Figure 1.
Identification of ERK2 as a novel Par3-interacting protein. A, Schematic representation of Par3. The numbers indicate the amino acids. CR1, Conserved region 1; PDZ, PSD-95/Dlg/ZO-1; aPKC BR, aPKC binding region. B, Validation of the results of the proteomic analysis. The eluates from affinity column chromatography were analyzed by immunoblotting using anti-ERK1/2, anti-JNK1, anti-p38α MAPK, anti-GSK-3β, and anti-Cdk5 antibodies. C, A coimmunoprecipitation assay was performed using rat brain lysate. Extracts of developing rat brain were incubated with rabbit IgG, anti-Par3, or anti-ERK1/2 antibody. The immunoprecipitates were analyzed by immunoblotting with rabbit anti-Par3 and mouse anti-ERK2, anti-JNK1, anti-p38α MAPK, anti-GSK-3β, and anti-Cdk5 antibodies. D, Super-resolution microscopy images of the cell body and the growth cone showing the colocalization of Par3 and ERK2. Hippocampal neurons were fixed at 3 DIV and then immunostained with specific antibodies against anti-ERK2 (green) and anti-Par3 (red). The peripheral regions of the growth cones were visualized by staining F-actin with Alexa-647-conjugated phalloidin (blue). Arrowheads in the enlarged images indicate the colocalization of the proteins. Scale bars, 5 μm and 0.5 μm for the enlarged images. E, GST-ERK2 was incubated with amylose-resin coated with MBP, MBP-Par3-1N, -2N, -3N, or -4N. The bound proteins were subjected to immunoblotting with anti-ERK2 antibody (top). The total amounts of MBP, MBP-Par3-1N, -2N, -3N, and -4N are shown with Coomassie Brilliant Blue (CBB) staining (bottom). Asterisks indicate intact MBP-fusion proteins. F, The putative ERK docking site KIM ((V/L)-X2-(R/K)-(R/K)-X3–6-L).