PROTEIN KINASES IN YEAST DNA CHECKPOINT PATHWAYS

Summary

Principal Investigator: David F Stern
Abstract: DNA damage response networks are common targets for carcinogenic mutations and may be excellent targets for response-modifying drugs. The central core of these networks consists of phosphatidyl inositol 3-kinase-related kinases (PIKKs), e.g. human Atm and Atr, which activate downstream pathways in response to DNA damage. PIKKs phosphorylate components of complexes that are recruited to damageinduced foci, including the Mrell/Rad50/Nbsl complex, 53BP1, and Brcal, and activate effector kinases Chkl and Chk2 that amplify the signal and help convey it to effector proteins. In budding yeast, the PIKK Mecl (Atm/Atr ortholog) is coupled to protein kinase Rad53 (Chk2 ortholog) through Mecl-dependent phosphorylation of the "mediator" Rad9 (unrelated to human Rad9). Mammalian BRCA1, 53BP1, and now MDC1/NFBD1 are candidate Rad9 orthologs that resemble Rad9 in having twin carboxyl terminal BRCT domains. Like Rad9, they undergo PIKK-dependent phosphorylation, form damage-induced complexes with other checkpoint proteins and are recruited to sites of DNA damage. MDC1/NFBD1 is intimately involved in DNA damage response mechanisms. The PIKK-Mediator-Chk core of DNA damage signaling will be investigated using both the simpler yeast cascade Mecl to Rad9 to Rad53, and the more complex interactions of ATM/ATR in regulation of NFBD1. Aim 1. A combination of biochemical and molecular methods will be used to determine the mechanism through which the mediator Rad9 and the PIKK Mecl regulate Rad53. Aim 2.. siRNA oligonucleotide knockdowns will be used to identify unique and overlapping functions of BRCA1, 53BP1, and NFBD1 in DNA checkpoint responses. Aim 3. Molecular techniques will be used for functional analysis of NFBD1/MDC1 in the activities identified in Aim 2.
Funding Period: 1999-07-01 - 2010-04-30
more information: NIH RePORT

Top Publications

  1. pmc Centrosomal Chk2 in DNA damage responses and cell cycle progression
    Amnon Golan
    Department of Pathology, Yale School of Medicine, New Haven, CT, USA
    Cell Cycle 9:2647-56. 2010
  2. pmc Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain
    Jinping Liu
    Beijing Key Laboratory of DNA Damage Response and College of Life Science, Capital Normal University, Beijing 100048, China
    Nucleic Acids Res 40:3898-912. 2012
  3. pmc NFBD1/MDC1 regulates Cav1 and Cav2 independently of DNA damage and p53
    Kathleen A Wilson
    Yale University, 333 Cedar Street, P O Box 208023, New Haven, CT 06520, USA
    Mol Cancer Res 9:766-81. 2011
  4. pmc Deciphering protein kinase specificity through large-scale analysis of yeast phosphorylation site motifs
    Janine Mok
    Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA
    Sci Signal 3:ra12. 2010
  5. pmc NFBD1/MDC1, 53BP1 and BRCA1 have both redundant and unique roles in the ATM pathway
    Kathleen A Wilson
    Department of Pathology and Graduate Program in Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA
    Cell Cycle 7:3584-94. 2008
  6. ncbi Regulation of the Rad53 protein kinase in signal amplification by oligomer assembly and disassembly
    Nianhan Jia-Lin Ma
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA
    Cell Cycle 7:808-17. 2008
  7. ncbi Activation of the checkpoint kinase Rad53 by the phosphatidyl inositol kinase-like kinase Mec1
    Jia Lin Ma
    Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA
    J Biol Chem 281:3954-63. 2006
  8. ncbi In situ detection of specific DNA double strand breaks using rolling circle amplification
    Jia Li
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA
    Cell Cycle 4:1767-73. 2005
  9. ncbi DNA damage regulates Chk2 association with chromatin
    Jia Li
    Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA
    J Biol Chem 280:37948-56. 2005
  10. ncbi Regulation of CHK2 by DNA-dependent protein kinase
    Jia Li
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA
    J Biol Chem 280:12041-50. 2005

Scientific Experts

  • Keqiong Ye
  • Kathleen A Wilson
  • Lyuben Tsvetkov
  • David F Stern
  • Jia Li
  • Jinping Liu
  • Amnon Golan
  • Janine Mok
  • Nianhan Jia-Lin Ma
  • Jia Lin Ma
  • Shukun Luo
  • Hongchang Zhao
  • Ji Liao
  • Chunying Yang
  • Bo Xu
  • Jing Li
  • Xingzhi Xu
  • Michael Snyder
  • Miri Jwa
  • Clarence S M Chan
  • Richelle Sopko
  • Claudio De Virgilio
  • Attila Remenyi
  • Benjamin E Turk
  • Hugo Y K Lam
  • Sirlester A Parker
  • Elisabetta Cameroni
  • Bruce Stillman
  • Stacy Piccirillo
  • Elah Pick
  • Hengyao Niu
  • Yasmine Nadler
  • Mark B Gerstein
  • Brenda J Andrews
  • Harriet Kluger
  • Holly E Sassi
  • Douglas L Sheridan
  • Jia Lin Nianhan Ma
  • Yi Jun Sheu
  • Philip M Kim
  • Xiuqiong Zhou
  • Nancy M Hollingsworth
  • Grace R Jeschke
  • Ved Desai
  • Matthew Good
  • Wendell A Lim
  • Soo Jung Lee
  • Jimmy K Duong
  • C S H Young
  • Paul M Lizardi

