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. 1997 Nov;17(11):6459–6464. doi: 10.1128/mcb.17.11.6459

Identification of a viral kinase that phosphorylates specific E2Fs and pocket proteins.

S Pajovic 1, E L Wong 1, A R Black 1, J C Azizkhan 1
PMCID: PMC232498  PMID: 9343408

Abstract

The transcription factor E2F and its regulation by pRB and related pocket proteins are central to cell cycle control in higher eukaryotes. Much of our knowledge of this regulation has come from studies using immediate-early proteins of DNA tumor viruses. Previously, we reported that the 72-kDa immediate-early region 1 gene product of the human cytomegalovirus, IE72, transactivates the dihydrofolate reductase promoter through the E2F site and that it physically interacts with E2F1 (M. J. Margolis, S. Pajovic, E. L. Wong, M. Wade, R. Jupp, J. A. Nelson, and J. C. Azizkhan, J. Virol. 69:7759-7767, 1995). In this study, we further characterized the mechanism by which IE72 modulates E2F-dependent transcription. In vitro phosphorylation reactions using gel-purified bacterially expressed proteins revealed that IE72 is a kinase that autophosphorylates and phosphorylates E2F1, -2, and -3 (but not E2F4 or -5) and the RB-related pocket proteins p130 and p107 (but not pRB). The region of IE72 spanning amino acids 173 to 197 shows a high level of homology to the ATP binding sites in over 500 kinases. The kinase-negative protein IE72deltaATP, from which this region has been deleted, cannot activate E2F-dependent transcription. The kinase activity of IE72 is also required for its ability to reduce the association of E2F4 with p107 and p130. Taken together, these data suggest that the kinase activity of IE72 is required for E2F-dependent transcriptional activation and that this is likely to result from phosphorylation of specific members of the E2F and pocket protein families by IE72.

