Supramolecular Complex Formation between Rad6 and Proteins of the p53 Pathway during DNA Damage-Induced Response

ABSTRACT The HR6A and -B genes, homologues of the yeast Rad6 gene, encode ubiquitin-conjugating enzymes that are required for postreplication repair of DNA and damage-induced mutagenesis. Using surface plasmon resonance, we show here that HR6 protein (referred as Rad6) physically interacts with p53. Analysis of proteins coimmunoprecipitated with Rad6 antibody from metabolically labeled normal MCF10A human breast epithelial cells not only confirmed Rad6-p53 interactions in vivo but also demonstrated for the first time that exposure of MCF10A cells to cisplatin or adriamycin (ADR) induces recruitment of p14ARF into Rad6-p53 complexes. Further analysis of ADR-induced p53 response showed that stable Rad6-p53-p14ARF complex formation is associated with a parallel increase and decrease in monoubiquitinated and polyubiquitinated p53, respectively, and arrest in G2/M phase of the cell cycle. Interestingly, the ADR-induced suppression of p53 polyubiquitination correlated with a corresponding decline in intact Hdm2 protein levels. Treatment of MCF10A cells with MG132, a 26S proteasome inhibitor, effectively stabilized monoubiquitinated p53 and rescued ADR-induced downregulation of Hdm2. These data suggest that ADR-induced degradation of Hdm2 occurs via the ubiquitin-proteasome pathway. Rad6 is present in both the cytoplasmic and nuclear compartments of normal MCF10A cells, although in response to DNA damage it is predominantly found in the nucleus colocalizing with ubiquitinated p53, whereas Hdm2 is undetectable. Consistent with in vivo data, results from in vitro ubiquitination assays show that Rad6 mediates addition of one (mono-) to two (multimono-) ubiquitin molecules on p53 and that inclusion of Mdm2 is essential for its polyubiquitination. The data presented in the present study suggest that Rad6-p53-p14ARF complex formation and p53 ubiquitin modification are important damage-induced responses that perhaps determine the fidelity of DNA postreplication repair.

[1]  L. Prakash,et al.  Recombination and mutagenesis in rad6 mutants of Saccharomyces cerevisiae: Evidence for multiple functions of the RAD6 gene , 2004, Molecular and General Genetics MGG.

[2]  L. Prakash.  Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations , 2004, Molecular and General Genetics MGG.

[3]  Boris Pfander,et al.  RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO , 2002, Nature.

[4]  H. Heng,et al.  Rad6 overexpression induces multinucleation, centrosome amplification, abnormal mitosis, aneuploidy, and transformation. , 2002, Cancer research.

[5]  L. Prakash,et al.  Requirement of RAD5 and MMS2 for Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae , 2002, Molecular and Cellular Biology.

[6]  D. Lane,et al.  Cocompartmentalization of p53 and Mdm2 is a major determinant for Mdm2-mediated degradation of p53. , 2001, Experimental cell research.

[7]  Curtis C. Harris,et al.  Functional Interaction of p53 and BLM DNA Helicase in Apoptosis* , 2001, The Journal of Biological Chemistry.

[8]  E. Appella,et al.  Post-translational modifications and activation of p53 by genotoxic stresses. , 2001, European journal of biochemistry.

[9]  W. Baek,et al.  The levels of MDM2 protein are decreased by a proteasome-mediated proteolysis prior to caspase-3-dependent pRb and PARP cleavages. , 2001, Journal of Korean medical science.

[10]  V. Rotter,et al.  Structural and functional involvement of p53 in BER in vitro and in vivo , 2001, Oncogene.

[11]  F. Sauer,et al.  Ubiquitin-activating/conjugating activity of TAFII250, a mediator of activation of gene expression in Drosophila. , 2000, Science.

[12]  W. Sumanasekera,et al.  The human RAD18 gene product interacts with HHR6A and HHR6B. , 2000, Nucleic acids research.

[13]  D. Lane,et al.  An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo , 2000, Oncogene.

[14]  E. Rosen,et al.  P53-independent down-regulation of Mdm2 in human cancer cells treated with adriamycin. , 2000, Molecular cell biology research communications : MCBRC.

[15]  M. Osley,et al.  Rad6-dependent ubiquitination of histone H2B in yeast. , 2000, Science.

[16]  Margaret Ashcroft,et al.  Regulation of p53 stability , 1999, Oncogene.

[17]  A. Levine,et al.  P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[18]  R. Govers,et al.  The ubiquitin-proteasome system and endocytosis. , 1999, Journal of cell science.

[19]  C. Pickart,et al.  Noncanonical MMS2-Encoded Ubiquitin-Conjugating Enzyme Functions in Assembly of Novel Polyubiquitin Chains for DNA Repair , 1999, Cell.

[20]  P. Herrlich,et al.  DNA damage induced p53 stabilization: no indication for an involvement of p53 phosphorylation , 1999, Oncogene.

[21]  R. Honda,et al.  Association of p19ARF with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53 , 1999, The EMBO journal.

