リン酸化によるRB蛋白質とp53の生理機能の制御

• Project/Area Number: 09254271
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas (A)
• Allocation Type: Single-year Grants
• Research Institution: National Cancer Center Research Institute and Research Center for Innovative Oncology, National Cancer Center Hospital East
• Principal Investigator: 田矢 洋一 国立がんセンター研究所, 生物学部, 室長 (60133641)
• Project Period (FY): 1997 – 1999
• Project Status: Completed (Fiscal Year 1999)
• Budget Amount: ¥18,000,000 (Direct Cost: ¥18,000,000); Fiscal Year 1999: ¥6,000,000 (Direct Cost: ¥6,000,000); Fiscal Year 1998: ¥6,000,000 (Direct Cost: ¥6,000,000); Fiscal Year 1997: ¥6,000,000 (Direct Cost: ¥6,000,000)
• Keywords: RB蛋白質 / p53 / Cdk4 / Cdk2 / E2F / カフェイン / ATM / Chk2 / サイクリンD1 / サイクリンE / ATR / MDM2 / アセチル化

Research Abstract

G1/S移行期には、サイクリンD1-Cdk4、サイクリンE-Cdk2の順で、少なくと2種類のキナーゼがRB蛋白質のリン酸化を行うと示唆されている。しかし、なぜ何種類ものキナーゼが関与するのかということと、それらがどういうように使い分けされているのかということは、ほとんどわかっていない。本研究はそれを解明することを目的とした。RB蛋白質上には約13ケ所のリン酸化部位があるが、われわれにはこれらの部位を特異的に認識する抗体をほぼすべて作製してきたので、これを積極的に活用した。そして、次の諸点を明らかにした。
1)RB蛋白質上のCdk4特異的部位がリン酸化されているとE2F-1との複合体形成が見られないのに対して、Cdk2特異的部位がリン酸化されていても複合体は形成される。 2)RB蛋白質のC末端側には、リン酸化に際してCdk2が先ず結合する部位が存在する。
一方、p53上にも約13ケ所のリン酸化部位がある。これらのリン酸化の生理的意義を研究するために、われわれはこれらの部位を特異的に認識する抗体をほぼすべて作製してきた。そして、すでに、Ser15のリン酸化がp53の活性化に重要であることを明らかにしてきたが、他の部位についてもこれらの抗体を用いて意義を明らかにすることを試みた。そして、次の諸点を明らかにした。
1)Ser20のリン酸化もp53の活性化に重要であり、かつ、そのリン酸化はDNA傷害や複製のチェックポイントに重要な働きをするChk2とChk1らしい。 2)カフェインが放射線に対する感受性を高めることは以前から知られていたが、これはATMとATRのキナーゼ活性を阻害することによって引き起こすらしいことを明らかにした。 3)p53のSer46のリン酸化がp53によるアポトーシス誘導能を制御していることを見いだした。