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Selected References

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  1. Albrecht T., Boldogh I., Fons M., Lee C. H., AbuBakar S., Russell J. M., Au W. W. Cell-activation responses to cytomegalovirus infection relationship to the phasing of CMV replication and to the induction of cellular damage. Subcell Biochem. 1989;15:157–202. [PubMed] [Google Scholar]
  2. Allende J. E., Allende C. C. Protein kinases. 4. Protein kinase CK2: an enzyme with multiple substrates and a puzzling regulation. FASEB J. 1995 Mar;9(5):313–323. doi: 10.1096/fasebj.9.5.7896000. [DOI] [PubMed] [Google Scholar]
  3. Bagchi S., Raychaudhuri P., Nevins J. R. Phosphorylation-dependent activation of the adenovirus-inducible E2F transcription factor in a cell-free system. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4352–4356. doi: 10.1073/pnas.86.12.4352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beijersbergen R. L., Kerkhoven R. M., Zhu L., Carlée L., Voorhoeve P. M., Bernards R. E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vivo. Genes Dev. 1994 Nov 15;8(22):2680–2690. doi: 10.1101/gad.8.22.2680. [DOI] [PubMed] [Google Scholar]
  5. Blake M. C., Azizkhan J. C. Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo. Mol Cell Biol. 1989 Nov;9(11):4994–5002. doi: 10.1128/mcb.9.11.4994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blake M. C., Jambou R. C., Swick A. G., Kahn J. W., Azizkhan J. C. Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter. Mol Cell Biol. 1990 Dec;10(12):6632–6641. doi: 10.1128/mcb.10.12.6632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  8. Chellappan S., Kraus V. B., Kroger B., Munger K., Howley P. M., Phelps W. C., Nevins J. R. Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4549–4553. doi: 10.1073/pnas.89.10.4549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cobrinik D., Whyte P., Peeper D. S., Jacks T., Weinberg R. A. Cell cycle-specific association of E2F with the p130 E1A-binding protein. Genes Dev. 1993 Dec;7(12A):2392–2404. doi: 10.1101/gad.7.12a.2392. [DOI] [PubMed] [Google Scholar]
  11. Fagan R., Flint K. J., Jones N. Phosphorylation of E2F-1 modulates its interaction with the retinoblastoma gene product and the adenoviral E4 19 kDa protein. Cell. 1994 Sep 9;78(5):799–811. doi: 10.1016/s0092-8674(94)90522-3. [DOI] [PubMed] [Google Scholar]
  12. Ginsberg D., Vairo G., Chittenden T., Xiao Z. X., Xu G., Wydner K. L., DeCaprio J. A., Lawrence J. B., Livingston D. M. E2F-4, a new member of the E2F transcription factor family, interacts with p107. Genes Dev. 1994 Nov 15;8(22):2665–2679. doi: 10.1101/gad.8.22.2665. [DOI] [PubMed] [Google Scholar]
  13. Hagemeier C., Caswell R., Hayhurst G., Sinclair J., Kouzarides T. Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein. EMBO J. 1994 Jun 15;13(12):2897–2903. doi: 10.1002/j.1460-2075.1994.tb06584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hayhurst G. P., Bryant L. A., Caswell R. C., Walker S. M., Sinclair J. H. CCAAT box-dependent activation of the TATA-less human DNA polymerase alpha promoter by the human cytomegalovirus 72-kilodalton major immediate-early protein. J Virol. 1995 Jan;69(1):182–188. doi: 10.1128/jvi.69.1.182-188.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Helin K., Lees J. A., Vidal M., Dyson N., Harlow E., Fattaey A. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell. 1992 Jul 24;70(2):337–350. doi: 10.1016/0092-8674(92)90107-n. [DOI] [PubMed] [Google Scholar]
  16. Hermiston T. W., Malone C. L., Witte P. R., Stinski M. F. Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter. J Virol. 1987 Oct;61(10):3214–3221. doi: 10.1128/jvi.61.10.3214-3221.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hiebert S. W., Blake M., Azizkhan J., Nevins J. R. Role of E2F transcription factor in E1A-mediated trans activation of cellular genes. J Virol. 1991 Jul;65(7):3547–3552. doi: 10.1128/jvi.65.7.3547-3552.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ivey-Hoyle M., Conroy R., Huber H. E., Goodhart P. J., Oliff A., Heimbrook D. C. Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F. Mol Cell Biol. 1993 Dec;13(12):7802–7812. doi: 10.1128/mcb.13.12.7802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jackson S. P. Regulating transcription factor activity by phosphorylation. Trends Cell Biol. 1992 Apr;2(4):104–108. doi: 10.1016/0962-8924(92)90014-e. [DOI] [PubMed] [Google Scholar]
  20. Kaelin W. G., Jr, Krek W., Sellers W. R., DeCaprio J. A., Ajchenbaum F., Fuchs C. S., Chittenden T., Li Y., Farnham P. J., Blanar M. A. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. doi: 10.1016/0092-8674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  21. Kitagawa M., Higashi H., Suzuki-Takahashi I., Segawa K., Hanks S. K., Taya Y., Nishimura S., Okuyama A. Phosphorylation of E2F-1 by cyclin A-cdk2. Oncogene. 1995 Jan 19;10(2):229–236. [PubMed] [Google Scholar]
  22. La Thangue N. B. DRTF1/E2F: an expanding family of heterodimeric transcription factors implicated in cell-cycle control. Trends Biochem Sci. 1994 Mar;19(3):108–114. doi: 10.1016/0968-0004(94)90202-x. [DOI] [PubMed] [Google Scholar]
  23. La Thangue N. B. E2F and the molecular mechanisms of early cell-cycle control. Biochem Soc Trans. 1996 Feb;24(1):54–59. doi: 10.1042/bst0240054. [DOI] [PubMed] [Google Scholar]
