Figure 5.

NLK inhibits the CBP-induced acetylation of A-Myb. (A) A-Myb binds to two regions of CBP. Top, schematic representation of the GST-CBP fusion proteins used. Top, the domain structure of CBP is shown schematically: C/H, cysteine- and histidine-rich domain; KIX, CREB-binding domain; Bromo, bromo domain. Structures of the three GST-CBP fusion proteins used are shown below. The results of binding assays shown below are indicated on the right. Bottom, GST pulldown assays. In the right panel, the GST-CBP fusion proteins bound by glutathione-Sepharose resin were analyzed by 10% SDS-PAGE followed by Coomassie Brilliant Blue staining. In the left panel, ³⁵S-labeled A-Myb was synthesized in vitro and mixed with various GST-CBP fusion proteins, and bound proteins were analyzed by SDS-PAGE and autoradiography. (B) A-Myb is acetylated by CBP in vivo. Left, CV-1 cells were transfected with the FLAG-A-Myb expression plasmid, with or without increasing amounts of the CBP-FLAG expression plasmid. Cell lysates were prepared and analyzed by SDS-PAGE and Western blotting using anti-acetylated lysine (top panel) or anti-FLAG antibody (middle and bottom panels). Right, comparison of the sequence surrounding the CBP-induced acetylation sites between mouse c-Myb and human A-Myb. Lys-438 and Lys-441 of mouse c-Myb, which are acetylated by CBP and indicated by bold letters, are conserved in human A-Myb. (C) Acetylation of A-Myb is inhibited by NLK. CV-1 cells were transfected with the FLAG-A-Myb and CBP-FLAG expression plasmids, with or without different amounts of the plasmid to express FLAG-tagged wild-type or kinase-negative NLK. Cell lysates were prepared and analyzed by a 4–12% gradient SDS-PAGE and Western blotting using anti-acetylated lysine (top panel) or anti-FLAG antibody (middle and bottom panels).