Interleukin-3 and granulocyte-macrophage colony-stimulating factor mediate rapid phosphorylation and activation of cytosolic c-raf.

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor induce the rapid phosphorylation of the c-raf protein in the growth factor-dependent FDC-P1 and DA-3 murine myeloid cell lines. Furthermore, immunoprecipitates of c-raf isolated from growth factor-stimulated cells demonstrate a marked increase in intrinsic protein kinase activity as measured in vitro. IL-3 and granulocyte-macrophage colony-stimulating factor induce phosphorylation of c-raf at both serine and tyrosine residues. Antiphosphotyrosine immunoprecipitates from IL-3-stimulated cells demonstrate the rapid and coordinate phosphorylation of both c-raf and a protein co-migrating with the 140-kDa putative IL-3 receptor component. Collectively, the findings of rapid and coordinate ligand-induced phosphorylation of a potential IL-3 growth factor receptor component and cytoplasmic c-raf with concomitant c-raf activation provide a cogent sequential molecular model for linking external growth stimuli to intracellular signal transduction events.     

Regulation of the interleukin 2 receptor complex tyrosine kinase activity in vitro.

Interleukin 2 (IL-2) has been shown to stimulate tyrosine phosphorylation of a number of proteins requiring only the p75 beta chain of the IL-2 receptor. Unlike the receptors for epidermal growth factor, insulin, and other growth factors, the p55-alpha and p75-beta chains of the IL-2 receptor have no tyrosine protein kinase domain suggesting that the IL-2 receptor complex activates protein kinases by a unique mechanism. The activation of tyrosine kinases by IL-2 in situ was studied and using a novel methodology has shown tyrosine kinase activity associated with the purified IL-2R complex in vitro. IL-2 stimulated the in situ tyrosine phosphorylation of 97 kDa and 58 kDa proteins which bound to poly(Glu,Tyr)4:1, a substrate for tyrosine protein kinases, suggesting these proteins had characteristics found in almost all tyrosine kinases. IL-2 was found to stimulate tyrosine protein kinase activity in receptor extracts partially purified from human T lymphocytes and the YT cell line. Biotinylated IL-2 was used to precipitate the high-affinity-receptor complex and phosphoproteins associated with it. The data indicated that the 97-kDa and 58-kDa phosphotyrosyl proteins were tightly associated with the IL-2 receptor complex. These proteins were phosphorylated on tyrosine residues by IL-2 stimulation of intact cells and ligand treatment of in vitro receptor extracts. Furthermore, the 97-kDa and 58-kDa proteins were found in streptavidin-agarose/biotinylated IL-2 purified receptor preparations and showed high affinity for tyrosine kinase substrate support matrixes. The experiments suggest that these two proteins are potential candidates for tyrosine kinases involved in the IL-2R complex signal transduction process.     

Granulocyte-macrophage colony-stimulating factor and steel factor induce phosphorylation of both unique and overlapping signal transduction intermediates in a human factor-dependent hematopoietic cell line.

Steel factor (SF), the ligand for the proto-oncogene c-kit, acts synergistically with GM-CSF or IL-3 to support the growth of normal human hematopoietic progenitor cells. We examined the effects of SF on GM-CSF or IL-3 induced proliferation of a human factor-dependent cell line, MO7. SF supported MO7 cell proliferation as well as IL-3 or GM-CSF alone, and its addition dramatically enhanced (three- to sixfold) maximal GM-CSF or IL-3 stimulated proliferation. SF did not increase the number or affinity of cell surface GM-CSF receptors. We examined several early events of signal transduction in an effort to elucidate the biochemical mechanisms of synergy of these factors. Since each of these three cytokines is believed to function in part through activation of a tyrosine kinase, we examined their effects on cellular phosphotyrosine containing proteins. Each cytokine induced rapid, transient, and concentration dependent tyrosine phosphorylation of a number of substrates. For GM-CSF and IL-3, these phosphoproteins were indistinguishable (150, 125, 106, 93, 80, 79, 73, 44, 42, and 36 kDa), while SF induced major or minor tyrosine phosphorylation of 205, 140-150, 116, 106, 94, 90, 80, 79, 73, 44, 42, 39, 36, 32 kDa phosphoproteins. Two other signal transduction intermediates known to be phosphorylated and activated by GM-CSF and IL-3, the 70-75 kDa Raf-1 kinase, and p42 mitogen-activated protein kinase-2 (MAPK), were also phosphorylated by SF. Combinations of GM-CSF or IL-3 with SF did not further increase the phosphorylation of Raf-1 or p42 MAPK when compared to any of the factors alone. In contrast SF, but not GM-CSF or IL-3, induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). These results indicate that SF and GM-CSF/IL-3 have partially overlapping effects on early signal transducing events, as well as striking differences, such as tyrosine phosphorylation of PLC-gamma. This cell line should provide a useful model system to investigate the complicated process of hematopoietic growth factor synergy.     

