator Zta (also referred to as BZLF1, EB1, and Zebra) is a sequence-specific DNA-binding protein related to the bZIP family of transcription factors, which plays a key role in the EBV replicative cycle (1Miller G. Fields B.N. Knipe D.M. Virology. Raven Press, New York1990: 1921-1958Google Scholar). By transactivating several early lytic cycle viral promoters, Zta initiates the ordered cascade of EBV gene expression that results in the induction of an estimated 100 or more viral replication associated genes and culminates in virus production (2Kieff E. Liebowitz D. Fields B.N. Knipe D.M. VirologyVirology. Raven Press, New York1990: 1889-1920Google Scholar). In addition to its role in EBV lytic gene expression (3Grogan E.J. Jenson J. Countryman J. Heston L. Gradoville L. Miller G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1332-1336Crossref PubMed Scopus (90) Google Scholar) and replication (4Schepers A. Pich D. Hammerschmidt W. EMBO J. 1993; 12: 3921-3929Crossref PubMed Scopus (123) Google Scholar, 5Fixman E.D. Hayward G.S. Hayward S.D. J. Virol. 1992; : 5030-5039Crossref PubMed Google Scholar), Zta can also regulate the expression of cellular factors such as the growth suppressive cytokine transforming growth factor-β (6Cayrol C. Flemington E.K. J. Virol. 1995; 69: 4206-4212Crossref PubMed Google Scholar).Zta is a member of the bZIP family of transcription factors and binds as a homodimer to multiple AP1 or ZRE (Zta-responsive elements) sites in the promoters of target genes (7Farrell P.J. Rowe D.T. Rooney C.M. Kouzarides T. EMBO J. 1989; 8: 127-132Crossref PubMed Scopus (241) Google Scholar). The carboxyl-terminal bZIP domain of Zta has significant amino acid homology with the basic DNA-binding and dimerization domains of c-Fos (7Farrell P.J. Rowe D.T. Rooney C.M. Kouzarides T. EMBO J. 1989; 8: 127-132Crossref PubMed Scopus (241) Google Scholar) and C/EBP (8Kouzarides T. Packham G. Cook A. Farrell P. Oncogene. 1991; 6: 195-204PubMed Google Scholar). The bZIP domain of Zta mediates homodimerization through a coiled-coil interaction, although Zta lacks the heptad repeat of leucine residues found in the leucine zipper proteins (8Kouzarides T. Packham G. Cook A. Farrell P. Oncogene. 1991; 6: 195-204PubMed Google Scholar, 9Flemington E. Speck S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9459-9463Crossref PubMed Scopus (86) Google Scholar, 10Chang Y.-N. Dong D.L.-Y. Hayward G.S. Hayward S.D. J. Virol. 1990; 64: 3358-3369Crossref PubMed Google Scholar). The amino-terminal region of Zta does not appear to influence dimerization or DNA binding, but plays a role in activation of transcription (11Giot J.-F. Mikaelion I. Buisson M. Manet E. Joab I. Nicolas J.-C. Sergeant A. Nucleic Acids Res. 1991; 19: 1251-1258Crossref PubMed Scopus (69) Google Scholar, 12Lieberman P.M. Berk A.J. Genes Dev. 1991; 5: 2441-2454Crossref PubMed Scopus (162) Google Scholar, 13Flemington E. Borras A.M. Lytle J.P. Speck S. J. Virol. 1992; 66: 922-929Crossref PubMed Google Scholar). The Zta activation domain has been shown to mediate association with the general transcription factor TFIIA (14Chi T. Carey M. Mol. Cell. Biol. 1993; 13: 7045-7055Crossref PubMed Scopus (59) Google Scholar, 15Lieberman P.M. Berk A.J. Genes Dev. 1994; 8: 995-1006Crossref PubMed Scopus (192) Google Scholar) and the TATA box-binding protein TBP (12Lieberman P.M. Berk A.J. Genes Dev. 1991; 5: 2441-2454Crossref PubMed Scopus (162) Google Scholar). Amino acids between 25 and 86 were shown to be critical for this latter interaction (12Lieberman P.M. Berk A.J. Genes Dev. 1991; 5: 2441-2454Crossref PubMed Scopus (162) Google Scholar).We have recently demonstrated that Zta inhibits proliferation by causing cell cycle arrest in G0/G1 in several epithelial tumor cell lines (16Cayrol C. Flemington E. EMBO J. 1996; 15: 2748-2759Crossref PubMed Scopus (159) Google Scholar). Zta-mediated G0/G1 arrest was found to result from induction of the tumor suppressor protein, p53, and the cyclin-dependent kinase (CDK) inhibitors, p21/WAF-1/CIP-1 and p27/KIP-1, two pleiotropic mediators of cell cycle arrest, that inhibit kinase activity of various cyclin-CDK complexes (17El-Diery W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7890) Google Scholar, 18Harper J.W. Adami G.R. Wei N. Deyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5216) Google Scholar, 19Polyak K. Lee M.