Document Detail


Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor.
MedLine Citation:
PMID:  11964165     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56(lck) and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-beta-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca(2+) concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts.
Authors:
Alexandre K Rouquette-Jazdanian; Claudette Pelassy; Jean-Philippe Breittmayer; Jean-Louis Cousin; Claude Aussel
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Biochemical journal     Volume:  363     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  2002 May 
Date Detail:
Created Date:  2002-04-19     Completed Date:  2002-06-10     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  England    
Other Details:
Languages:  eng     Pagination:  645-55     Citation Subset:  IM    
Affiliation:
INSERM U343, Hôpital de l'Archet I, 151 Route de Saint Antoine de Ginestière, B.P. 79, 06202 Nice Cedex 3, France.
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MeSH Terms
Descriptor/Qualifier:
Antigens, CD3 / metabolism,  physiology*
Arachidonic Acid / metabolism
Calcium / metabolism
Cell Membrane / ultrastructure
Cholesterol / metabolism
Cyclodextrins / pharmacology
Diglycerides / biosynthesis
Glycerophospholipids / metabolism
Humans
Jurkat Cells
Membrane Lipids / metabolism
Palmitic Acid / metabolism
Protein Kinase C / metabolism
Signal Transduction*
beta-Cyclodextrins*
Chemical
Reg. No./Substance:
0/1,2-diacylglycerol; 0/Antigens, CD3; 0/Cyclodextrins; 0/Diglycerides; 0/Glycerophospholipids; 0/Membrane Lipids; 0/beta-Cyclodextrins; 0/methyl-beta-cyclodextrin; 506-32-1/Arachidonic Acid; 57-10-3/Palmitic Acid; 57-88-5/Cholesterol; 7440-70-2/Calcium; EC 2.7.11.13/Protein Kinase C
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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