BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

[1]  J. Pines,et al.  Cyclins and cyclin-dependent kinases: take your partners , 1993 .

[2]  F. Collins,et al.  Somatic mutations in the BRCA1 gene in sporadic ovarian tumours , 1995, Nature Genetics.

[3]  James Steele,et al.  The Salk Institute. , 1968, The Journal of practical nursing.

[4]  W. Jiang,et al.  Cdc2-mediated phosphorylation of the gap junction protein, connexin43, during mitosis. , 1998, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[5]  E. Nigg,et al.  Cyclin‐dependent protein kinases: Key regulators of the eukaryotic cell cycle , 1995, BioEssays : news and reviews in molecular, cellular and developmental biology.

[6]  P. Polakis,et al.  Induction of phosphorylation on BRCA1 during the cell cycle and after DNA damage. , 1997, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[7]  J. D. Thompson,et al.  BRCA1 mutations in a population-based sample of young women with breast cancer. , 1996, The New England journal of medicine.

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

[9]  B. Lewin.  Driving the cell cycle: M phase kinase, its partners, and substrates , 1990, Cell.

[10]  N. Shao,et al.  Induction of apoptosis by the tumor suppressor protein BRCA1. , 1996, Oncogene.

[11]  M. King,et al.  Human, canine and murine BRCA1 genes: sequence comparison among species. , 1996, Human molecular genetics.

[12]  D. Easton,et al.  Genetic linkage analysis in familial breast and ovarian cancer: results from 214 families. The Breast Cancer Linkage Consortium. , 1993, American journal of human genetics.

[13]  M. Provencio,et al.  Germ-line BRCA1 mutations in women with sporadic breast cancer: clinical correlations. , 1998, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[14]  D. Birnbaum,et al.  Loss of heterozygosity in human breast carcinomas in the ataxia telangiectasia, Cowden disease and BRCA1 gene regions , 1997, Oncogene.

[15]  G. Sourvinos,et al.  Decreased BRCA1 expression levels may arrest the cell cycle through activation of p53 checkpoint in human sporadic breast tumors. , 1998, Biochemical and biophysical research communications.

[16]  James E. Thomas,et al.  Subcellular Localization and Analysis of Apparent 180-kDa and 220-kDa Proteins of the Breast Cancer Susceptibility Gene, BRCA1* , 1996, The Journal of Biological Chemistry.

[17]  S. Elledge,et al.  Cdk inhibitors in development and cancer. , 1996, Current opinion in genetics & development.

[18]  T. Hunter,et al.  Phosphopeptide Mapping and Phosphoamino Acid Analysis on Cellulose Thin-Layer Plates , 1998 .

[19]  M. Wigler,et al.  DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J. Mornon,et al.  From BRCA1 to RAP1: a widespread BRCT module closely associated with DNA repair , 1997, FEBS letters.

[21]  D. F. Easton,et al.  Genetic linkage analysis in familial breast and ovarian cancer: Results from 214 families , 1993 .

[22]  I. Verma,et al.  Transcriptional activation by BRCA1 , 1996, Nature.

[23]  H. Ruffner,et al.  CBP/p300 interact with and function as transcriptional coactivators of BRCA1. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[24]  T. Hunter,et al.  Cyclins and cancer II: Cyclin D and CDK inhibitors come of age , 1994, Cell.

[25]  T. Hunter,et al.  Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. , 1991, Methods in enzymology.

[26]  Hui Tian,et al.  BRCA1 physically associates with p53 and stimulates its transcriptional activity , 1998, Oncogene.

[27]  F. Couch,et al.  Localization of BRCA1 and a splice variant identifies the nuclear localization signal , 1997, Molecular and cellular biology.

[28]  N Risch,et al.  Genetic analysis of breast cancer in the cancer and steroid hormone study. , 1991, American journal of human genetics.

[29]  Peer Bork,et al.  A superfamily of conserved domains in DNA damage‐ responsive cell cycle checkpoint proteins , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[30]  K. Cocke,et al.  Identification of a BRCA1-associated kinase with potential biological relevance , 1998, Oncogene.

[31]  R. Fisher.  CDKs and cyclins in transition(s). , 1997, Current opinion in genetics & development.

[32]  R. Beijersbergen,et al.  Cell cycle regulation by the retinoblastoma family of growth inhibitory proteins. , 1996, Biochimica et biophysica acta.

[33]  Steven E. Bayer,et al.  A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. , 1994, Science.

[34]  C. Osborne,et al.  Location of BRCA1 in Human Breast and Ovarian Cancer Cells , 1996, Science.

[35]  S F Altschul,et al.  BRCA1 protein products ... Functional motifs... , 1996, Nature genetics.

[36]  B. Weber,et al.  Relationship of p215BRCA1 to tyrosine kinase signaling pathways and the cell cycle in normal and transformed cells , 1997, Oncogene.

[37]  B. Gusterson,et al.  Combined loss of BRCA1/BRCA2 in grade 3 breast carcinomas , 1996, The Lancet.

[38]  P. Nurse,et al.  Animal cell cycles and their control. , 1992, Annual review of biochemistry.

[39]  C. Liu,et al.  Inactivation of the mouse Brca1 gene leads to failure in the morphogenesis of the egg cylinder in early postimplantation development. , 1996, Genes & development.

[40]  H. Ruffner,et al.  BRCA1 is a cell cycle-regulated nuclear phosphoprotein. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[41]  A. Berchuck,et al.  BRCA1 expression is induced before DNA synthesis in both normal and tumor-derived breast cells. , 1996, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[42]  K. Kinzler,et al.  Cancer-susceptibility genes. Gatekeepers and caretakers. , 1997, Nature.