-H. Erdjument-Bromage H. Koff A. Roberts J.M. Temps P. Massagué J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2048) Google Scholar, 20Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1927) Google Scholar, 21Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3205) Google Scholar). Inactivation of the retinoblastoma tumor suppressor protein (pRb), a known target of cyclin-dependent kinases (22Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4295) Google Scholar), was shown to overcome Zta-mediated G0/G1 arrest, indicating that pRb or pRb-related proteins are involved in the pathway of growth suppression induced by Zta (16Cayrol C. Flemington E. EMBO J. 1996; 15: 2748-2759Crossref PubMed Scopus (159) Google Scholar).Here we report on the ability of Zta mutants to cause G0/G1 growth arrest and demonstrate that growth arrest is independent of the transactivation function of Zta. These results suggest a key role for the bZIP domain and flanking sequences in Zta-mediated growth arrest and induction of p21.DISCUSSIONSeveral conclusions can be derived from this limited genetic analysis. First, a tight correlation exists between growth arrest and induction of p21. We showed previously that induction of p21 by Zta is mediated through p53 (16Cayrol C. Flemington E. EMBO J. 1996; 15: 2748-2759Crossref PubMed Scopus (159) Google Scholar), and, indeed, induction of p53 follows the same genetics as that of p21 (data not shown). Therefore, this pathway is likely to play an important role in transmitting Zta growth arrest signals.These studies also demonstrate that a carboxyl-terminal region of Zta encompassing the bZIP domain is sufficient to cause a G0/G1 growth arrest. Deletion of the amino-terminal half of Zta does not significantly affect its capacity to block G1/S progression since the mutant, ZΔ1-128, inhibited the percentage of cells in S phase, as efficiently as wild type Zta (respectively 3.9% and 5.7% versus 31% in untransfected cells). We note, however, that the G2/M population is not reduced in ZΔ1-128 expressing cells indicating an inability of cells to traverse through G2/M. This suggests that sequences between amino acids 1 and 128 are required for progression through the G2/M checkpoint in the context of other Zta-mediated alterations in cellular growth control pathways. This apparent G2/M arrest is not evident in Zdbm1-transfected cells indicating that the inability of ZΔ1-128 to transactivate ZRE containing cellular promoters is not the sole defect leading to a block in G2/M progression. Instead, it is possible that these sequences contribute to some functional interactions with factors involved in controlling the G2/M checkpoint control.The ability of the Zta DNA-binding mutant, Zdbm1, to efficiently induce cellular growth arrest provides additional support for the idea that the transactivation function of Zta is not required for inducing growth arrest. We have previously reported that Zta DNA binding mutants, including Zdbm1, can activate certain promoters in transient reporter assays probably through protein-protein interactions (23Flemington E.K. Lytle J.P. Cayrol C. Borras A.M. Speck S.H. Mol. Cell. Biol. 1994; 14: 3041-3052Crossref PubMed Scopus (40) Google Scholar). However, in these studies, we found that Zdbm1 and wild type Zta elicited transcriptional activation through distinct promoter elements and they may therefore have distinct promoter specificities. Although we cannot rule out the possibility that transcriptional activation plays a role in eliciting cellular growth arrest, we favor a model whereby Zta induces growth arrest through protein-protein interactions with key cell cycle control proteins. Previous studies demonstrated an interaction between p53 and the dimerization domain of Zta (26Zhang Z. Gutsch D. Kenney S. J. Virol. 