Signal transduction of human interleukin 3 and granulocyte-macrophage colony-stimulating factor through serine and tyrosine phosphorylation.

To elucidate the rapid events in signal transduction of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL 3), we examined phosphorylation of proteins on both serine and tyrosine residues in a cytokine-stimulated human myeloid cell line. We found increases in tyrosine phosphorylation within 30 s of stimulation with GM-CSF or IL 3, with peak responses occurring within 2 min. IL 3 and GM-CSF also induced serine phosphorylation, though 10 min of stimulation was required for maximum phosphate incorporation. Interestingly, both IL 3 and GM-CSF stimulated phosphate incorporation in identical substrates, a 68 kDa seryl-phosphoprotein (p68) and a 140 kDa tyrosyl-phosphoprotein (p140). Treatment of AML 193 cells with phorbol myristate acetate resulted in serine phosphorylation of p68; however, p140 was not phosphorylated on tyrosine. Depletion of protein kinase C isoenzymes with high concentrations of phorbol myristate acetate resulted in p68 phosphorylation, which was not further increased by IL 3 or GM-CSF. In contrast, cytokine-induced phosphorylation on tyrosine of p140 was observed after protein kinase C depletion. These data demonstrate the co-ordinate yet independent serine and tyrosine phosphorylation in IL 3- and GM-CSF-treated human myeloid cells, and thus suggest a common set of protein kinases stimulated by each separate ligand.     

Entamoeba histolytica: tyrosine kinase activity induced by fibronectin through the beta1-integrin-like molecule

Previously, we characterized a 140-kDa protein from Entamoeba histolytica as a beta1-integrin-like molecule that binds fibronectin. In this work we present data showing that the amoebic receptor is associated with another surface molecule, the 220-kDa lectin, and with protein tyrosine kinase activity. By immunoprecipitation with the alphabeta1Eh antibody, we demonstrated by immune complex assays for tyrosine protein kinases that the amoebic fibronectin receptor was associated with two phosphorylated proteins of 50 and 70 kDa when internal membranes were used as the source of antigen. When cells were stimulated with fibronectin, two proteins of 55 and 90 kDa were tyrosine phosphorylated, as shown by Western blot with alphaPY20, its phosphorylation being time dependent after fibronectin stimulation. However, when the actin cytoskeleton of fibronectin-stimulated trophozoites was stabilized with phalloidin, the level and the pattern of phosphorylated proteins were different. In this case, a high-molecular-weight component, heavily phosphorylated, was present, which may include the 220-kDa lectin. We also present data confirming that the signaling pathway that is activated by fibronectin is specific. This was demonstrated by comparing the pattern of phosphoproteins obtained in immune complexes prepared with alphabeta1Eh, alphaL220, and alphaPY20 from total extracts obtained in the presence of phalloidin, from cells that had been exposed to fibronectin, soluble concanavalin A, or concanavalin-A-coated substrate. The presence of tyrosine kinases associated with the beta1-integrin-like amoebic molecule was confirmed by immunoprecipitation assays along with the combined use of a tyrosine kinase-specific substrate, the peptide RR-SRC, and a tyrosine kinase inhibitor, genistein.     

