RESUMO
The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitination, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Other functions of the APC/C are, however, less well defined. Here we show that two APC/C components, APC5 and APC7, interact directly with the coactivators CBP and p300 through protein-protein interaction domains that are evolutionarily conserved in adenovirus E1A. This interaction stimulates intrinsic CBP/p300 acetyltransferase activity and potentiates CBP/p300-dependent transcription. We also show that APC5 and APC7 suppress E1A-mediated transformation in a CBP/p300-dependent manner, indicating that these components of the APC/C may be targeted during cellular transformation. Furthermore, we establish that CBP is required in APC/C function; specifically, gene ablation of CBP by RNA-mediated interference markedly reduces the E3 ubiquitin ligase activity of the APC/C and the progression of cells through mitosis. Taken together, our results define discrete roles for the APC/C-CBP/p300 complexes in growth regulation.
Assuntos
Proteína de Ligação a CREB/metabolismo , Ciclo Celular/fisiologia , Regulação da Expressão Gênica , Transcrição Gênica , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Subunidade Apc5 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc7 do Ciclossomo-Complexo Promotor de Anáfase , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/genética , Linhagem Celular , Transformação Celular Neoplásica , Sequência Conservada , Humanos , Mitose , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
Human papillomavirus (HPV) antigens are expressed in epithelial cells at different stages of differentiation, and this may affect how they are handled by the immune system. We assessed the relative immunogenicities of four different HPV type 1 proteins: E6 and E7, which are made early in basal or parabasal cells; E4, which is made suprabasally in differentiating cells; and L1, a late protein which appears in the highly differentiated upper spinous layers. Pools of 15-mer peptides covering the primary sequences of all four proteins were used to screen 15 normal donors in enzyme-linked immunospot assays of gamma interferon release for both CD4(+)- and CD8(+)-T-cell reactivities. CD8(+)-T-cell responses were detected to the L1 protein in 7 of the 15 samples examined. No responses to E6, E7, or E4 were detected. CD4(+)-T-cell reactivities were again detected in 7 of the 15 donors. A broader spectrum of responses to E6 (three of seven), E4 (six of seven), and L1 (three of seven) was apparent, but there was no reactivity to E7. The predominant CD4(+) response was to E4. Reactivities were seen in some cases to corresponding regions on other common HPV types but were probably due to a multiple infection rather than to a cross-reaction. Antibodies to HPV1 virus-like particles were detected in 12 of the 15 (80%) donors, but antibody status did not correlate with T-cell reactivity. The differences in the relative immunogenicities of the four proteins revealed in this study are discussed in relation to how they may be processed and presented to the immune system by differentiating epithelial cells.