RESUMO
The E6 oncoprotein from high-risk genus alpha human papillomaviruses (α-HPVs), such as HPV 16, has been well characterized with respect to the host-cell proteins it interacts with and corresponding signaling pathways that are disrupted due to these interactions. Less is known regarding the interacting partners of E6 from the genus beta papillomaviruses (ß-HPVs); however, it is generally thought that ß-HPV E6 proteins do not interact with many of the proteins known to bind to α-HPV E6. Here we identify p300 as a protein that interacts directly with E6 from both α- and ß-HPV types. Importantly, this association appears much stronger with ß-HPV types 5 and 8-E6 than with α-HPV type 16-E6 or ß-HPV type 38-E6. We demonstrate that the enhanced association between 5/8-E6 and p300 leads to p300 degradation in a proteasomal-dependent but E6AP-independent manner. Rather, 5/8-E6 inhibit the association of AKT with p300, an event necessary to ensure p300 stability within the cell. Finally, we demonstrate that the decreased p300 protein levels concomitantly affect downstream signaling events, such as the expression of differentiation markers K1, K10 and Involucrin. Together, these results demonstrate a unique way in which ß-HPV E6 proteins are able to affect host-cell signaling in a manner distinct from that of the α-HPVs.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Diferenciação Celular , Proteína p300 Associada a E1A/genética , Marcadores Genéticos , Humanos , Queratinócitos/metabolismo , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Papillomaviridae/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Transcrição GênicaRESUMO
OBJECTIVE: The first objective of this study was to examine the differences in levels of adducin (ADD1, ADD2, ADD3) mRNA expression during human erythropoiesis. The second objective was to determine whether the rapid induction of ADD2 expression could be attributed to a novel erythroid-specific promoter. METHODS: Expression of mRNA was quantified using real-time RT-PCR. Primary erythroid precursors were isolated from normal human bone marrow using fluorescence-activated cell sorting. Two model systems were compared: CD34(+) hematopoietic stem cells induced to differentiate with erythropoietin and HEL cells induced to differentiate with hemin. 5'RACE analysis was performed using primary human erythroblasts as starting material. RESULTS: All three adducin genes showed different patterns of expression during erythropoietic differentiation of cultured CD34(+) stem cells. Levels of ADD3 mRNA were higher than levels of ADD2 mRNA at early stages of erythropoiesis. Expression of ADD2 was induced to very high levels (100 times baseline) in erythropoietin-stimulated cultures. 5'RACE analysis identified a novel starting exon and putative erythroid promoter for ADD2. CONCLUSION: These results suggest that expression of each adducin gene is regulated in a gene-specific manner during erythropoiesis. The early expression of ADD3 suggests that it may have a role in erythroblasts but is replaced by ADD2 in later stages of erythropoiesis. The very high levels of expression of ADD2 suggest that its promoter may be useful for directing erythroid-specific gene expression.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Proteínas do Citoesqueleto/genética , Eritropoese/genética , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Antígenos CD/análise , Antígenos CD34/análise , Sequência de Bases , Biópsia por Agulha , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Linhagem Celular Tumoral , Éxons , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Íntrons , Leucemia Eritroblástica Aguda , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Adducins are a family of cytoskeletal proteins encoded by 3 genes (alpha, beta, and gamma). Platelets express alpha and gamma adducins, in contrast to red blood cells that express alpha and beta adducins. During platelet activation with thrombin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate, alpha and gamma adducins were phosphorylated by protein kinase C (PKC) as detected by an antibody specific for a phosphopeptide sequence in the highly conserved carboxy terminus. Platelet activation also led to adducin proteolysis; inhibition by calpeptin suggests that the protease was calpain. The kinase inhibitor staurosporine inhibited PKC phosphorylation of adducin and also inhibited proteolysis of adducin. Experiments with recombinant alpha adducin demonstrated that the PKC-phosphorylated form was proteolyzed at a significantly faster rate than the unphosphorylated form. The concentration of adducin in platelets was estimated at 6 microM, similar to the concentration of capping protein. Fractionation of platelets into high-speed supernatant (cytosol) and pellet (membrane and cytoskeleton) revealed a shift of PKC-phosphorylated adducin to the cytosol during platelet activation. Platelet aggregation detected turbidometrically was decreased in the presence of staurosporine and was completely inhibited by calpeptin. Thrombin-induced changes in morphology were assessed by confocal microscopy with fluorescein phalloidin and were not prevented by staurosporine or calpeptin. Our results suggest that regulation of adducin function by PKC and calpain may play a role in platelet aggregation.