Tyrosine-specific phosphorylation of gpIIIa in platelet membranes

In vitro phosphorylation of platelet subcellular fractions revealed that most of the alkali-resistant phosphoproteins and the majority of pp60c-src were in the surface membrane fraction. An alkali-resistant phosphoprotein of about 100 kDa was also immune precipitated by an anti-phosphotyrosine antibody and comigrated with gpIIIa. The phosphorylation of gpIIIa, but not gpIIb, was confirmed by the comparison of reduced and non-reduced gels, and this protein was phosphorylated exclusively on tyrosine. In contrast, both gpIIb and gpIIIa were phosphorylated when the purified complex was added to immunopurified, immobilised pp60c-src. A synthetic peptide with partial homology to a putative tyrosine phosphorylation site in the cytoplasmic domain of gpIIIa was phosphorylated by antibody-purified pp60c-src. Our results indicate that tyrosine-specific phosphorylation of gpIIIa by pp60c-src may play a role in the regulation of platelet function.     

Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins

Platelet-derived growth factor (PDGF) stimulates the phosphorylation of proteins at tyrosine when added to quiescent 3T3 cells, as evidenced by the increase in the amount of phosphotyrosine, relative to phosphoserine and phosphothreonine, in cellular proteins. The increase was detected within 1 min of adding PDGF and was maximal by 5 min. This effect showed the same dependence on PDGF concentration as did association of 125I-PDGF with the cells. In different 3T3 cell lines the magnitude of the increase was approximately proportional to the number of PDGF receptors per cell. A number of proteins phosphorylated at tyrosine in response to PDGF have been detected by two-dimensional gel electrophoresis. They include a pair of related 45 kilodalton phosphoproteins, a pair of related 43 kilodalton phosphoproteins and a 42 kilodalton phosphoprotein. Similar changes were noted when quiescent 3T3 cells were incubated with epidermal growth factor. Possibly, these phosphoproteins are primary substrates of the tyrosine protein kinases activated by the receptors for PDGF and epidermal growth factor, and are involved in physiological effects common to the two growth factors.     

Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis.

A role for tyrosine phosphorylation in the signal-transducing mechanisms of several hematopoietic growth factors has been hypothesized. To extend these observations, we have examined the effects of erythropoietin (Epo) on tyrosine phosphorylation in an Epo-responsive cell that was obtained by transfecting the murine erythropoietin receptor (EpoR) into an interleukin-3 (IL-3)-dependent cell line. By two-dimensional analysis of phosphotyrosine-containing proteins isolated with a monoclonal antibody (1G2) against phosphotyrosine, Epo and IL-3 were found to rapidly induce tyrosine phosphorylation of comparable substrates of 92, 70, and 56 kDa. In addition, Epo uniquely induced phosphorylation of a 72-kDa substrate while IL-3 uniquely induced phosphorylation of a 140-kDa substrate. Immunoprecipitation and mixing experiments indicated that the 72-kDa substrate may represent a small fraction of the EpoR. To explore the significance of tyrosine phosphorylation, we generated two mutants of the EpoR that lacked 108 or 146 amino acids at their carboxyl termini. In addition we constructed an internally deleted mutant that lacked 20 amino acids in a region of sequence homology with the IL-2 receptor beta chain. Although all mutants were expressed at comparable levels and had comparable binding affinities for Epo, only the mutant lacking 108 amino acids at the carboxyl terminus retained significant mitogenic activity or the ability to induce tyrosine phosphorylation.     

Nerve growth factor promotes the activation of phosphatidylinositol 3-kinase and its association with the trk tyrosine kinase.

