Cholesterol depletion disrupts lipid rafts and modulates the activity of multiple signaling pathways in T lymphocytes.
Lipid rafts are specialized plasma membrane microdomains, in which glycosphingolipids and cholesterol are major structural components. In T lymphocytes, several signaling proteins are associated with lipid rafts including the protein tyrosine kinase LCK and the adapter protein LAT. To investigate their importance in T cell signaling, lipid rafts were disrupted by depleting cholesterol with methyl-beta-cyclodextrin (MbetaCD). This transiently induced tyrosine phosphorylation of multiple proteins, including the ZAP-70 tyrosine kinase, its associated T cell antigen receptor zeta chain, LAT and phospholipase Cgamma1. Tyrosine phosphorylation was dependent on expression of LCK in lipid rafts. Depletion of cholesterol also resulted in activation of the Ras-ERK pathway. This was largely dependent on phorbol ester-sensitive protein kinase C (PKC) and the PKC-theta isoform translocated to the plasma membrane following MbetaCD treatment. MbetaCD did not stimulate intracellular Ca2+ fluxes; however, consistent with its ability to stimulate Ras, MbetaCD synergized with a Ca2+ ionophore to induce formation of the transcription factor NF-AT. These data indicate a crucial role for cholesterol in the regulation of signaling pathways in T cells, which is likely to reflect its importance in the formation of plasma membrane lipid rafts.
Kabouridis
P S
PS
Division of Membrane Biology, National Institute for Medical Research, London, GB.
Janzen
J
J
Magee
A L
AL
Ley
S C
SC
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017252:CD3 Complex / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D000074121:Protein Kinase C-theta; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D047392:beta-Cyclodextrins
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D003505:Cyclodextrins; D007476:Ionophores; D007527:Isoenzymes; D008563:Membrane Lipids; D008565:Membrane Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D014443:Tyrosine; D002784:Cholesterol; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D020928:Mitogen-Activated Protein Kinases