Detail Information

Publications12

  1. pmc Centrosomal Chk2 in DNA damage responses and cell cycle progression
    Amnon Golan
    Department of Pathology, Yale School of Medicine, New Haven, CT, USA
    Cell Cycle 9:2647-56. 2010
    ..These and other data suggest a model in which binding of Chk2 to the centrosome at multiple cell cycle junctures controls co-localization of Chk2 with other cell cycle and centrosomal regulators...
  2. pmc Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain
    Jinping Liu
    Beijing Key Laboratory of DNA Damage Response and College of Life Science, Capital Normal University, Beijing 100048, China
    Nucleic Acids Res 40:3898-912. 2012
    ..Our results suggest a novel mechanism for the regulation of MDC1 function through T4 phosphorylation and FHA-mediated dimerization...
  3. pmc NFBD1/MDC1 regulates Cav1 and Cav2 independently of DNA damage and p53
    Kathleen A Wilson
    Yale University, 333 Cedar Street, P O Box 208023, New Haven, CT 06520, USA
    Mol Cancer Res 9:766-81. 2011
    ..Furthermore, like Cav1-depletion, NFBD1 shRNA increases Erk phosphorylation. Thus, Cav1 could act as a mediator of the DNA-damage independent effects of NFBD1 in mitogenic signaling...
  4. pmc Deciphering protein kinase specificity through large-scale analysis of yeast phosphorylation site motifs
    Janine Mok
    Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA
    Sci Signal 3:ra12. 2010
    ..Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of previously unknown kinase substrates across eukaryotes...
  5. pmc NFBD1/MDC1, 53BP1 and BRCA1 have both redundant and unique roles in the ATM pathway
    Kathleen A Wilson
    Department of Pathology and Graduate Program in Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA
    Cell Cycle 7:3584-94. 2008
    ..We extend previous findings in cancer cells and mouse cells that NFBD1 is upstream of 53BP1 and BRCA1 to primary human cells. Furthermore, NFBD1 promotes BRCA1 IRIF through both 53BP1-dependent and 53BP1-independent mechanisms...
  6. ncbi Regulation of the Rad53 protein kinase in signal amplification by oligomer assembly and disassembly
    Nianhan Jia-Lin Ma
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA
    Cell Cycle 7:808-17. 2008
    ..The results support the conclusions that Rad53/Chk2 homo-oligomerization is an evolutionarily conserved mechanism that drives Rad53/Chk2 activation and promotes signal amplification in DNA damage responses...
  7. ncbi Activation of the checkpoint kinase Rad53 by the phosphatidyl inositol kinase-like kinase Mec1
    Jia Lin Ma
    Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA
    J Biol Chem 281:3954-63. 2006
    ..Moreover, this work has substantiated a model for PIKK-independent amplification of Rad53 activation (and by extension, activation of other Chk2 orthologs) mediated by inter-Rad53 phosphorylation...
  8. ncbi In situ detection of specific DNA double strand breaks using rolling circle amplification
    Jia Li
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA
    Cell Cycle 4:1767-73. 2005
    ..This methodology provides a novel approach for investigation of DNA recombination, DNA repair, and checkpoint controls in mammalian cells...
  9. ncbi DNA damage regulates Chk2 association with chromatin
    Jia Li
    Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA
    J Biol Chem 280:37948-56. 2005
    ....
  10. ncbi Regulation of CHK2 by DNA-dependent protein kinase
    Jia Li
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA
    J Biol Chem 280:12041-50. 2005
    ..Ionizing radiation-induced Chk2 phosphorylation was wortmannin-sensitive in ATM-defective cells with depleted ATR. These results suggest that DNA-PK augments ATM and ATR in activation of Chk2 by DNA damage...
  11. ncbi Interaction of chromatin-associated Plk1 and Mcm7
    Lyuben Tsvetkov
    Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut 06511, USA
    J Biol Chem 280:11943-7. 2005
    ..The strongest interaction between endogenous Plk1 and Mcm7 was detected in a soluble chromatin fraction. These findings suggest a new function for Plk1 in coordination of DNA replication and mitotic events...
  12. ncbi Phosphorylation of Plk1 at S137 and T210 is inhibited in response to DNA damage
    Lyuben Tsvetkov
    Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut 06510, USA
    Cell Cycle 4:166-71. 2005
    ..These data suggest a participation of DNA damage checkpoints in regulation of the signaling pathways upstream of Plk1...