A) Histograms showing the binding profile of 88 artificial ligands to PDZ domains of Mint1. Axis X indicates identification (i.d.) numbers of individual artificial ligands (see A). Axis Y indicates the normalized relative affinity. For normalization, the values of slopes of individual ELISA kinetics were divided by the highest slope value in each of the two sets shown. The number 46 indicates the last peptide in the set of peptides isolated for PDZ-1. Grey bars indicate peptides containing Val at position “0”. Red bars indicate negative controls (phage library aliquots). A horizontal threshold line was drawn at the value equal to double the value of the highest negative control. B) A graphical representation of an amino acid multiple sequence alignment (consensus logo) for either PDZ domain. The logos were generated using the WebLogo application at http://weblogo.berkeley.edu.

A) Histograms showing the binding profile of 89 C-terminal proteomic fragments (natural ligands) to both PDZ domains. Axis X indicates identification (i.d.) numbers of individual natural ligands. Axis Y indicates the normalized relative affinity. The last four bars on the histogram indicate control peptides. Positive controls: positions 87 and 88 (ASYCKQRNIVWL and GHLSESSQVRWL, respectively) are synthetic artificial peptides isolated from a phage-displayed library, which showed relatively high affinity for both PDZ domains; position 89 is copper chaperone for superoxide dismutase (CCS) (GRKESAQPPAHL). Negative control: position 90 (gridded bar), GP41 peptide (CWFSITNWLWYI). Grey colored bars represent peptides with C-terminal Val residue. B) Pie graph showing percentage of different cellular functionalities associated with Mint1, discovered in this study.

(A) Co-IP of TAZ and APP in the presence and absence of Mint1 and Mint3. (B-E) TAZ and YAP potently stimulate transcription mediated by APP fused to Gal4 DNA binding domain in the presence of Mint3. (B-C) Potent transactivation of transcription is achieved with APP fused to Gal4 DNA binding domains only when both Mint3 and TAZ(B)/YAP(C) are present. Note the inhibition of stimulation by mutation of the APP Mint3 binding site. (D-E) This effect is specific for Mint3 because in the presence of Mint1 or Mint2, TAZ(D)/YAP(E) does not induce high-level transactivation. (F-G) Co-expressed Mint1 or Mint2 over APP-Gal4-Mint3-TAZ(F)/YAP(G) inhibited Mint3-TAZ(F)/YAP(G) directed transactivation. Diagrams exhibit representative experiments in which cells were cotransfected with a Gal4-luciferase reporter plasmid (to measure transactivation), a β-galactosidase plasmid (to normalize for transfection efficiency), and the test plasmids identified below the bars. The APP*-Gal4 construct contains a point mutation in the NPTY sequence of the APP cytoplasmic tail that prevents Mint3 binding. The normalized luciferase activity is expressed as fold induction over transcription by APP-Gal4 alone.

Histograms showing the effects of alanine substitutions at positions −1 to −5 on binding to PDZ domains of Mint1. Two representative peptides, which interact with both PDZ domains (TAKCAARFPTWL) or PDZ-2 only (GVPARTQVEVWV), were chosen for the mutagenesis studies.

(A-B) Co-IP of TAZ with Mint1 (A) and Mint3 (B). 293T cells transiently expressing full length FLAG- tagged TAZ and Mint1(A) or Mint3(B) were lysed and the crude lysates were subjected to immunoprecipitation with M2 anti-FLAG beads. Cells expressing Mint1 (A) or Mint3 (B) only were used as controls. Immunoprecipitated samples were subjected to SDS-PAGE and analyzed by immunoblotting with anti-Mint1(A) or anti-Mint3(B) antibodies. Total cell lysates were used as input controls. (C-F) TAZ colocalizes with Mint1. Double labeling of 293T cells cotransfected with Mint1 (green) and TAZ (red) constructs. (G-J) TAZ colocalizes with Mint3. Double labeling of 293T cells cotransfected with Mint3 (green) and TAZ (red) constructs. (K-N) YAP colocalizes with Mint1. Double labeling of 293T cells cotransfected with Mint1 (red) and YAP (green) constructs. (O-R) YAP colocalizes with Mint3. Double labeling of 293T cells cotransfected with Mint3 (red) and YAP (green) constructs. DAPI staining (blue) was used to visualize nuclei. Images were captured on a laser-scanning confocal microscope (Zeiss LSM510).

(A-B) TAZ- and YAP-directed APP-Gal4/Mint3 transactivation was inhibited by the γ-secretase inhibitor DAPT. Potent transactivation of transcription was achieved with APP fused to the Gal4 DNA binding domain when both Mint3 and TAZ(A)/YAP(B) were present, and the transactivations were abolished by DAPT treatment. (C-D) TAZ- and YAP-directed Gal4-Mint3 transactivation was inhibited by coexpression of intact APP. Potent transactivation of transcription was achieved with Mint3 fused to Gal4 DNA binding domain in the presence of TAZ(C)/YAP(D). Coexpression of full-length APP inhibited the transactivation, while DAPT treatment further decreased the transactivation. Diagrams exhibit representative experiments in which cells were cotransfected with a Gal4-luciferase reporter plasmid (to measure transactivation), a β-galactosidase plasmid (to normalize for transfection efficiency), and the test plasmids identified below the bars. The normalized luciferase activity is expressed as fold induction over transcription by APP-Gal4 alone (A-B), or as fold induction over Mint3-Gal4 alone (C-D). (E) Schematic model indicating that full length APP molecules trap Mint3 and TAZ /YAP in the cytoplasm, while γ-secretase cleavage releases the triple protein complexes of AICD-Mint3-TAZ/YAP and allows them to translocate into the nucleus and induce transcription of target genes.