RESUMO
An effective new platform for phosphosite mapping and subsequent functional screening was developed to analyze the targeted protein-protein interactions of p300 and CBP with ß-catenin. Two novel functional phosphosites, Ser12 of p300 and Ser92 of CBP, were revealed to modulate p300/ß-catenin and CBP/ß-catenin interactions, respectively.
Assuntos
Fosfoproteínas/química , Proteoma/química , Técnicas do Sistema de Duplo-Híbrido , Sequência de Aminoácidos , Animais , Proteína de Ligação a CREB/biossíntese , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/isolamento & purificação , Linhagem Celular , Cromatografia Líquida , Humanos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Fosforilação , Proteômica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de Proteína , Espectrometria de Massas em Tandem , beta Catenina/biossíntese , beta Catenina/química , beta Catenina/isolamento & purificação , Fatores de Transcrição de p300-CBP/biossíntese , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/isolamento & purificaçãoRESUMO
CREB-binding protein (CBP) is an important coactivator of basal transcription machinery and a critical regulator of cellular proliferation, differentiation, and apoptosis. It is hypothesized that CBP function is regulated by post-translational modifications, such as phosphorylation and methylation. Specific kinase-mediated phosphorylation of CBP has been shown to affect not only intrinsic histone acetyl transferase activity, but also transcriptional activity of various target promoters and interaction with binding partners. While most of the identified CBP phosphorylation sites have been mapped to the N-terminus of the protein, based on previous studies of the CBP homolog (p300), protein kinase B/Akt is predicted to phosphorylate the C-terminus of CBP. However, there is no direct evidence of Akt-mediated phosphorylation of CBP. Here we report the first purification procedure of recombinant fragment of CBP, encompassing the cysteine/histidine-rich domain 3 (CH3) and glutamine-rich (Q) domain of the protein, which is suitable for structural and interaction studies. We provide the first evidence of protein-protein interaction between the full-length Akt1 and the C-terminus of CBP by fluorescence spectroscopy and the subsequent phosphorylation of CBP by in vitro phosphorylation assay. Our results suggest that Akt signaling may have important implications on the in vivo molecular interaction of CBP with various transcription factors and modulation of cellular responses.