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First published online 19 October 2004
doi: 10.1242/jcs.01472

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Bcr (breakpoint cluster region) protein binds to PDZ-domains of scaffold protein PDZK1 and vesicle coat protein Mint3

¹ Department of Medical Biochemistry, Göteborg University, Medicinaregatan 9A, 413 90 Gothenburg, Sweden
² Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, S. C. Johnson Medical Research Center, 13400 E. Shea Boulevard, Scottsdale, AZ 85259, USA

View larger version (69K): [in a new window]   Fig. 1. Bcr is bound by the first PDZ domain of PDZK1. (A) GST fusion proteins with each of the four PDZ domains of PDZK1 immobilized on glutathione-Sepharose beads and incubated with lysates from Calu-3 cells. Bound products were eluted, separated by SDS-PAGE and visualized with Coomassie Blue staining. (B) The peptide masses of the trypsinized major band pulled down with PDZ 1 (marked with arrows in A) identified by MALDI-TOF mass spectrometry. The peptides identified were distributed over the entire Bcr protein. (C) Bcr in several human epithelial cell lines. Lysates from human epithelial cells were separated by SDS-PAGE and Bcr was detected by western blotting using the C20 anti-Bcr antibody.

View larger version (42K): [in a new window]   Fig. 2. Confirmation and specificity of the PDZK1-Bcr interaction. GST-PDZ domain fusion proteins from PDZK1 (A) as well as EBP50 and GOPC (B) were bound to glutathione-Sepharose beads, incubated with Calu-3 cell lysate, and the bound products separated by SDS-PAGE followed by western blotting using the anti-Bcr C20 antibody. (C) Confirmation of expression of GST fusions with PDZ domains of EBP50 and GOPC. (D) Bcr was immunoprecipitated from lysates prepared from BHK-21 cells transiently expressing Bcr and FLAG-tagged PDZK1, separated by SDS-PAGE followed by western blotting. (E) FLAG-tagged PDZK1-M2 was transiently expressed in BHK-21 cells with or without Bcr fused to GST. Lysates prepared from these cells were incubated with glutathione-Sepharose beads and the material pulled down was separated by SDS-PAGE. PDZK1 was detected by western blotting with the mAb anti-FLAG M2. (F) Bcr, Bcr-V1271A and Bcr-delTEV were transiently expressed in BHK 21 cells and lysates prepared from these cells were incubated with PDZK1 PDZ 1-GST bound to glutathione-Sepharose beads. The bound material was separated by SDS-PAGE and Bcr was visualized by western blotting using the anti-Bcr N20 antibody.

View larger version (100K): [in a new window]   Fig. 3. Cellular localization of recombinantly and endogenously expressed BCR. (A) BHK-21 cells transiently expressing Bcr were immunostained with rabbit anti-Bcr and mouse anti-plasma membrane calcium ATPase (PMCA-ATPase) antibodies as described in Materials and methods and viewed by confocal microscopy. (B) Endogenous Bcr, ß-COP and clathrin were visualized in HEK293 cells by immunofluorescence microscopy using rabbit anti-Bcr and mouse anti-ß-COP or mouse anti-clathrin heavy chain antibodies. (C) Endogenous Bcr and CFTR were detected by immunofluorescence staining in polarized Calu-3 cells, grown at an air-liquid interface using rabbit anti-Bcr and mouse anti-CFTR antibodies.

View larger version (115K): [in a new window]   Fig. 4. Bcr binds the first PDZ domain of Mint3. Twenty-eight different PDZ domain-containing proteins were screened for Bcr binding using a TransSignal PDZ Domain Array. Detergent lysates from Bcr-transfected BKH-21 cells were added to the membrane and the bound Bcr visualized with the anti-Bcr C20 antibody. The X and Y coordinates of the array are indicated by letters and numerals, respectively. Mint2 PDZ1 (A1), Mint3 PDZ1 (B1), Mint3 PDZ2 (C1), Mint1 PDZ2 (E1), GIPC (F3), ZO-2 (F6), RGS12 (A7).

View larger version (27K): [in a new window]   Fig. 5. Confirmation of the Bcr-Mint3 interaction. (A) Mint3 and Bcr-GST were transiently expressed in BHK-21 cells. Lysates prepared from these cells were incubated with glutathione-Sepharose beads and bound products were separated by SDS-PAGE. Mint3 was detected by western blotting using the rabbit anti-Mint3 antibody. The control is from untransfected parental BHK cells. (B) Similar experiment employing Bcr with deletion of 3 C-terminal amino acids. (C) Mint3 is detected in an immunoprecipitate with a Bcr antibody in lysate from parental HEK293 cells. Controls using an irrelevant antibody or immunoblotting with only secondary antibody, were negative for this band.

View larger version (87K): [in a new window]   Fig. 6. Cellular localization of endogenous and recombinantly expressed Mint3. Endogenous Mint3 and Bcr were visualized by confocal immunofluorescence microscopy in polarized Calu-3 layers (A) and HEK293 cells (B) as described in Materials and Methods. (C) BHK-21 cells were transiently transfected with Bcr/pcDNA3.1 and Mint3/pCMV5 using LipofectAMINE Plus and proteins were detected 30 hours after transfection.