Phosphorylation/dephosphorylation of high-affinity IgE receptors: a mechanism for coupling/uncoupling a large signaling complex.

Engagement of high-affinity IgE receptors leads to activation of tyrosine and serine/threonine kinases and the immediate phosphorylation of receptor beta (serine and tyrosine) and gamma (threonine and tyrosine) chains. Receptor disengagement leads to dephosphorylation of beta and gamma chains via the action of undefined phosphatases. Here we have identified five distinct polypeptides associated with the high-affinity IgE-receptor tetrameric complex, which apparently become phosphorylated and dephosphorylated in sequence with the beta and gamma chains. Like beta chain, polypeptides pp180, pp48, pp42, and pp28 are phosphorylated on serine and tyrosine, whereas pp125 is only phosphorylated on serine. The phosphorylation of each of these receptor-associated polypeptides is antigen-dose dependent and is restricted to activated receptor complexes. Furthermore the physical association between pp125 and the receptor is quantitatively affected by receptor phosphorylation and dephosphorylation, indicating a coupling-uncoupling mechanism. Finally, in vitro kinase experiments show that activated receptor complexes are also physically associated with tyrosine and serine/threonine kinases as part of a larger complex containing the phosphorylated polypeptides

IL-2 stimulates the production of IL-1 alpha and IL-1 beta by human peripheral blood mononuclear cells.

Abstract Human PBMC were cultured in medium containing human rIL-2, and the supernatants and cell lysates were analyzed for IL-1 alpha and IL-1 beta using specific RIA. IL-2, but not the excipient detergents included in the rIL-2 preparation, induced the synthesis of both cytokines. The concentrations of IL-1 alpha and IL-1 beta in the cell lysates and supernatants depended on both the concentration of rIL-2 in the culture medium and the duration of the incubation. After 24 h of stimulation, IL-2-induced IL-1 alpha remained almost entirely cell-associated. In contrast, IL-1 beta was present in both cell lysates and supernatants and was more abundant in the latter. SDS-PAGE analysis after radioimmunoprecipitation with anti-IL-1 antibodies indicates that cell-associated IL-1 resulting from IL-2 stimulation was in the form of the 35 kDa IL-1 precursor whereas secreted IL-1 was almost entirely in the form of the mature 18 kDa product. Depletion of monocytes from the PBMC culture substantially reduced IL-2-induced IL-1 production. In addition, Leu M3+ monocytes obtained through FACS, but not CD16+ NK cells, produced both IL-1 alpha and IL-1 beta in response to IL-2. The low level of endotoxin present in the IL-2 preparation used in our studies and the selective inhibition by polymyxin B of LPS-induced, but not IL-2-induced, IL-1 production by PBMC indicate that IL-2-induced IL-1 production was not due to endotoxin contamination. Furthermore, an anti-IL-2 antiserum selectively inhibited IL-1 production in response to IL-2 stimulation. We conclude that IL-2 is a potent inducer of IL-1 synthesis and secretion in vitro and propose that IL-1 may be generated in vivo in patients undergoing IL-2 immunotherapy.</jats:p

Regulation by CD45 of the tyrosine phosphorylation of high affinity IgE receptor beta- and gamma-chains.

Abstract Previous studies using tyrosine phosphatase inhibitors have implicated tyrosine phosphatases in the signal transduction pathway initiated by aggregation of Fc epsilon RI, the high affinity receptor for IgE. To define more precisely a role for the tyrosine phosphatase CD45 in Fc epsilon RI-mediated signaling, we have transfected the three subunits of Fc epsilon RI into wild-type Jurkat and a CD45-deficient Jurkat derivative. Here we demonstrate that CD45 is necessary for the initiation of calcium flux through the transfected Fc epsilon RI. In contrast to the effect of phosphatase inhibitors, the tyrosine phosphorylation levels of beta and gamma after aggregation of Fc epsilon RI are surprisingly reduced, relative to wild-type Jurkat, in the CD45-deficient cells. After reconstitution of the CD45-deficient cells with a chimeric molecule containing the cytoplasmic phosphatase domains of CD45, both the base line and activation-induced tyrosine phosphorylation levels are increased. By examining Lck autophosphorylation, we find that Fc epsilon RI aggregation induces an increase in Lck enzymatic activity only in wild-type Jurkat and the CD45-deficient Jurkat reconstituted with chimeric CD45. This regulation of src-family tyrosine kinase activity may be the means by which CD45 controls aggregation-induced receptor phosphorylation.</jats:p