Tyrosine phosphorylation is an obligatory event in IL-2 secretion.
Activation of T lymphocytes leads to the production of the T cell growth factor IL-2 that regulates T cell proliferation. This activation is associated with several potential intracellular signalling events including increased activity of phospholipase C (PLC) and resultant increases in production of inositol phosphates and diacylglycerols. In addition, phosphorylation of specific intracellular proteins on serine, threonine, and tyrosine residues increases. The role of each of these events in IL-2 production is unclear. Using Western blotting with antiphosphotyrosine antibodies, we demonstrate that activation of murine T cells with mitogenic lectins or anti-CD3 antibodies leads to a rapid increase in tyrosine phosphorylation of proteins of 120, 72, 62, 55, and 40 kDa. Similar patterns of antiphosphotyrosine antibodies reactivity were observed in splenocytes, a T cell hybridoma, and a T lymphoma. Tyrosine phosphorylation was detectable within minutes of addition of mitogenic lectins and persisted for at least 6 h. Pretreatment of the cells with pertussis toxin did not inhibit tyrosine phosphorylation indicating that a pertussis toxin-sensitive G protein is not involved in signal transduction. Neither increasing cytosolic-free calcium nor activating protein kinase C mimicked the effects of mitogenic lectins suggesting that tyrosine phosphorylation was not a consequence of activation of PLC. This was confirmed by demonstrating that mitogenic lectins induced similar patterns of tyrosine phosphorylation in cells in which activation of the TCR leads to increased PLC activity and in cells in which PLC is not stimulated. To test whether tyrosine phosphorylation is linked to IL-2 secretion, we determined the effect of three specific tyrosine kinase inhibitors (tyrphostins) on tyrosine phosphorylation, IL-2 secretion, and cellular proliferation. The concentration dependence of inhibition of tyrosine phosphorylation and IL-2 production were similar. However, higher concentrations of the tyrphostins were required to inhibit constitutive proliferation of the T cell line indicating that inhibition of IL-2 secretion was not secondary to nonspecific toxic effects of the tyrphostins. Addition of the tyrphostins after mitogenic lectin decreased the amount of tyrosine phosphorylation and IL-2 secretion in parallel. This indicates that both tyrosine kinases and phosphatases are activated and that continuous tyrosine phosphorylation is likely required for IL-2 secretion. Therefore, tyrosine phosphorylation appears to represent an obligatory event in the transmembrane signaling processes that lead to IL-2 secretion.
Toronto General Hospital, Ontario, Canada.
J B
JB
Stanley
R
R
Gorczynski
C K
CK
Huang
J
J
Love
G B
GB
Mills
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D003208:Concanavalin A / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D007376:Interleukin-2 / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D008213:Lymphocyte Activation; D051379:Mice; D008970:Molecular Weight; D037342:Pertussis Toxin; D010766:Phosphorylation; D019000:Phosphotyrosine; D010835:Phytohemagglutinins / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013601:T-Lymphocytes / Q000502:physiology*; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007376:Interleukin-2; D010835:Phytohemagglutinins; D010566:Virulence Factors, Bordetella; D003208:Concanavalin A; D019000:Phosphotyrosine; D014443:Tyrosine; D015759:Ionomycin; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins; D002118:Calcium