RESUMO
We have previously shown that ASPP1 and ASPP2 are specific activators of p53; one mechanism by which wild-type p53 is tolerated in human breast carcinomas is through loss of ASPP activity. We have further shown that 53BP2, which corresponds to a C-terminal fragment of ASPP2, acts as a dominant negative inhibitor of p53 (ref. 1). Hence, an inhibitory form of ASPP resembling 53BP2 could allow cells to bypass the tumor-suppressor functions of p53 and the ASPP proteins. Here, we characterize such a protein, iASPP (inhibitory member of the ASPP family), encoded by PPP1R13L in humans and ape-1 in Caenorhabditis elegans. iASPP is an evolutionarily conserved inhibitor of p53; inhibition of iASPP by RNA-mediated interference or antisense RNA in C. elegans or human cells, respectively, induces p53-dependent apoptosis. Moreover, iASPP is an oncoprotein that cooperates with Ras, E1A and E7, but not mutant p53, to transform cells in vitro. Increased expression of iASPP also confers resistance to ultraviolet radiation and to cisplatin-induced apoptosis. iASPP expression is upregulated in human breast carcinomas expressing wild-type p53 and normal levels of ASPP. Inhibition of iASPP could provide an important new strategy for treating tumors expressing wild-type p53.
Assuntos
Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Osteossarcoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas E1A de Adenovirus/fisiologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose , Western Blotting , Neoplasias da Mama/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Transformação Celular Neoplásica , Cisplatino/farmacologia , Resistência a Medicamentos/genética , Feminino , Citometria de Fluxo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Genes ras/fisiologia , Humanos , Técnicas In Vitro , Microscopia de Fluorescência , Mutação , Oligonucleotídeos Antissenso/farmacologia , Osteossarcoma/genética , Interferência de RNA , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Domínios de Homologia de src/fisiologiaRESUMO
We demonstrate here that the E2F1 induced by DNA damage can bind to and promote the apoptotic function of p53 via the cyclin A binding site of E2F1. This function of E2F1 does not require its DP-1 binding, DNA binding, or transcriptional activity and is independent of mdm2. All the cyclin A binding E2F family members can interact and cooperate with p53 to induce apoptosis. This suggests a novel role for E2F in regulating apoptosis in response to DNA damage. Cyclin A, but not cyclin E, prevents E2F1 from interacting and cooperating with p53 to induce apoptosis. However, in response to DNA damage, cyclin A levels decrease, with a concomitant increase in E2F1-p53 complex formation. These results suggest that the binding of E2F1 to p53 can specifically stimulate the apoptotic function of p53 in response to DNA damage.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular , Ciclina A/metabolismo , Dano ao DNA , Proteínas Nucleares , Proteínas Proto-Oncogênicas c-bcl-2 , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Separação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Citometria de Fluxo , Humanos , Proteínas de Neoplasias/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Fatores de Transcrição/química , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Raios Ultravioleta , Proteína X Associada a bcl-2RESUMO
Despite the wealth of information on the regulation of wild-type p53 function by phosphorylation, nothing is known about the biological effect of phosphorylation on mutant p53. Here we show that p53H175 is phosphorylated like wild-type p53 in cells of the same background. Ser(392) nonphosphorylatable p53 mutants p53H175A392 and p53W248A392 more potently transformed rat embryo fibroblasts in cooperation with the ras oncogene than p53H175S392 and p53W248S392. p53H175A392 also had an enhanced ability to confer cellular resistance to the cytotoxic effect of cisplatin and UV radiation. This correlated with p53H175A392 being a more potent dominant negative mutant than p53H175 in inhibiting the apoptotic functions of wild-type p53. Moreover, p53H175E392, which mimics the phosphorylated form of p53H175, was less able to confer cellular resistance to DNA-damaging agents. p53H175 and p53W248 are phosphorylated like wild-type p53 in cells of the same background. Ser(392) nonphosphorylated p53 was present in human breast tumors expressing mutant p53 including p53H175. Together, these results demonstrated a novel function of Ser(392) phosphorylation in regulating the oncogenic function of mutant p53.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação , Osteossarcoma/genética , Osteossarcoma/metabolismo , Fosforilação , Ratos , Serina/metabolismo , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Raios UltravioletaRESUMO
We show here that the cell cycle-dependent DNA-binding and transcriptional activity of p53 correlates with E2F expression in human primary fibroblasts. E2F1 binds and stimulates DNA-binding, transactivation and apoptotic functions of p53 but not p63 and p73. E2F1 binds residues 347-370 of p53 and enhances nuclear retention of Ser315 phosphorylated p53. This regulation of p53 by E2F1 is cell cycle dependent, as the cellular distribution of Ser315 phosphorylated p53 is associated with the periodic expression of E2F and cyclin A throughout the cell cycle. This is the first demonstration that the activities of p53 are regulated during the cell cycle by E2F/p53 interactions and that phosphorylation of p53 at Ser315 is required for this regulation.