Temporal proteomics profiling of lipid rafts in CCR6-activated T cells reveals the integration of actin cytoskeleton dynamics.
Chemokines orchestrate leukocyte migration toward sites of inflammation and infection and target leukocytes via chemokine receptors such as CCR6, a subfamily of the seven-transmembrane G-protein-coupled receptors. Lipid rafts are cholesterol and sphingolipid-enriched liquid-ordered membrane microdomains thought to serve as scaffolding platforms for signal transduction. To globally understand the dynamic changes of proteins within lipid rafts upon CCR6 activation in T cells, we quantitatively analyzed the time-dependent changes of lipid raft proteome using our recently reported membrane proteomics strategy combining gel-assisted digestion, iTRAQ labeling and LC-MS/MS. To our knowledge, the error-free identification of 852 proteins represents the first data set of the raft proteome in T cells upon chemokine receptor activation, including 354 previously annotated raft proteins and 85 dynamically recruited proteins that are potential raft-associated proteins. The temporal profiles revealed that many proteins involved in the actin cytoskeleton rearrangement are actively recruited into lipid rafts upon CCR6 activation. We further confirmed the proteomics results by Western blotting and used small interfering RNA-mediated knockdown to evaluate their roles upon CCR6 activation. In sum, we employed quantitative proteomic strategy to analyze raft proteome and identified many molecules actively involved in the control of actin assembly and disassembly regulating CCR6 activation-induced cell migration.
Lin
Shu-Ling
SL
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.
Chien
Chih-Wei
CW
Han
Chia-Li
CL
Chen
Eric S-W
ES
Kao
Shao-Hsuan
SH
Chen
Yu-Ju
YJ
Liao
Fang
F
D000199:Actins / Q000378:metabolism*; D015153:Blotting, Western; D054418:Chemokine CCL20; D002633:Chemotaxis; D003599:Cytoskeleton / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D054399:Receptors, CCR6 / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D000199:Actins; C108355:CCL20 protein, human; C106311:CCR6 protein, human; D054418:Chemokine CCL20; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C047262:LCP1 protein, human; D008840:Microfilament Proteins; D054399:Receptors, CCR6; C079373:STIP1 protein, human