RESUMO
Two-hybrid searches with the tumor suppressor MMAC1/PTEN isolated the proteins hDLG and hMAST205. Further two-hybrid analysis and microtiter plate binding assays localized the sites of interaction to PDZ domains from hDLG and hMAST205 and the PDZ binding domain at the COOH terminus of MMAC1/PTEN. A synthetic peptide derived from the MMAC1/PTEN PDZ binding domain (MMAC1/PTEN-PDZBD) was used to coprecipitate proteins from A431 human cell lysate. The recovered proteins were resolved by SDS-PAGE and immobilized on a nitrocellulose membrane. Treatment of this membrane with an anti-hDLG antibody identified a Mr 140,000 band, consistent with the size of hDLG. Treatment of this membrane with the MMAC1/PTEN-PDZBD peptide identified a single prominent band of slightly larger than Mr 200,000 (Mr 200,000 kDa). Threonine phosphorylation of the MMAC1/ PTEN-PDZBD peptide inhibited both microtiter plate binding to the hDLG and hMAST205 PDZ domains and coprecipitation of the Mr 140,000 and > 200,000 proteins, but promoted coprecipitation of proteins of approximately Mr 90,000 and Mr 120,000 from A431 cell lysate. This result suggests phosphorylation of the MMAC1/PTEN PDZ binding domain can both inhibit and promote PDZ interactions.
Assuntos
Proteínas de Transporte/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas/metabolismo , Treonina/metabolismo , Proteínas Supressoras de Tumor , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteína 1 Homóloga a Discs-Large , Guanilato Quinases , Humanos , Proteínas de Membrana , Camundongos , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Células Tumorais CultivadasRESUMO
There are several lines of evidence indicating that the carboxy-terminal region of the tumor suppressor protein BRCA1 is a functionally significant domain. Using the yeast two-hybrid and in vitro biochemical assays, we show that a protein, CtIP, interacts specifically with the carboxy-terminal segment of human BRCA1 from residues 1602-1863. A germ line truncation mutation, Y1853ter, that removes the last 11 amino acids from the carboxy-terminus of BRCA1, abolishes not only its transcriptional activation function, but also binding to CtIP. The function of CtIP is unknown, but its reported association with a transcriptional repressor CtBP lends further support that it may have a role in transcription. A sequence based screen of a panel of 89 tumor cell line cDNAs for mutations in the CtIP coding region identified five missense variants. In the pancreatic carcinoma cell line, BxPC3, the non-conservative lysine to glutamic acid change at codon 337 is accompanied with apparent loss of heterozygosity or non-expression of the wild type allele. Thus it is plausible that CtIP may itself be a tumor suppressor acting in the same pathway as BRCA1.
Assuntos
Proteína BRCA1/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Sequência de Bases , Genes Reporter , Mutação em Linhagem Germinativa , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Recent work has shown that the murine BRCA2 tumor suppressor protein interacts with the murine RAD51 protein. This interaction suggests that BRCA2 participates in DNA repair. Residues 3196-3232 of the murine BRCA2 protein were shown to be involved in this interaction. Here, we report the detailed mapping of additional domains that are involved in interactions between the human homologs of these two proteins. Through yeast two-hybrid and biochemical assays, we demonstrate that the RAD51 protein interacts specifically with the eight evolutionarily conserved BRC motifs encoded in exon 11 of brca2 and with a similar motif found in a Caenorhabditis elegans hypothetical protein. Deletion analysis demonstrates that residues 98-339 of human RAD51 interact with the 59-residue minimal region that is conserved in all BRC motifs. These data suggest that the BRC repeats function to bind RAD51.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Proteína BRCA2 , Predisposição Genética para Doença , Humanos , Ligação Proteica , Rad51 Recombinase , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiaeRESUMO
Os autores analisam 137 pacientes portadores de doenca litiasica vesicular e coledociana, operados no periodo de janeiro de 1975 a dezembro de 1979. Os pacientes foram divididos em grupos: o Grupo I formado por 106 pacientes, apresentava colelitiase isoladamente; o Grupo II formado por 31 pacientes, apresentava colelitiase com coledocolitiase ou papilite associada.Os pacientes foram analisados, segundo faixa etaria, sexo, incisao operatoria, tipos de cirurgia e complicacoes imediatas e tardias.Foram revistos no pos-operatorio tardio (um ano ou mais apos a cirurgia) 75 pacientes dos dois grupos. Estes pacientes foram classificados, em funcao das queixas em A, B, C, tendo sido rotulados 61 pacientes (81,33%) no grupo A, 7 (9,33%) no grupo B e 7 (9,33%) no grupo C