RESUMO
The retinoblastoma protein (Rb) inhibits both cell division and apoptosis, but the mechanism by which Rb alternatively regulates these divergent outcomes remains poorly understood. Cyclin-dependent kinases (Cdks) promote cell division by phosphorylating and reversibly inactivating Rb by a hierarchical series of phosphorylation events and sequential conformational changes. The stress-regulated mitogen-activated protein kinase p38 also phosphorylates Rb, but it does so in a cell cycle-independent manner that is associated with apoptosis rather than with cell division. Here, we show that p38 phosphorylates Rb by a novel mechanism that is distinct from that of Cdks. p38 bypasses the cell cycle-associated hierarchical phosphorylation and directly phosphorylates Rb on Ser567, which is not phosphorylated during the normal cell cycle. Phosphorylation by p38, but not Cdks, triggers an interaction between Rb and the human homolog of murine double minute 2 (Hdm2), leading to degradation of Rb, release of E2F1 and cell death. These findings provide a mechanistic explanation as to how Rb regulates cell division and apoptosis through different kinases, and reveal how Hdm2 may functionally link the tumor suppressors Rb and p53.
Assuntos
Apoptose , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Etoposídeo/farmacologia , Humanos , Immunoblotting , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Purinas/farmacologia , Interferência de RNA , Proteína do Retinoblastoma/genética , Roscovitina , Serina/genética , Serina/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
A Vibrio tubiashii hemagglutinin, a protease, was purified by ammonium sulfate precipitation, gel filtration, and hydrophobic interaction chromatography. It agglutinates sheep, chicken, bovine, rabbit, guinea pig, and human erythrocytes. It has a molecular mass of 35 kDa, isoelectric points of 3.5 and 3.7, and is inhibited by ortho-phenanthro line, phosphoramidon, and Zincov. The N-terminal amino acid sequence (Ala-Gln-Ala-Thr-Gly-Thr-Gly- Pro-Gly-Gly-Asn-Gln-Lys-Thr-Gly-Gln- Tyr-Asn-Phe-Gly) has strong homology to other Vibrio proteases.
Assuntos
Metaloproteases/isolamento & purificação , Metaloproteases/metabolismo , Vibrio/enzimologia , Sequência de Aminoácidos , Sulfato de Amônio , Animais , Fracionamento Químico/métodos , Cromatografia/métodos , Glicopeptídeos/farmacologia , Hemaglutinação , Humanos , Ácidos Hidroxâmicos/farmacologia , Ponto Isoelétrico , Metaloproteases/química , Metaloproteases/genética , Dados de Sequência Molecular , Peso Molecular , Fenantrolinas/farmacologia , Inibidores de Proteases/farmacologia , Homologia de Sequência de Aminoácidos , Vibrio/genética , Zinco/análiseRESUMO
An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.