1994; 14: 1929-1938Google Scholar). Moreover, in this study, it was suggested that the Zta-mediated post-transcriptional induction of p53 might arise through masking of p53 sequences involved in targeting it for ubiquitin-dependent degradation (26Zhang Z. Gutsch D. Kenney S. J. Virol. 1994; 14: 1929-1938Google Scholar). Since the results presented here point to a critical role for Zta's bZIP domain in eliciting G0/G1 growth arrest and p21 induction, this is a reasonable possibility.Results presented here suggest that sequences upstream from the bZIP domain, amino acids 128 to 167, are also required for p21 induction and for cellular growth arrest. At this time we don't know whether this sequence might contribute to or stabilize interactions with key cellular proteins or whether this region provides conformational contributions to the bZIP structure.The finding that the carboxyl-terminal region of Zta encompassing the bZIP domain is sufficient to block cell proliferation further emphasizes the critical role of bZIP domains and bZIP transcription factors in the control of cell proliferation. Other bZIP factors have previously been implicated in affecting cell growth control pathways. For example, the AP1 family bZIP factors, c-Fos and c-Jun, promote cell proliferation in some settings and are associated with differentiation in several cellular differentiation model systems (27Szabo E. Preis L.H. Birrer M.J. Cell Growth Differ. 1994; 5: 439-446PubMed Google Scholar, 28Gandarillas A. Watt F.M. Mamm. Genome. 1995; 6: 680-682Crossref PubMed Scopus (14) Google Scholar, 29Gandarillas A. Watt F.M. Oncogene. 1995; 11: 1403-1407PubMed Google Scholar, 30de Groot R.P. Kruyt F.A. van der Saag P.T. Kruijer W. EMBO J. 1990; 9: 1831-1837Crossref PubMed Scopus (97) Google Scholar, 31de Groot R.P. Kruijer W. Cell Growth Differ. 1991; 2: 631-636PubMed Google Scholar). The Caenorhabditis elegans bZIP protein Ces-2 has been shown to induce programmed cell death (32Metzstein M.M. Hengartner M.O. Tsung N. Ellis R.E. Horvitz H.R. Nature. 1996; 382: 545-547Crossref PubMed Scopus (139) Google Scholar). Further investigations into mechanisms driving Zta-mediated G0/G1 growth arrest should reveal additional insights into the role of bZIP transcription factors in cell proliferation. INTRODUCTIONThe Epstein-Barr virus (EBV) 1The abbreviations used are: EBVEpstein-Barr virusbZIPbasic leucine zipperTBPTATA box-binding proteinCDKcyclin-dependent kinaseRbretinoblastoma proteinZREZta-responsive elementsPBSphosphate-buffered salineFACSfluorescence-activated cell sorting. lytic switch transactivator Zta (also referred to as BZLF1, EB1, and Zebra) is a sequence-specific DNA-binding protein related to the bZIP family of transcription factors, which plays a key role in the EBV replicative cycle (1Miller G. Fields B.N. Knipe D.M. Virology. Raven Press, New York1990: 1921-1958Google Scholar). By transactivating several early lytic cycle viral promoters, Zta initiates the ordered cascade of EBV gene expression that results in the induction of an estimated 100 or more viral replication associated genes and culminates in virus production (2Kieff E. Liebowitz D. Fields B.N. Knipe D.M. VirologyVirology. Raven Press, New York1990: 1889-1920Google Scholar). In addition to its role in EBV lytic gene expression (3Grogan E.J. Jenson J. Countryman J. Heston L. Gradoville L. Miller G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1332-1336Crossref PubMed Scopus (90) Google Scholar) and replication (4Schepers A. Pich D. Hammerschmidt W. EMBO J. 1993; 12: 3921-3929Crossref PubMed Scopus (123) Google Scholar, 5Fixman E.D. Hayward G.S. Hayward S.D. J. Virol. 1992; : 5030-5039Crossref PubMed Google Scholar), Zta can also regulate the expression of cellular factors such as the growth suppressive cytokine transforming growth factor-β (6Cayrol C. Flemington E.K. J. Virol. 