We investigated the involvement of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in the initiation of signal transduction by nerve growth factor (NGF) in the rat pheochromocytoma PC12 cell line. PtdIns 3-kinase catalyzes the formation of phosphoinositides with phosphate in the D-3 position of the inositol ring and previously has been found to associate with other activated protein tyrosine kinases, including growth factor receptor tyrosine kinases. Anti-phosphotyrosine immunoprecipitates had PtdIns 3-kinase activity that reached a maximum (9 times the basal activity) after a 5-min exposure of PC12 cells to NGF (100 ng/ml). Since NGF activates the tyrosine kinase activity of gp140trk, the protein product of the trk proto-oncogene, we also examined the association of PtdIns 3-kinase with gp140trk. Anti-gp140trk immunoprecipitates from NGF-stimulated PC12 cells had increased PtdIns 3-kinase activity compared to that of unstimulated cells, and larger increases were detected in cells overexpressing gp140trk, indicating that PtdIns 3-kinase associates with gp140trk. NGF produced large increases in [32P]phosphatidylinositol 3,4-bisphosphate and [32P]phosphatidylinositol 3,4,5-trisphosphate in PC12 cells labeled with [32P]orthophosphate, indicating an increase in PtdIns 3-kinase activity in intact cells. Using an anti-85-kDa PtdIns 3-kinase subunit antibody, we found that NGF promoted the tyrosine phosphorylation of an 85-kDa protein and two proteins close to 110 kDa. These studies demonstrate that NGF activates PtdIns 3-kinase and promotes its association with gp140trk and also show that NGF promotes the tyrosine phosphorylation of the 85-kDa subunit of PtdIns 3-kinase. Thus, PtdIns 3-kinase activation appears to be involved in differentiation as well as mitogenic responses.     

Association between B-lymphocyte membrane immunoglobulin and multiple members of the Src family of protein tyrosine kinases.

Treatment of B lymphocytes with antibodies to membrane immunoglobulin (Ig) stimulates protein tyrosine phosphorylation. We have examined the phosphorylation in vitro of proteins associated with membrane Ig. The Src family protein tyrosine kinases p53/56lyn, p59fyn, and p56lck are associated with membrane Ig in spleen B cells and B-cell lines and undergo phosphorylation in vitro. The pattern of expression of Src family protein tyrosine kinases in B cells varied. Our studies suggest that multiple kinases can potentially interact with membrane Ig and that within any one B-cell type, all of the Src family kinases expressed can be found in association with membrane Ig. We also observed that the Ig-associated Ig alpha protein, multiple forms of Ig beta, and proteins of 100 and 25 kDa were tyrosine phosphorylated in vitro. The 100- and 25-kDa proteins remain unidentified.     

The human granulocyte-macrophage colony-stimulating factor receptor is capable of initiating signal transduction in NIH3T3 cells.

The ability of the receptor for the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to function in non-hematopoietic cells is unknown. NIH3T3 fibroblasts were transfected with cDNAs encoding the alpha and beta subunit of the human GM-CSF receptor and a series of stable transformants were isolated that bound GM-CSF with either low (KD = 860 - > 1000 pM) or high affinity (KD = 20-80 pM). Low affinity receptors were not functional. However, the reconstituted high affinity receptors were found to be capable of activating a number of signal transduction pathways, including tyrosine kinase activity, phosphorylation of Raf-1, and the transient induction of c-fos and c-myc mRNAs. The activation of protein tyrosine phosphorylation by GM-CSF in NIH3T3 cells was rapid (< 1 min) and transient (peaking at 5-20 min) and resulted in the phosphorylation of proteins of estimated molecular weights of 42, 44, 52/53 and 58-60 kDa. Some of these proteins co-migrated with proteins from myeloid cells that were phosphorylated on tyrosine residues in response to GM-CSF. In particular, p42 and p44 were identified as mitogen-activated protein kinases (MAP kinases), and the phosphorylation on tyrosine residues of p42 and p44 MAP kinases occurred at the same time as the phosphorylation of Raf-1. However, despite evidence for activation of many mitogenic signal transduction molecules, GM-CSF did not induce significant proliferation of transfected NIH3T3 cells. These results suggest that murine fibroblasts contain signal transducing molecules that can effectively interact with the human GM-CSF receptor, and that are sufficient to activate at least some of the same signal transduction pathways this receptor activates in myeloid cells, including activation of one or more tyrosine kinase(s). However, the level of activation of signal transduction is either below a threshold of necessary activity or at least one mitogenic signal necessary for proliferation is missing.     

The platelet-derived growth factor beta-receptor kinase insert confers specific signaling properties to a chimeric fibroblast growth factor receptor.

