Demonstration of BACE (beta-secretase) phosphorylation and its interaction with GGA1 in cells by fluorescence-lifetime imaging microscopy.
beta-Secretase (BACE) carries out the first of two proteolysis steps to generate the amyloid-beta peptides that accumulate in the senile plaques in Alzheimer's disease (AD). Because most BACE activity occurs in endosomes, signals regulating its trafficking to these compartments are important to an understanding of AD pathogenesis. A DISLL sequence near the BACE C-terminus mediates binding of BACE to the VHS domains of Golgi-localized gamma-ear-containing ARF-binding (GGA) proteins, which are involved in the sorting of proteins to endosomes. Phosphorylation of the motif's serine residue regulates BACE recycling back to the cell surface from early endosomes and enhances the interaction of BACE with GGA proteins in isolated protein assays. We found that BACE phosphorylation influences BACE-GGA interactions in cells using a new fluorescence-resonance-energy-transfer-based assay of protein proximity, fluorescence lifetime imaging. Although serine-phosphorylated BACE was distributed throughout the cell, interaction of GGA1 with the wild-type protein occurred in juxtanuclear compartments. Pseudo-phosphorylated and non-phosphorylated BACE mutants remained localized with GGA1 in the Golgi body, but the latter mutation diminished the two proteins' FRET signal. Because BACE phosphorylated at serine residues can be identified in human brain, these data suggest that serine phosphorylation of BACE is a physiologically relevant post-translational modification that regulates trafficking in the juxtanuclear compartment by interaction with GGA1.
von Arnim
Christine A F
CA
Alzheimer Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical School, 114 16th Street, Charlestown, MA 02129, USA.
Tangredi
Michelle M
MM
Peltan
Ithan D
ID
Lee
Bonny M
BM
Irizarry
Michael C
MC
Kinoshita
Ayae
A
Hyman
Bradley T
BT
D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000544:Alzheimer Disease / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D010450:Endopeptidases / Q000378:metabolism* / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051379:Mice; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D014162:Transfection
D033942:Adaptor Proteins, Vesicular Transport; C407306:GGA adaptor proteins; D049452:Green Fluorescent Proteins; D010744:Phosphoric Monoester Hydrolases; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C509282:Bace1 protein, mouse; D020727:ADP-Ribosylation Factors