Interleukin‐4 (IL‐4) was shown to induce a potent mitogenic response in the IL‐3‐dependent myeloid progenitor cell line, FDCP‐2. Although IL‐4 could not sustain long‐term growth of FDCP‐2 cells, it enhanced their growth in serum‐free medium containing IL‐3. IL‐4 triggered prominent tyrosine phosphorylation of a substrate(s) migrating at 170 kDa and less striking phosphorylation of several other proteins, including the IL‐4 receptor. By contrast, IL‐3 induced distinct tyrosine phosphorylation of proteins migrating at 145, 97, 70, 55 and 52 kDa in the same cell line. IL‐4 treatment of FDCP‐2 cells caused a dramatically strong association of phosphatidylinositol 3‐kinase (PI 3‐kinase) both with the 170 kDa tyrosine phosphorylated substrate and with the IL‐4 receptor itself. By contrast, IL‐3 triggered only weak association of PI 3‐kinase activity with the 97 kDa substrate. While IL‐4 did not affect cellular raf, IL‐3 stimulation did induce a shift in its mobility presumably due to serine/threonine phosphorylation. Taken together, our results indicate that IL‐4 and IL‐3 activate distinct phosphorylation cascades in the same cell background; this may reflect a difference in the biological function of these two cytokines.

Interleukin 4 (IL-4), insulin, and insulin-like growth factor I (IGF-I) efficiently induced DNA synthesis in the IL-3-dependent murine myeloid cell lines FDC-P1 and FDC-P2. Although these factors could not individually sustain long-term growth of these lines, a combination of IL-4 with either insulin or IGF-I did support continuous growth. The principal tyrosine-phosphorylated substrate observed in FDC cells stimulated with IL-4, previously designated 4PS, was of the same size (170 kDa) as the major substrate phosphorylated in response to insulin or IGF-I. These substrates had phosphopeptides of the same size when analyzed by digestion with Staphylococcus aureus V8 protease, and each tightly associated with the 85-kDa component of phosphatidylinositol 3-kinase after factor stimulation. IRS-1, the principal substrate phosphorylated in response to insulin or IGF-I stimulation in nonhematopoietic cells, is similar in size to 4PS. However, anti-IRS-1 antibodies failed to efficiently precipitate 4PS, and some phosphopeptides generated by V8 protease digestion of IRS-1 were distinct in size from the phosphopeptides of 4PS. Nevertheless, IL-4, insulin, and IGF-I were capable of stimulating tyrosine phosphorylation of IRS-1 in FDC cells that expressed this substrate as a result of transfection. These rmdings indicate that (i) IL-4, insulin, and IGF-I use signal transduction pathways in FDC lines that have at least one major feature in common, the rapid tyrosine phosphorylation of 4PS, and (it) insulin and IGF

Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

IL‐3 dependent mast cell lines produce cytokines in response to Fc receptor cross‐linkage or to ionomycin. In this study we have observed that cells pre‐cultured in IL‐3 produce 10–100 times more cytokine after receptor cross‐linkage in comparison with IL‐4 pre‐cultured cells. Although several hematopoietin receptors, including those for IL‐3, IL‐4 and EPO, do not contain tyrosine kinase domains, their occupancy with ligand causes tyrosine phosphorylation of specific cellular substrates. Therefore, the contribution of tyrosine kinase activation to the ability of an IL‐3 dependent mast cell line, CFTL‐15, to produce cytokines was analyzed. The CFTL‐15 cells were transfected with growth factor receptors containing ligand‐inducible tyrosine kinase domains (EGFR and PDGFR, and CSF‐IR) or with the EPOR. All of the transfectants were able to proliferate in response to IL‐3 or to their respective growth factor and to produce IL‐3 in response to IgE receptor cross‐linkage. Stimulation of the EGFR and PDGFR transfectants with their respective ligands resulted in the production of IL‐3, IL‐6, and GM‐CSF. Stimulation of the CSF‐1R or EPOR transfectants with growth factor alone failed to induce cytokine production. However, in co‐stimulation assays each of the growth factors enhanced the amount of cytokine produced in response to Fc epsilon RI cross‐linkage. The ability of these stimuli to induce tyrosine phosphorylation in the transfectants was analyzed. Fc epsilon RI cross‐linkage in the transfectants routinely induced the tyrosine phosphorylation of 145, 86 and 72 kDa proteins, with occasional phosphorylation of 55, 52, and 40 kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)