Cholesterol Depletion Disrupts Lipid Rafts and Modulates the Activity of Multiple Signaling Pathways in T Lymphocytes

Eur J Immunol. 2000 Mar;30(3):954-63. doi: 10.1002/1521-4141(200003)30:3<954::AID-IMMU954>3.0.CO;2-Y.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / pharmacology
  • CD3 Complex / metabolism
  • Calcium Signaling / drug effects
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cholesterol / metabolism*
  • Cyclodextrins / pharmacology
  • Enzyme Activation / drug effects
  • Humans
  • Ionophores / pharmacology
  • Isoenzymes / metabolism
  • Jurkat Cells
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / metabolism
  • Membrane Lipids / metabolism*
  • Membrane Proteins / metabolism
  • Mitogen-Activated Protein Kinases / metabolism
  • Phosphorylation
  • Protein Kinase C / metabolism
  • Protein Kinase C-theta
  • Signal Transduction
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism*
  • Tyrosine / metabolism
  • beta-Cyclodextrins*

Substances

  • Antibodies, Monoclonal
  • CD3 Complex
  • Cyclodextrins
  • Ionophores
  • Isoenzymes
  • Membrane Lipids
  • Membrane Proteins
  • beta-Cyclodextrins
  • methyl-beta-cyclodextrin
  • Tyrosine
  • Cholesterol
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
  • PRKCQ protein, human
  • Protein Kinase C
  • Protein Kinase C-theta
  • Mitogen-Activated Protein Kinases