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 glycosph ingolipids and cholesterol are major structural components. In T lymphocyte s, several signaling proteins are associated with lipid rafts including the protein tyrosine kinase LCK and the adapter protein LAT. To investigate th eir importance in T cell signaling, lipid rafts were disrupted by depleting cholesterol with methyl-beta-cyclodextrin (M beta CD). This transiently in duced tyrosine phosphorylation of multiple proteins, including the ZAP-70 t yrosine kinase, its associated T cell antigen receptor zeta, chain, LAI and phospholipase Cy1. Tyrosine phosphorylation was dependent on expression of LCK in lipid rafts. Depletion of cholesterol also resulted in activation o f the Ras-ERK pathway. This was largely dependent on phorbol ester-sensitiv e protein kinase C (PKC) and the PKC-theta isoform translocated to the plas ma membrane following M beta CD treatment. M beta CD did not stimulate intr acellular Ca2+ fluxes: however, consistent with its ability to stimulate Pa s, M beta CD synergized with a Ca2+ ionophore to induce formation of the tr anscription factor NF-AT. These data indicate a crucial role for cholestero l in the regulation of signaling pathways in T cells, which is likely to re flect its importance in the formation of plasma membrane lipid rafts.