[43]  B. Koller,et al.  BRCA1 required for transcription-coupled repair of oxidative DNA damage. , 1998, Science.

[44]  Ralph Scully,et al.  Dynamic Changes of BRCA1 Subnuclear Location and Phosphorylation State Are Initiated by DNA Damage , 1997, Cell.

[45]  C. Osborne,et al.  Aberrant Subcellular Localization of BRCA1 in Breast Cancer , 1995, Science.

[46]  D. Slamon,et al.  Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-Δ11b , 1997, Oncogene.

[47]  Y. Chen,et al.  BRCA1 is a 220-kDa nuclear phosphoprotein that is expressed and phosphorylated in a cell cycle-dependent manner. , 1996, Cancer research.

[48]  T. Hunter,et al.  Cytoplasmic displacement of cyclin E-cdk2 inhibitors p21Cip1 and p27Kip1 in anchorage-independent cells , 1998, Oncogene.

[49]  Erich A. Nigg,et al.  Cellular substrates of p34cdc2 and its companion cyclin-dependent kinases , 1993 .

[50]  G. Wahl,et al.  PRC1: a human mitotic spindle-associated CDK substrate protein required for cytokinesis. , 1998, Molecular cell.

[51]  B. Koller,et al.  Brca1 deficiency results in early embryonic lethality characterized by neuroepithelial abnormalities , 1996, Nature Genetics.

[52]  F. Couch,et al.  Localization of BRCA 1 and a Splice Variant Identifies the Nuclear Localization Signal , 1996 .

[53]  Junzhe Xu,et al.  The developmental pattern of Brca1 expression implies a role in differentiation of the breast and other tissues , 1995, Nature Genetics.

[54]  David L. Page,et al.  Decreased expression of BRCA1 accelerates growth and is often present during sporadic breast cancer progression , 1995, Nature Genetics.

[55]  W. Sellers,et al.  Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors , 1996, Molecular and cellular biology.

[56]  J. Parvin,et al.  BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A , 1998, Nature Genetics.

[57]  P. Leder,et al.  Expression of Brca1 is associated with terminal differentiation of ectodermally and mesodermally derived tissues in mice. , 1995, Genes & development.

[58]  E. Lees.  Cyclin dependent kinase regulation. , 1995, Current opinion in cell biology.

[59]  D. Kelsell,et al.  Genetic analysis of the BRCA1 region in a large breast/ovarian family: refinement of the minimal region containing BRCA1. , 1993, Human molecular genetics.

[60]  S. Bull,et al.  Preferential allelic expression can lead to reduced expression of BRCA1 in sporadic breast cancers , 1998, International journal of cancer.

[61]  C. Osborne,et al.  Response: Location of BRCA1 in Human Breast and Ovarian Cancer Cells , 1996, Science.

[62]  S. Neuhausen,et al.  Loss of heterozygosity in familial tumors from three BRCA1-linked kindreds. , 1994, Cancer Research.

[63]  Y. Houvras,et al.  Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDK-inhibitor p21WAF1/CiPl , 1997, Nature.

[64]  M. Skolnick,et al.  BRCA1 mutations in primary breast and ovarian carcinomas. , 1994, Science.

[65]  S. van den Heuvel,et al.  Distinct roles for cyclin-dependent kinases in cell cycle control. , 1993, Science.

[66]  J. E. Celis,et al.  Cell Biology: A Laboratory Handbook , 1997 .

[67]  H. Nguyen,et al.  Cell cycle regulation of BRCA1 messenger RNA in human breast epithelial cells. , 1996, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[68]  C. Mathew,et al.  Identification of missense and truncating mutations in the BRCA1 gene in sporadic and familial breast and ovarian cancer , 1998, Genes, chromosomes & cancer.

[69]  T. Ouchi,et al.  BRCA1 regulates p53-dependent gene expression. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[70]  B. Karlan,et al.  Localization of human BRCA1 and its loss in high-grade, non-inherited breast carcinomas , 1999, Nature Genetics.

[71]  Huichen Wang,et al.  BRCA1 proteins are transported to the nucleus in the absence of serum and splice variants BRCA1a, BRCA1b are tyrosine phosphoproteins that associate with E2F, cyclins and cyclin dependent kinases , 1997, Oncogene.

[72]  J. Rossant,et al.  The Tumor Suppressor Gene Brca1 Is Required for Embryonic Cellular Proliferation in the Mouse , 1996, Cell.

[73]  H. Hanafusa,et al.  Evidence for a transcriptional activation function of BRCA1 C-terminal region. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[74]  Yonghong Xiao,et al.  Association of BRCA1 with Rad51 in Mitotic and Meiotic Cells , 1997, Cell.

[75]  B. Weber,et al.  Mouse Brca1: localization sequence analysis and identification of evolutionarily conserved domains. , 1995, Human molecular genetics.

[76]  L. Meijer,et al.  Chemical inhibitors of cyclin-dependent kinases , 1997 .

[77]  R. Young,et al.  BRCA1 is a component of the RNA polymerase II holoenzyme. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[78]  B. Schulman,et al.  Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[79]  Yumay Chen,et al.  The Nuclear Localization Sequences of the BRCA1 Protein Interact with the Importin-α Subunit of the Nuclear Transport Signal Receptor* , 1996, The Journal of Biological Chemistry.

[80]  P. Sismondi,et al.  Identification of a possible somatic BRCA1 mutation affecting translation efficiency in an early-onset sporadic breast cancer patient. , 1998, Journal of the National Cancer Institute.

[81]  F. Couch,et al.  Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. , 1998, Molecular cell.