  24. Lees J. A., Saito M., Vidal M., Valentine M., Look T., Harlow E., Dyson N., Helin K. The retinoblastoma protein binds to a family of E2F transcription factors. Mol Cell Biol. 1993 Dec;13(12):7813–7825. doi: 10.1128/mcb.13.12.7813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lukac D. M., Manuppello J. R., Alwine J. C. Transcriptional activation by the human cytomegalovirus immediate-early proteins: requirements for simple promoter structures and interactions with multiple components of the transcription complex. J Virol. 1994 Aug;68(8):5184–5193. doi: 10.1128/jvi.68.8.5184-5193.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lukas J., Petersen B. O., Holm K., Bartek J., Helin K. Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression. Mol Cell Biol. 1996 Mar;16(3):1047–1057. doi: 10.1128/mcb.16.3.1047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Margolis M. J., Pajovic S., Wong E. L., Wade M., Jupp R., Nelson J. A., Azizkhan J. C. Interaction of the 72-kilodalton human cytomegalovirus IE1 gene product with E2F1 coincides with E2F-dependent activation of dihydrofolate reductase transcription. J Virol. 1995 Dec;69(12):7759–7767. doi: 10.1128/jvi.69.12.7759-7767.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moberg K., Starz M. A., Lees J. A. E2F-4 switches from p130 to p107 and pRB in response to cell cycle reentry. Mol Cell Biol. 1996 Apr;16(4):1436–1449. doi: 10.1128/mcb.16.4.1436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moran E. DNA tumor virus transforming proteins and the cell cycle. Curr Opin Genet Dev. 1993 Feb;3(1):63–70. doi: 10.1016/s0959-437x(05)80342-9. [DOI] [PubMed] [Google Scholar]
  30. Nevins J. R. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science. 1992 Oct 16;258(5081):424–429. doi: 10.1126/science.1411535. [DOI] [PubMed] [Google Scholar]
  31. Pearson B. E., Nasheuer H. P., Wang T. S. Human DNA polymerase alpha gene: sequences controlling expression in cycling and serum-stimulated cells. Mol Cell Biol. 1991 Apr;11(4):2081–2095. doi: 10.1128/mcb.11.4.2081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Peeper D. S., Keblusek P., Helin K., Toebes M., van der Eb A. J., Zantema A. Phosphorylation of a specific cdk site in E2F-1 affects its electrophoretic mobility and promotes pRB-binding in vitro. Oncogene. 1995 Jan 5;10(1):39–48. [PubMed] [Google Scholar]
  33. Poma E. E., Kowalik T. F., Zhu L., Sinclair J. H., Huang E. S. The human cytomegalovirus IE1-72 protein interacts with the cellular p107 protein and relieves p107-mediated transcriptional repression of an E2F-responsive promoter. J Virol. 1996 Nov;70(11):7867–7877. doi: 10.1128/jvi.70.11.7867-7877.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reichel R. R. Regulation of E2F/cyclin A-containing complex upon retinoic acid-induced differentiation of teratocarcinoma cells. Gene Expr. 1992;2(3):259–271. [PMC free article] [PubMed] [Google Scholar]
  35. Sidle A., Palaty C., Dirks P., Wiggan O., Kiess M., Gill R. M., Wong A. K., Hamel P. A. Activity of the retinoblastoma family proteins, pRB, p107, and p130, during cellular proliferation and differentiation. Crit Rev Biochem Mol Biol. 1996 Jun;31(3):237–271. doi: 10.3109/10409239609106585. [DOI] [PubMed] [Google Scholar]
  36. Slansky J. E., Farnham P. J. Introduction to the E2F family: protein structure and gene regulation. Curr Top Microbiol Immunol. 1996;208:1–30. doi: 10.1007/978-3-642-79910-5_1. [DOI] [PubMed] [Google Scholar]
  37. Smith E. J., Nevins J. R. The Rb-related p107 protein can suppress E2F function independently of binding to cyclin A/cdk2. Mol Cell Biol. 1995 Jan;15(1):338–344. doi: 10.1128/mcb.15.1.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Spector D. J., Tevethia M. J. Identification of a human cytomegalovirus virus DNA segment that complements an adenovirus 5 immediate early mutant. Virology. 1986 Jun;151(2):329–338. doi: 10.1016/0042-6822(86)90053-x. [DOI] [PubMed] [Google Scholar]
  39. Tevethia M. J., Spector D. J. Complementation of an adenovirus 5 immediate early mutant by human cytomegalovirus. Virology. 1984 Sep;137(2):428–431. doi: 10.1016/0042-6822(84)90236-8. [DOI] [PubMed] [Google Scholar]
  40. Thalmeier K., Synovzik H., Mertz R., Winnacker E. L., Lipp M. Nuclear factor E2F mediates basic transcription and trans-activation by E1a of the human MYC promoter. Genes Dev. 1989 Apr;3(4):527–536. doi: 10.1101/gad.3.4.527. [DOI] [PubMed] [Google Scholar]
  41. Wade M., Kowalik T. F., Mudryj M., Huang E. S., Azizkhan J. C. E2F mediates dihydrofolate reductase promoter activation and multiprotein complex formation in human cytomegalovirus infection. Mol Cell Biol. 1992 Oct;12(10):4364–4374. doi: 10.1128/mcb.12.10.4364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Xu M., Sheppard K. A., Peng C. Y., Yee A. S., Piwnica-Worms H. Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation. Mol Cell Biol. 1994 Dec;14(12):8420–8431. doi: 10.1128/mcb.14.12.8420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zheng J., Knighton D. R., ten Eyck L. F., Karlsson R., Xuong N., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. Biochemistry. 1993 Mar 9;32(9):2154–2161. doi: 10.1021/bi00060a005. [DOI] [PubMed] [Google Scholar]

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