RESUMO
The Bcl-2 oncoprotein is a potent inhibitor of apoptosis and is overexpressed in a variety of different malignancies. Bcl-2 function is regulated through heterodimerization with other members of the Bcl-2 protein family. In addition, several proteins that are not members of the Bcl-2 family can bind to Bcl-2, including BAG-1 protein. In this study, we screened for proteins that bind to Bcl-2, and isolated two additional members of the BAG-1 protein family, BAG-3 and BAG-4. The BAG-4 protein that we cloned also corresponds to the recently isolated suppressor of death domains (SODD) protein, a molecule that binds and inhibits signaling by tumor necrosis factor receptor 1 (TNFR1). Both BAG-3 and BAG-4/SODD were found to physically associate with Bcl-2, and both proteins are well conserved from human to mouse. A region of homology, comprising 68 amino acids, is present in the carboxyl termini of BAG-3 and BAG-4/SODD, and this region corresponds with sequences termed BAG domains that are found in other members of the BAG-1 protein family. In BAG-3 and BAG-4/SODD, the BAG domains appear to constitute the Bcl-2 binding regions of these molecules. BAG-3 and BAG-4/SODD, like BAG-1, were also shown to bind to Hsp70 inside the cell. Moreover, BAG-3 overexpression modestly inhibited apoptosis resulting from cytokine deprivation of IL-3-dependent 32D cells. Together, our findings demonstrate that other members of the BAG-1 protein family, namely BAG-3 and BAG-4/SODD, bind to Bcl-2 and provide a potential link between pathways regulated by Bcl-2 and pathways regulated by Hsp70, as well as TNFR1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Proteínas Reguladoras de Apoptose , Baculoviridae/metabolismo , Proteínas de Transporte/isolamento & purificação , Linhagem Celular , Clonagem Molecular , Sequência Conservada , DNA Complementar/metabolismo , Proteínas de Ligação a DNA , Biblioteca Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Insetos , Interleucina-3/metabolismo , Camundongos , Dados de Sequência Molecular , Família Multigênica , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Transcrição , TransfecçãoRESUMO
The Bcl-2 oncoprotein is an integral membrane protein localized primarily to the outer membrane of the mitochondria. The precise molecular mechanism responsible for the antiapoptotic action of Bcl-2 remains unknown. Two cysteine residues are found in Bcl-2 and these residues are well-conserved across species. The first cysteine (cys(155)) is located in the alpha5 domain, a region important for the ion channel properties of Bcl-2, while the second cysteine (cys(226)) is located in the carboxyl-terminal membrane anchor domain. In this study, we found that replacement of both cysteines with serine residues generated a mutant protein that retained the ability to homodimerize and heterodimerize with proapoptotic Bax protein in vitro. In whole cells, the mutant protein efficiently heterodimerized with Bax, but exhibited impaired homodimerizationrelative to wild-type Bcl-2. The mutant protein was also less efficient than wild-type Bcl-2 at suppressing caspase activation, DNA fragmentation, and loss of viability during IL-3 withdrawal-induced apoptosis. Together, the data indicate that the cysteine residues in Bcl-2 contribute, but are not absolutely essential, to the ability of Bcl-2 to homodimerize, heterodimerize with Bax, and suppress apoptosis.
