RESUMO
Members of the Cool protein family contain SH3, Dbl, and pleckstrin homology domains and are binding partners for the p21-activated kinase (PAK). Using the yeast two-hybrid screen, we identified Cbl-b as a Cool family binding partner. We co-immunoprecipitated endogenous Cool and Cbl-b from a variety of breast cancer cell lines. The Cool-Cbl-b interaction requires the SH3 domain of Cool and competes with the binding of PAK to Cool proteins. Expression of Cbl-b effectively blocks the ability of Cool-2 to stimulate PAK, thus providing an additional mechanism, aside from catalyzing receptor ubiquitination, by which Cbl-b acts as a negative regulator for signaling activities requiring PAK activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Animais , Ligação Competitiva , Neoplasias da Mama/metabolismo , Células COS , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Dados de Sequência Molecular , Fosfoproteínas/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Fatores de Troca de Nucleotídeo Guanina Rho , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido , Quinases Ativadas por p21 , Domínios de Homologia de src/fisiologiaRESUMO
OBJECTIVE: Resveratrol, a constituent found in grapes and various other plants, has been shown to have chemo-preventive activity against cancer, and specifically demonstrated to induce apoptosis by p53-dependent pathways in murine cells. The goal of this research was to identify the role of p53-dependent or p53-independent pathways in the induction of apoptosis in human breast cancer cells by this natural product. DESIGN: A number of human breast cancer cell lines, as well as a control of a wild-type line (astrocytoma N 1321N1), were investigated for induction of apoptosis by resveratrol using both microscopic evaluation and DNA fragmentation assays. Concurrently, we established the p53 gene status (wild-type or mutant) of each cell line by Western blot using p53-specific antibody. RESULTS: Apoptosis induced by resveratrol was found to occur only in breast cancer cells expressing wild-type p53 but not in mutant p53-expressing cells. CONCLUSIONS: We therefore conclude that the natural product, resveratrol, induces apoptosis in breast cancer cells via p53-dependent pathways.