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First published online 5 October 2004
doi: 10.1242/jcs.01422

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Demonstration of BACE (ß-secretase) phosphorylation and its interaction with GGA1 in cells by fluorescence-lifetime imaging microscopy

Alzheimer Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical School, 114 16th Street, Charlestown, MA 02129, USA

View larger version (59K): [in a new window]   Fig. 1. Phosphorylation-state-dependent localization of BACE and GGA1. BACE-V5 [wild-type (WT), S498D and S498A] and GGA1-Myc co-transfected N2a cells grown on glass coverslips were immunostained with anti-V5 mAb (visualized using Cy3) and rabbit anti-Myc Ab (visualized using FITC). Notice that the subcellular localization of BACE in juxtanuclear compartments and the partial colocalization with GGA1 is the same in WT, S498D and S498A transfected cells, whereas distribution in the periphery of the cell predominates in the non-phosphorylated BACE mutant (S498A).

View larger version (73K): [in a new window]   Fig. 2. FLIM analysis of the proximity of wild-type BACE and GGA1 within cells. N2a cells were transfected with BACE-V5 (labeled with FITC) and GGA1-Myc (labeled with Cy3). Only the donor fluorophore (FITC) was applied for the negative control (A,B). The intensity image shows the standard immunostaining pattern for BACE (A,C). The color-coded FLIM image shows the lifetimes (in picoseconds) of FITC in the absence (B) or presence (D) of the acceptor Cy3. The shorter FITC lifetimes reflecting proximity between GGA1 and BACE appear only in juxtanuclear compartments (TGN) (D). The negative control exhibits a single population of lifetimes for FITC in the absence of an acceptor, represented by a homogenous blue-green pseudocolor image.

View larger version (44K): [in a new window]   Fig. 3. Phosphorylation-dependent interaction of GGA1 with BACE. Intensity images of GGA1 (A,C,E,G) and pseudocolored FLIM images when co-transfected with wild-type BACE (WT-BACE; D) or the BACE-S498D (F) and BACE-S498A (H) mutants in N2a cells. In the absence of an acceptor or Cy3-tagged, non-phosphorylated BACE mutants (S498A), no change in fluorescence lifetime was detected. WT-BACE and pseudo-phosphorylated BACE mutants (S498D) showed a significant decrease of fluorescence lifetime in the juxtanuclear compartment.

View larger version (93K): [in a new window]   Fig. 4. Localization of BACE-GFP and endogenous phosphoserine residues. N2a cells were transfected with BACE-GFP, treated with OA and stained with an antibody specific to phosphorylated serine residues, labeled by Cy3. BACE-GFP shows the standard immunostaining pattern for BACE. Phosphoserine residues show a homogenous staining throughout the cell.

View larger version (61K): [in a new window]   Fig. 5. FLIM analysis of BACE phosphorylation. N2a cells were transfected with BACE-GFP, treated with 20 nM OA and stained with an antibody specific to phosphorylated serine residues for the analysis. Negative controls were only transfected with BACE-GFP and treated with OA (A,B). The intensity image shows the standard immunostaining pattern for BACE (A,C). The color-coded FLIM image shows the lifetimes (in picoseconds) of GFP in the presence of acceptor Cy3. The lifetime reflects the proximity between BACE-C-terminus and phosphoserine residues, demonstrated in pseudocolor. The FLIM image suggests that there is a close proximity between the BACE C-terminus and phosphoserine residues not only in juxtanuclear compartments (TGN), but throughout the cell (D). The chart shows mean changes in lifetime and statistically shorter lifetimes in the presence of Cy3-labeled endogenous phosphoserine (P<0.001).

View larger version (58K): [in a new window]   Fig. 6. Immunoprecipitation of BACE phosphorylated at its Serine residue in human brain tissue. (Lane 1) AD human brain. Immunoprecipitation (IP) with ms anti-phosphoserine, probe with rb anti-BACE (N-terminus). (Lane 2) Normal human brain. IP with ms anti-phosphoserine, probe with rb anti-BACE (N-terminus). (Lane 3) Normal human brain. IP with rb anti-BACE (C-terminus), probe with ms anti-phosphoserine. (Lane 4) Normal human brain. IP with ms anti-BACE (C-terminus), probe with rb anti-BACE (N-terminus) (positive control). (Lane 5) BACE standard. Probe with rb anti-BACE (N-terminus) (positive control). (Lane 6) Buffer. IP with ms anti-phosphoserine, probe with rb anti-BACE (N-terminus) (negative control). (Lane 7) Normal human brain. IP with ms anti-phosphotyrosine, probe with rb anti-BACE (N-terminus) (negative control).

View larger version (15K): [in a new window]   Fig. 7. APP shedding assay. HEK293 cells were transfected with SEAP-APP, ß-gal and empty vector (Topo) or with a plasmid encoding wild-type BACE, a catalytically inactive BACE mutant (BACE D93/289A), non-phosphorylated BACE mutant (BACE S498A), pseudophosphorylated BACE mutant (BACE S498D) or BACE dileucine mutant (LL 499/500 AA). The alkaline phosphatase activity in the conditioned medium is shown, normalized to ß-gal activity, with the means and SD after both transfection and measurement were carried out in triplicate, representing two independent assays. **, P<0.001 (ANOVA, Fisher's PLSD post hoc test).

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