[22]  C. Prives.  Signaling to p53 Breaking the MDM2–p53 Circuit , 1998, Cell.

[23]  Kevin Ryan,et al.  The alternative product from the human CDKN2A locus, p14ARF, participates in a regulatory feedback loop with p53 and MDM2 , 1998, The EMBO journal.

[24]  Yue Xiong,et al.  ARF Promotes MDM2 Degradation and Stabilizes p53: ARF-INK4a Locus Deletion Impairs Both the Rb and p53 Tumor Suppression Pathways , 1998, Cell.

[25]  R. Stein,et al.  Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. , 1998, Biochemistry.

[26]  Hirofumi Tanaka,et al.  Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53 , 1997, FEBS letters.

[27]  H. Wang,et al.  Altered P53 conformation. , 1997, International journal of oncology.

[28]  Yoichi Taya,et al.  DNA Damage-Induced Phosphorylation of p53 Alleviates Inhibition by MDM2 , 1997, Cell.

[29]  C. Harris,et al.  Interaction of p53 with the human Rad51 protein. , 1997, Nucleic acids research.

[30]  S. Lauder,et al.  Yeast DNA Repair Proteins Rad6 and Rad18 Form a Heterodimer That Has Ubiquitin Conjugating, DNA Binding, and ATP Hydrolytic Activities* , 1997, The Journal of Biological Chemistry.

[31]  Satya Prakash,et al.  Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein , 1997, Molecular and cellular biology.

[32]  P. Howley,et al.  Physical Interaction between Specific E2 and Hect E3 Enzymes Determines Functional Cooperativity* , 1997, The Journal of Biological Chemistry.

[33]  Stephen N. Jones,et al.  Regulation of p53 stability by Mdm2 , 1997, Nature.

[34]  M. Oren,et al.  Mdm2 promotes the rapid degradation of p53 , 1997, Nature.

[35]  A. Levine.  p53, the Cellular Gatekeeper for Growth and Division , 1997, Cell.

[36]  P. Howley,et al.  Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation , 1997, Molecular and cellular biology.

[37]  L. Prakash,et al.  Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[38]  M. Oren,et al.  p53 in growth control and neoplasia. , 1996, Biochimica et biophysica acta.

[39]  A. Fornace,et al.  The two faces of tumor suppressor p53. , 1996, The American journal of pathology.

[40]  K Tanaka,et al.  Structure and functions of the 20S and 26S proteasomes. , 1996, Annual review of biochemistry.

[41]  C. Lawrence,et al.  DNA polymerase zeta and the control of DNA damage induced mutagenesis in eukaryotes. , 1996, Cancer surveys.

[42]  M. Oren,et al.  Biochemical properties and biological effects of p53. , 1995, Current opinion in genetics & development.

[43]  M. Hochstrasser.  Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. , 1995, Current opinion in cell biology.

[44]  Aaron Ciechanover,et al.  The ubiquitin-proteasome proteolytic pathway , 1994, Cell.

[45]  D. Ecker,et al.  Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant , 1994, Molecular and cellular biology.

[46]  P. Sung,et al.  Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. , 1994, Genes & development.

[47]  C. Lawrence.  The RAD6 DNA repair pathway in Saccharomyces cerevisiae: What does it do, and how does it do it? , 1994, BioEssays : news and reviews in molecular, cellular and developmental biology.

[48]  Bert Vogelstein,et al.  Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53 , 1993, Nature.

[49]  P. Sung,et al.  DNA repair genes and proteins of Saccharomyces cerevisiae. , 1993, Annual review of genetics.

[50]  P. Meltzer,et al.  Amplification of a gene encoding a p53-associated protein in human sarcomas , 1992, Nature.

[51]  A. Hagemeijer,et al.  Localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to chromosomes Xq24-q25 and 5q23-q31. , 1992, Genomics.

[52]  B. Vogelstein,et al.  Participation of p53 protein in the cellular response to DNA damage. , 1991, Cancer research.

[53]  J. Hoeijmakers,et al.  Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[54]  P. Sung,et al.  Stable ester conjugate between the Saccharomyces cerevisiae RAD6 protein and ubiquitin has no biological activity. , 1991, Journal of molecular biology.

[55]  P. Sung,et al.  Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N‐end‐recognizing E3 enzyme. , 1991, The EMBO journal.

[56]  F. Fabre,et al.  A similar defect in UV-induced mutagenesis conferred by the rad6 and rad18 mutations of Saccharomyces cerevisiae. , 1991, Mutation research.

[57]  J. Russo,et al.  Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. , 1990, Cancer research.

[58]  P. Sung,et al.  Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[59]  D. Ecker,et al.  A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. , 1989, Science.

[60]  P. Sung,et al.  The RAD6 protein of Saccharomyces cerevisiae polyubiquitinates histones, and its acidic domain mediates this activity. , 1988, Genes & development.

[61]  Alexander Varshavsky,et al.  The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme , 1987, Nature.

[62]  H. Busch.  2 – The Eukaryotic Nucleus , 1977 .