RESUMO
Accessibility of the genome to DNA-binding transcription factors is regulated by proteins that control the acetylation of amino-terminal lysine residues on nucleosomal histones. Specifically, histone deacetylase (HDAC) proteins repress transcription by deacetylating histones. To date, the only known regulatory mechanism of HDAC1 function is via interaction with associated proteins. Although the control of HDAC1 function by protein interaction and recruitment is well precedented, we were interested in exploring HDAC1 regulation by post-translational modification. Human HDAC1 protein was analyzed by ion trap mass spectrometry, and two phosphorylated serine residues, Ser(421) and Ser(423), were unambiguously identified. Loss of phosphorylation at Ser(421) and Ser(423) due to mutation to alanine or disruption of the casein kinase 2 consensus sequence directing phosphorylation reduced the enzymatic activity and complex formation of HDAC1. Deletion of the highly charged carboxyl-terminal region of HDAC1 also decreased its deacetylase activity and protein associations, revealing its requirement in maintaining HDAC1 function. Our results reinforce the importance of protein associations in modulating HDAC1 function and provide the first step toward characterizing the role of post-translational modifications in regulating HDAC activity in vivo.
Assuntos
Histona Desacetilases/metabolismo , Proteínas Repressoras , Alanina/química , Sequência de Aminoácidos , Sítios de Ligação , Caseína Quinase II , Divisão Celular , Deleção de Genes , Ácido Glutâmico/química , Histona Desacetilase 1 , Histona Desacetilase 2 , Humanos , Células Jurkat , Luciferases/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serina/química , Transcrição Gênica , TransfecçãoRESUMO
Although previous work has shown that the hepatitis B virus X protein (pX) stabilizes complexes between basic region leucine zipper (bZIP) proteins and target DNA, the relationship between enhanced binding and transcriptional activation has not been established. Here we show that interactions between CREB and pX, which coincidentally enhance DNA affinity, are necessary but not sufficient for increased transcriptional potency. Further, we show that transcriptional activation by pX requires a form of CREB in which Ser-133 is not phosphorylated. By stimulating the transcriptional potency of unphosphorylated CREB, pX can up-regulate the expression of cAMP-responsive genes implicated in hepatocyte proliferation, leading ultimately to the development of liver cancer after viral infection.
