NK-LAK induction with IL-2 is regulated by tyrosine phosphorylation of a 105-110 kDa protein.
Tyrosine phosphorylation of the cellular proteins of IL-2-stimulated NK cells was determined by anti-phosphotyrosine immunoblotting. IL-2 induced tyrosine phosphorylation of a 105-110 kDa protein in a dose-dependent manner. The tyrosine phosphorylation took place within 5 min after the addition of IL-2 to NK cells, and reached a maximal level in 15 min. The degree of the tyrosine phosphorylation correlated with IL-2-induced LAK activity. Staurosporine and pertussis toxin, which slightly suppressed LAK induction, did not inhibit tyrosine phosphorylation of the 105-110 kDa protein. Genistein, TMB-8 and EGTA completely inhibited LAK induction; however, the calcium channel blocker and chelator did not prevent the protein tyrosine phosphorylation. Anti-IL-2R beta mAb almost completely suppressed tyrosine phosphorylation of the 105-110 kDa protein, but anti-IL-2R alpha mAb only slightly suppressed it; this result correlated with that of the suppression of LAK activity. No further suppression of the tyrosine phosphorylation was induced even when both mAbs were added. Western blotting of the immunoprecipitates revealed no association of PLC-gamma 1 or IL-2R beta with the 105-110 kDa protein. These results suggest that both tyrosine phosphorylation of the 105-110 kDa protein and translocation of [Ca++]i are essential for NK-LAK induction, and the tyrosine phosphorylation plays a critical role in the early stage of IL-2 signalling from the IL-2R beta chain.
Yoneda
K
K
Department of Oral Surgery, Kochi Medical School Kohasu, Japan.
Osaki
T
T
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D015979:Killer Cells, Lymphokine-Activated / Q000187:drug effects / Q000378:metabolism*; D007694:Killer Cells, Natural / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*
D007376:Interleukin-2; D015375:Receptors, Interleukin-2; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D002118:Calcium