Characterization of an interleukin-2 (IL-2)-induced tyrosine phosphorylated 116-kDa protein associated with the IL-2 receptor beta-subunit.
In this paper we have extended previous results on interleukin-2 receptor (IL2-R) signal transduction and focused on the interleukin-2 (IL-2)-stimulated tyrosine phosphorylation of a 116-kDa protein (p116) observed in IL-2 responsive cells. This protein exhibited rapid and transient phosphorylation kinetics in both human T-lymphocytes and the YT cell line, attaining maximum tyrosine phosphorylation within 5 min of stimulation with IL-2. Tyrosine phosphorylated p116 co-purified with activated IL-2 receptor beta-chain (IL2-R beta) when IL2-R complexes were covalently stabilized with the membrane-permeable cleavable cross-linking agent dithiobis(succimidyl propionate) prior to detergent cell lysis and immunoprecipitation with monoclonal anti-IL2-R beta antibodies. Under these conditions comparable amounts of tyrosine-phosphorylated p116 were immunoprecipitated with either anti-IL2-R beta antibodies or anti-phosphotyrosine antibodies, suggesting that a major portion of tyrosine phosphorylated p116 is associated with the IL2-R beta subunit. Furthermore, unphosphorylated p116 was also associated with unactivated IL2-R beta, based on the observation that p116 from unstimulated YT cells underwent tyrosine phosphorylation in IL2-R beta immune-complex tyrosine kinase assay as demonstrated by anti-phosphotyrosine immunoblotting. The presence of tyrosine kinase activity in affinity-purified IL2-R beta complexes supports the notion of a preformed receptor-kinase complex. The co-association of both p116 and tyrosine kinase activity with the IL2-R beta supports the critical role of the beta-chain in IL2-R signal transduction and suggests that p116 may have a role in the dynamics of IL2-R activation.
Kirken
R A
RA
Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21702-1201.
Rui
H
H
Evans
G A
GA
Farrar
W L
WL
D002460:Cell Line; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D007700:Kinetics; D008213:Lymphocyte Activation; D046911:Macromolecular Substances; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015375:Receptors, Interleukin-2 / Q000302:isolation & purification / Q000378:metabolism*; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D007376:Interleukin-2; D046911:Macromolecular Substances; D010750:Phosphoproteins; D015375:Receptors, Interleukin-2; D019000:Phosphotyrosine; D014443:Tyrosine; D020794:Receptor Protein-Tyrosine Kinases