Proteome analysis of lipid rafts in Jurkat cells characterizes a raft subset that is involved in NF-kappaB activation.
Lipid rafts are detergent-insoluble membrane domains that play a key role in signal transduction by the T-cell antigen receptor. Proteome analysis revealed the presence of amidosulfobetaine-soluble signal transducing, integral membrane, cytoskeletal, heat shock, and GTP-binding proteins in rafts prepared from Jurkat cells. Several of these proteins were recruited to rafts by CD3/CD28 costimulation. Of particular interest is the inducible association of activated IkappaB kinase complexes with raft vesicles that could be captured with anti-flotillin-1 antibodies. Following amidosulfobetaine solubilization, flotillin-beta and IKKbeta underwent reciprocal co-immunoprecipitation. Treatment of Jurkat cells with methyl-beta-cyclodextrin disrupted the assembly and activation of this raft complex and also interfered in CD3/ CD28-induced activation of a NF-kappaB response element in the IL-2 promoter.
Tu
Xiaolin
X
Division of Clinical Immunology and Allergy, Department of Medicine, The Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, California 90095, USA.
Huang
Aaron
A
Bae
David
D
Slaughter
Ndaisha
N
Whitelegge
Julian
J
Crother
Timothy
T
Bickel
Perry E
PE
Nel
Andre
A
D018106:CD28 Antigens / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D051550:I-kappa B Kinase; D047468:Immunoprecipitation; D007376:Interleukin-2 / Q000235:genetics; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020543:Proteome; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D047392:beta-Cyclodextrins / Q000494:pharmacology*
D018106:CD28 Antigens; D017252:CD3 Complex; D007376:Interleukin-2; D008565:Membrane Proteins; D020543:Proteome; D047392:beta-Cyclodextrins; C106354:flotillins; C108732:methyl-beta-cyclodextrin; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human