Quantitation of glycogen synthase kinase-3 sensitive proteins in neuronal membrane rafts.
We report a quantitative proteomic study to investigate the changes induced in membrane rafts by the inhibition of glycogen synthase kinase-3. Sensitive quantitation of membrane raft proteins using isobaric tagging chemistries was enabled by a novel hybrid proteomic method to isolate low-microgram (10-30 microg) membrane raft protein preparations as unresolved bands in a low-density acrylamide gel. Samples were in-gel digested, differentially tagged and combined for 2-D LC and quantitative MS. Analysis of hippocampal membrane preparations using this approach resulted in a sixfold increase in sensitivity and a threefold increase in the number of quantifiable proteins compared with parallel processing using a traditional in-solution method. Quantitative analysis of membrane raft preparations from a human neuronal cell line treated with glycogen synthase kinase-3 inhibitors SB415286 or lithium chloride, that have been reported to modulate processing of the Alzheimer amyloid precursor protein, identified several protein changes. These included decreases in lamin B1 and lamin B receptor, as well as increases in several endosome regulating rab proteins, rab5, rab7 and rab11 that have been implicated in processing of the amyloid precursor protein in Alzheimer's disease.
Thompson
Andrew J
AJ
Department of Neuroscience, MRC Centre for Neurodegeneration Research, King's College London, Institute of Psychiatry, London, UK. a.thompson@iop.kcl.ac.uk
Williamson
Ritchie
R
Schofield
Emma
E
Stephenson
John
J
Hanger
Diane
D
Anderton
Brian
B
D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D038362:Glycogen Synthase Kinase 3 / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D016014:Linear Models; D008297:Male; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D008811:Mice, Inbred DBA; D009474:Neurons / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity
D008565:Membrane Proteins; D038362:Glycogen Synthase Kinase 3