1995; 69: 4206-4212Crossref PubMed Google Scholar).Zta is a member of the bZIP family of transcription factors and binds as a homodimer to multiple AP1 or ZRE (Zta-responsive elements) sites in the promoters of target genes (7Farrell P.J. Rowe D.T. Rooney C.M. Kouzarides T. EMBO J. 1989; 8: 127-132Crossref PubMed Scopus (241) Google Scholar). The carboxyl-terminal bZIP domain of Zta has significant amino acid homology with the basic DNA-binding and dimerization domains of c-Fos (7Farrell P.J. Rowe D.T. Rooney C.M. Kouzarides T. EMBO J. 1989; 8: 127-132Crossref PubMed Scopus (241) Google Scholar) and C/EBP (8Kouzarides T. Packham G. Cook A. Farrell P. Oncogene. 1991; 6: 195-204PubMed Google Scholar). The bZIP domain of Zta mediates homodimerization through a coiled-coil interaction, although Zta lacks the heptad repeat of leucine residues found in the leucine zipper proteins (8Kouzarides T. Packham G. Cook A. Farrell P. Oncogene. 1991; 6: 195-204PubMed Google Scholar, 9Flemington E. Speck S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9459-9463Crossref PubMed Scopus (86) Google Scholar, 10Chang Y.-N. Dong D.L.-Y. Hayward G.S. Hayward S.D. J. Virol. 1990; 64: 3358-3369Crossref PubMed Google Scholar). The amino-terminal region of Zta does not appear to influence dimerization or DNA binding, but plays a role in activation of transcription (11Giot J.-F. Mikaelion I. Buisson M. Manet E. Joab I. Nicolas J.-C. Sergeant A. Nucleic Acids Res. 1991; 19: 1251-1258Crossref PubMed Scopus (69) Google Scholar, 12Lieberman P.M. Berk A.J. Genes Dev. 1991; 5: 2441-2454Crossref PubMed Scopus (162) Google Scholar, 13Flemington E. Borras A.M. Lytle J.P. Speck S. J. Virol. 1992; 66: 922-929Crossref PubMed Google Scholar). The Zta activation domain has been shown to mediate association with the general transcription factor TFIIA (14Chi T. Carey M. Mol. Cell. Biol. 1993; 13: 7045-7055Crossref PubMed Scopus (59) Google Scholar, 15Lieberman P.M. Berk A.J. Genes Dev. 1994; 8: 995-1006Crossref PubMed Scopus (192) Google Scholar) and the TATA box-binding protein TBP (12Lieberman P.M. Berk A.J. Genes Dev. 1991; 5: 2441-2454Crossref PubMed Scopus (162) Google Scholar). Amino acids between 25 and 86 were shown to be critical for this latter interaction (12Lieberman P.M. Berk A.J. Genes Dev. 1991; 5: 2441-2454Crossref PubMed Scopus (162) Google Scholar).We have recently demonstrated that Zta inhibits proliferation by causing cell cycle arrest in G0/G1 in several epithelial tumor cell lines (16Cayrol C. Flemington E. EMBO J. 1996; 15: 2748-2759Crossref PubMed Scopus (159) Google Scholar). Zta-mediated G0/G1 arrest was found to result from induction of the tumor suppressor protein, p53, and the cyclin-dependent kinase (CDK) inhibitors, p21/WAF-1/CIP-1 and p27/KIP-1, two pleiotropic mediators of cell cycle arrest, that inhibit kinase activity of various cyclin-CDK complexes (17El-Diery W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7890) Google Scholar, 18Harper J.W. Adami G.R. Wei N. Deyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5216) Google Scholar, 19Polyak K. Lee M.-H. Erdjument-Bromage H. Koff A. Roberts J.M. Temps P. Massagué J. Cell. 1994; 78: 59-66Abstract Full Text PDF PubMed Scopus (2048) Google Scholar, 20Toyoshima H. Hunter T. Cell. 1994; 78: 67-74Abstract Full Text PDF PubMed Scopus (1927) Google Scholar, 21Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3205) Google Scholar). Inactivation of the retinoblastoma tumor suppressor protein (pRb), a known target of cyclin-dependent kinases (22Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4295) Google Scholar), was shown to overcome Zta-mediated G0/G1 arrest, indicating that pRb or pRb-related proteins are involved in the pathway of growth suppression induced by Zta (16Cayrol C. Flemington E. EMBO J. 1996; 15: 2748-2759Crossref PubMed Scopus (159) Google Scholar).Here we report on the ability of Zta mutants to cause G0/G1 growth arrest and demonstrate that growth arrest is independent of the transactivation function of Zta. These results suggest a key role for the bZIP domain and flanking sequences in Zta-mediated growth arrest and induction of p21.