Research Products

• [Publications] Tibbetts, R.S.et al.: "A role for ATR in the DNA damage-induced phosphorylation of p53"Genes & Dev.,. 13. 152-157 (1999)
• [Publications] Adams, P.D.et al.: "The retinoblastoma protein contains a C-terminal motif that targets it for phosphorylation by cyclin/cdk2 complexes"Mol. Cell. Biol.. 19. 1068-1080 (1999)
• [Publications] Shieh, S.-Y.et al.: "DNA damage-inducible phosphorylation at N-terminal sites including a novel site, serine 20, requires oligomerization of p53"EMBO J.. 18. 1815-1823 (1999)
• [Publications] Sarkaria, J.N.et al.: "Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine"Cancer Res.. 59. 4375-4382 (1999)
• [Publications] Sugimoto, M.et al.: "Regulation of CDK4 activity by a novel CDK4 binding protein, p34SEI-1"Genes & Dev.. 13. 3027-3033 (1999)
• [Publications] Shieh, S-Y.et al.: "The human homologues of checkpoint kinases Chk1 and Cds1 (Chk2) phosporylate p53 at multiple DNA damage inducible sites"Genes & Dev.. 14. 289-300 (2000)
• [Publications] Duckett, D.R.et al.: "hMutSα-and hMutLα-dependent phosphorylation of p53 in response to DNA methylator damage"Proc. Natl. Acad. Sci. USA. 96. 12384-12388 (1999)
• [Publications] Ariumi, Y.et al.: "HTLV-1 Tax oncoprotein represses the p53-mediated transactivation function through coactivator CBP sequestration"Oncogene. (in press). (2000)
• [Publications] Ashcroft, M.et al.: "Stress signal utilize multiple pathways to stabilize p53 : Involvement of MDM2 expressio, cytoplasmic localization of p53 and ARF independent nucleolar sequestration of MDM2"Mol. Cell. Biol.. (in press). (2000)
• [Publications] Sanchez Prieto: "A role for the p38^<MAPK> pathway in the transcriptional activation of p53 upon genotoxic stress by chemotherapeutic agents"Cancer Res.. (in press).
• [Publications] Pandita, T.et al.: "Ionizing radiation activates the ATM kinase throughout the cell cycle"Oncogene. (in press). (2000)
• [Publications] Kapoor, M.et al.: "Cooperative phosphorylation at multiple sites is required to activate p53 in response to UV radiation"Oncogene. 19. 358-364 (2000)
• [Publications] Cuddihy, A.R.et al.: "Double-strand-RNA-activated protein kinase PKR enhances transcriptional activation by tumor suppressor p53"Mol. Cell. Biol.. 19. 2475-2484 (1999)
• [Publications] Nakagawa, K.et al.: "Requirement of ATM in the phosphorylation of the human p53 protein at serine 15 following DNA double-strand breaks"Mol. Cell. Biol.. 19. 2828-2834 (1999)
• [Publications] Brugarolas, J.et al.: "Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma proteoin-mediated G1 arrest after γ-irradiation"Proc. Natl. Acad. Sci. USA. 96. 1002-1007 (1999)
• [Publications] Nagata, Y.et al.: "The stabilization mechanism of mutant-type p53 by impared ubiquitinatopn : the loss of wild-type p53 function and the hsp90 association"Oncogene. 18. 6037-6049 (1999)
• [Publications] Burma, S.et al.: "DNA-dependent protein kinase-independent activation of p53 in response to DNA damage"J. Biol. Chem.. 274. 17139-17143 (1999)
• [Publications] Araki, R.et al.: "Enhanced phosphorylation of Ser18 on p53 following DNA damage in DNA-PKcs-deficient cells"Cancer Res.. 59. 3543-3546 (1999)
• [Publications] Banin,S.et al.: "Enhanced phosphorylation of p53 by ATM in response to DNA damage." Science. 281. 1674-1677 (1998)
• [Publications] Canman,C.E.et al.: "Activation of the ATM kinase by ionizing radiation and phosphorylation of p53." Science. 281. 1677-1679 (1998)
• [Publications] Khanna,K.K.et al.: "ATM associates with and phosphorylates p53: mapping the region of interaction." Nature Genet.20. 398-400 (1998)
• [Publications] Lu,H.et al.: "Ultraviolet radiation,but not gamma radiation or etoposide-induced DNA damage,results in the phosphorylation of the murine p53 protein at serine-389" Proc.Natl.Acad.Sci.USA. 95. 6399-6402 (1998)
• [Publications] Tibbetts,R.S.et al.: "ATR is a DNA damage-responsive protein kinase that phosphorylates the p53 tumor suppressor protein." Genes & Dev.13. 152-157 (1999)
• [Publications] Adams,P.D.et al.: "The retinoblastoma protein contains a C-terminal motif that targets it for phosphorylation by cyclin/cdk2 complexes." Mol.Cell.Biol.19. 1068-1080 (1999)
• [Publications] Cuddihy,A.R.et al.: "The interferon-inducible protein kinase PKR mediates the transcriptional activation of the tumor suppressor p53." Mol.Cell.Biol.19. 2475-2484 (1999)
• [Publications] Nakagawa,K.et al.: "Requirement of ATM in the phosphorylation of the human p53 protein at serine 15." Mol.Cell.Biol.19. 2828-2834 (1999)
• [Publications] Brugarolas,J.et al.: "Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G1 arrest after g-irradiation." Proc.Natl.Acad.Sci.USA. 96. 1002-1007 (1999)
• [Publications] Shieh,S.-Y.et al.: "DNA damage-inducible phosphorylation at N-terminal sites including a novel site,serine 20,requiresoliqomerization of p53." EMBO J.(in press). (1999)
• [Publications] Nagata,Y.et al.: "The stabilization mechanism of mutant-type p53 by impaired ubiquitination: the loss of wild-type p53 function and the hsp90 association" Oncogene. (in press). (1999)
• [Publications] Taya,Y.: "RB-kinases and RB-binding proteins:new points of view." Trends Biochem.Sci.22. 14-17 (1997)
• [Publications] Shieh,S-Y.et al.: "DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2." Cell. 91. 325-334 (1997)
• [Publications] Siliciano,J.D.et al.: "DNA damage induces phosphorylation of the amino terminus of p53." Genes Dev.11. 3471-3481 (1997)
• [Publications] Ko,L.J.et al.: "p53 is phosphorylated by CDK7/cyclinH in a p36/MAT1 dependent manner." Mol.Cell.Biol.17. 7220-7229 (1997)
• [Publications] Taya,Y.et al.: "Cdk4-cyclin D1 and Cdk2-cyclin E/A phosphorylate different sites in the RB protein." Genomic Instability and Immortality in Cancer. 229-231 (1997)
• [Publications] Lu,H.et al.: "Ultraviolet rediation,but not gamma radiation or etoposide-induced DNA damage,results in the phosphorylation of the murine p53 protein at serine-389" Proc.Natl.Acad.Sci.USA. (in press).