RESUMO
A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins.
Assuntos
Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Cromossomos Humanos Par 3 , Proteínas Fúngicas/genética , Filogenia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/química , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Sequência Conservada , Dano ao DNA , Primers do DNA , DNA Complementar , Drosophila melanogaster/genética , Feminino , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Biblioteca Gênica , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Gravidez , Proteínas Quinases/biossíntese , Proteínas Quinases/química , Recombinação Genética , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Homologia de Sequência de Aminoácidos , Linfócitos TAssuntos
Ciclo Celular , Reparo do DNA , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Fosfotransferases/fisiologia , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , 1-Fosfatidilinositol 4-Quinase , Sequência de Aminoácidos , Animais , Dano ao DNA , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Humanos , Dados de Sequência Molecular , Fosfotransferases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Filogenia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
When complexed with the intracellular protein FKBP12, rapamycin is a potent immunosuppressant and an inhibitor of a mitogen-stimulated signalling pathway that leads to activation of p70 S6 kinase (p70S6k) and cyclin-dependent kinases (CDKs). A recently cloned FKBP12-rapamycin-associated protein (FRAP/RAFT) is the likely mediator of these effects. Using FRAP variants that do not bind FKBP12-rapamycin, we demonstrate here that FRAP is a rapamycin-sensitive regulator of p70S6k in vivo and that the kinase activity of FRAP is required for this regulation. In addition, we show that FRAP autophosphorylates in vitro. Consistent with an essential role for FRAP kinase activity in vivo, autophosphorylation of FRAP is inhibited by FKBP12-rapamycin. Deletion studies indicate that the kinase activity of FRAP alone is not sufficient for control of p70S6k and that an amino-terminal domain in FRAP is also required.
Assuntos
Imunofilinas , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Proteínas de Choque Térmico/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Polienos/farmacologia , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases S6 Ribossômicas , Sirolimo , Proteínas de Ligação a TacrolimoRESUMO
The structurally related natural products rapamycin and FK506 bind to the same intracellular receptor, FKBP12, yet the resulting complexes interfere with distinct signalling pathways. FKBP12-rapamycin inhibits progression through the G1 phase of the cell cycle in osteosarcoma, liver and T cells as well as in yeast, and interferes with mitogenic signalling pathways that are involved in G1 progression, namely with activation of the protein p70S6k (refs 5, 11-13) and cyclin-dependent kinases. Here we isolate a mammalian FKBP-rapamycin-associated protein (FRAP) whose binding to structural variants of rapamycin complexed to FKBP12 correlates with the ability of these ligands to inhibit cell-cycle progression. Peptide sequences from purified bovine FRAP were used to isolate a human cDNA clone that is highly related to the DRR1/TOR1 and DRR2/TOR2 gene products from Saccharomyces cerevisiae. Although it has not been previously demonstrated that either of the DRR/TOR gene products can bind the FKBP-rapamycin complex directly, these yeast genes have been genetically linked to a rapamycin-sensitive pathway and are thought to encode lipid kinases.
