The PH domain of PDK1 binds to the CD of AMIGO2. (A and B) The CD of AMIGO2 interacts with PDK1. HEK293 cells were transfected with Flag-PDK1 and GFP-tagged AMIGO2 or domain deletion mutants. Lysates were immunoprecipitated with GFP (A) or Flag (B) antibodies and blotted with the GFP or Flag antibody. PDK1 was immunoprecipitated with the Flag antibody and detected with the anti-GFP antibody. A2, AMIGO2^WT; ΔLRR, AMIGO2^ΔLRR; ΔIgG, AMIGO2^ΔIgG; ΔCD, AMIGO2^ΔCD; CD, CD domain of AMIGO2. (C) Schematic of 491 aa and 457 aa of the AMIGO2 constructs. (D) PDK1 associates with the region between 491 aa and 457 aa of AMIGO2. HEK293 cells were transfected with Flag-PDK1– and GFP-tagged AMIGO2 or domain deletion mutants. Lysates were immunoprecipitated with GFP antibody and blotted with the Flag and GFP antibodies. (E) Each His-tagged PDK1^kinase and His-tagged PDK1^PH was incubated with purified GST-AMIGO2^CD. Mixtures of His-tagged PDK1^kinase or PDK1^PH proteins and GST-AMIGO2^CD proteins were pulled down with GST resin and analyzed by Western blotting using anti-His and -GST antibodies. His-kinase, His-PDK1^kinase; His-PH, His-PDK1^PH; GST-CD, GST-AMIGO2^CD. (F) Mixtures of His-PDK1^PH and GST-CD proteins were incubated with PIP3-coated or control beads and treated with DM-PIT-1. The PIP3-bound proteins were analyzed with His and GST antibodies. (G) Multiple sequence alignments of the AMIGO family CD domains were performed. An asterisk indicates that the alignment contains identical amino acid residues in all sequences (or identical bases if DNA sequences are aligned); a colon indicates that the alignment contains different but highly conserved (very similar) amino acids; a period indicates that the alignment contains different amino acids that are somewhat similar; and a blank space indicates that the alignment contains dissimilar amino acids or gaps (or different bases if DNA sequences are aligned). (H) Diagram showing the protein sequences of cytosolic-truncated mutants. The total length of the cytosolic tail is 103 aa. (I) Far-western analysis was performed for each truncated mutant. Coomassie blue staining revealed purified GST-tagged truncated mutants that were previously loaded for far-western analysis. (J) Mixtures of purified GST-tagged truncated mutants and His-tagged PH domain proteins were pulled down with GST beads and eluted, and the Western blot was performed using His and GST antibodies. HC, heavy chain; IP, immunoprecipitation.