Lipid rafts as the signaling scaffold for NK cell activation: tyrosine phosphorylation and association of LAT with phosphatidylinositol 3-kinase and phospholipase C-gamma following CD2 stimulation.
Natural killer (NK) cells participate in both innate and adaptive immunity through the prompt secretion of cytokines and ability to lyse virally infected cells or tumor cells. Although it has been well understood that lipid rafts (rafts) and a raft-associated linker for activation of T cells (LAT) plays a central role in TCR signal transduction, there are still great gaps in our knowledge of the molecular events involved in NK cell activation. We show here that CD2 and rafts became polarized to the site of NK cell activation by CD2 cross-linking or target cell binding using confocal microscopy, and LAT and a significant amount of CD2 colocalized in raft fractions of sucrose-density gradient from an NK cell line, NK3.3. CD2 cross-linking strongly induced tyrosine phosphorylation of LAT, resulting in increased association with phosphatidylinositol 3-kinase (PI 3-K) and phospholipase C-gamma1 (PLC-gamma1). In vitro binding studies using glutathione S-transferase fusion proteins demonstrated that a large portion of the association between LAT and PI 3-K or PLC-gamma1 was mediated through their SH2 domains in tyrosine phosphorylation-dependent manner. Furthermore, disruption of lipid rafts by cholesterol depletion from cell membranes using methyl-beta-cyclodextrin markedly reduced LAT tyrosine phosphorylation and NK cell functions, including cytotoxicity and granule exocytosis. These results document that modulation of raft integrity by aggregation of NK cell activating receptors, which leads to the formation of complexes of LAT with PI 3-K and PLC-gamma1, is essential for the NK cell lytic mechanisms.
Inoue
Hiroshi
H
Department of Internal Medicine, Osaka Dental University, Osaka, Japan.
Miyaji
Michihiko
M
Kosugi
Atsushi
A
Nagafuku
Masakazu
M
Okazaki
Toshiro
T
Mimori
Tsuneyo
T
Amakawa
Ryuichi
R
Fukuhara
Shirou
S
Domae
Naochika
N
Bloom
Eda T
ET
Umehara
Hisanori
H
D048868:Adaptor Proteins, Signal Transducing; D018801:CD2 Antigens / Q000032:analysis / Q000502:physiology*; D002352:Carrier Proteins / Q000032:analysis / Q000502:physiology*; D002460:Cell Line; D002784:Cholesterol / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000502:physiology*; D007694:Killer Cells, Natural / Q000276:immunology*; D008213:Lymphocyte Activation; D021962:Membrane Microdomains / Q000502:physiology*; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000032:analysis / Q000502:physiology*; D010766:Phosphorylation; D010738:Type C Phospholipases / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D018801:CD2 Antigens; D002352:Carrier Proteins; D007527:Isoenzymes; C110810:LAT protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D014443:Tyrosine; D002784:Cholesterol; D019869:Phosphatidylinositol 3-Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma