title: Purification and characterization of recombinant CH3 domain fragment of the CREB-binding protein.
abstract: 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.
authors.affiliation: Department of Medicine, Duke University Medical Center, Box 2628, Durham, NC 27710, USA. ibarr001@mc.duke.edu
authors.forename: Catherine Ibarra
authors.initials: CI
authors.lastname: Drendall
authors.forename: Quang H
authors.initials: QH
authors.lastname: Pham
authors.forename: Eric C
authors.initials: EC
authors.lastname: Dietze
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D050882:CREB-Binding Protein / Q000737:chemistry / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification*; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
chemical_list: D010446:Peptide Fragments; D050882:CREB-Binding Protein; C494363:CREBBP protein, human