Signal transduction by tyrosine kinase growth factor receptors involves ligand-induced phosphorylation of substrates for the kinase, resulting in mediation of common or receptor-specific biological signals. We have compared signal transduction pathways for the fibroblast growth factor receptor-1 (FGFR-1), the platelet-derived growth factor beta-receptor (PDGFR-beta), and a chimeric FGFR-1 molecule, FGFRchim, in which the FGFR-1 kinase insert was replaced with that of the PDGFR-beta. The different receptors were characterized and found to be functional as ligand-stimulatable kinases, after expression of the respective human cDNAs in porcine aortic endothelial cells. Substrates for the receptors were analyzed by ligand stimulation of [32P]orthophosphate-labeled cells and immunoprecipitation with phosphotyrosine antiserum. A number of phosphoproteins were induced in all the different types of cells, but components specifically induced after stimulation of FGFR-1 and PDGFR-beta expressing cells could also be detected. Examination of receptor-associated substrates by in vitro kinase assays revealed phosphoproteins of 65 and 85 kDa, which were associated with PDGFR-beta and FGFRchim, but not with FGFR-1. The 85-kDa phosphoprotein could correspond to the regulatory subunit of phosphatidylinositol 3' kinase (PI3-K), since phosphatidylinositol 3' kinase activity was detected after ligand stimulation of FGFRchim- and PDGFR-beta- but not FGFR-1-expressing cells. In addition, ligand stimulation of FGFRchim- and PDGFR-beta-expressing cells, but not FGFR-1-expressing cells, led to induction of actin reorganization in the form of circular membrane ruffling. Thus, replacement of a discrete segment of the intracellular domain of the FGFR-1 with the corresponding stretch from the PDGFR-beta resulted in transfer of PDGFR-beta-specific signaling properties to the chimeric molecule.     

Phosphoproteins and protein kinases of the Golgi apparatus membrane

Incubation of a highly purified fraction derived from rat liver Golgi apparatus with [gamma-32P]ATP results in phosphorylation of several endogenous phosphoproteins. One phosphoprotein with an apparent Mr of 48,300 is radiolabeled to an apparent extent at least 5-fold higher than any other phosphoprotein as part of either the Golgi apparatus or highly purified rat liver fractions derived from the rough endoplasmic reticulum, mitochondria, plasma membrane, coated vesicles, cytosol, and total homogenate. Approximately 70% of the 48.3-kDa phosphoprotein appears to be a specific extrinsic Golgi membrane protein with the phosphorylated amino acid being threonine. The protein kinase which phosphorylates the 48.3-kDa protein is an intrinsic Golgi membrane protein and is dependent on Mg2+, independent of Ca2+, calmodulin, and cAMP, and is inhibited by N-ethylmaleimide. Preliminary evidence suggests that there are also intrinsic membrane protein kinases in the Golgi apparatus which are dependent on Ca2+ and cAMP. The physiological role of the above phosphoproteins and protein kinases is not known.     

Proliferative action of erythropoietin is associated with rapid protein tyrosine phosphorylation in responsive B6SUt.EP cells

Erythropoietin is a prime regulator of the growth and terminal differentiation of erythroid blood cells. However, little is understood concerning its molecular mechanism of action. Presently it is shown in the responsive, factor-dependent murine cell line B6SUt.EP that erythropoietin induces the tyrosine phosphorylation of six plasma membrane-associated proteins in a time- and concentration-dependent fashion (i.e. phosphoproteins PY153, PY140, PY100, PY93, PY74, and PY54). Among these, PY153 was prominent. For all proteins, maximal levels of phosphorylation were induced within 3-7 min at low factor concentrations (100-500 pM). These findings establish tyrosine kinase activation as a novel candidate pathway of erythropoietin-induced proliferation. In addition, the tyrosine phosphorylation of six proteins with identical Mr, as well as a Mr 104,000 protein, was induced in B6SUt.EP cells by interleukin 3. In contrast, no induced tyrosine phosphorylation was detectable in the erythropoietin-responsive, leukemic erythroid cell line. Rauscher Red 1, yet proteins of Mr 153,000 and 54,000 were shown to be phosphorylated constitutively at relative levels greater than those observed in B6SUt.EP cells. A possible role for these phosphoproteins in hematopoietic cell transformation is considered.     

Growth hormone stimulates the tyrosine phosphorylation of 42- and 45-kDa ERK-related proteins.

Growth hormone (GH) influences a number of tissue-specific biological activities in diverse cell types. However, little is known about the biochemical pathway by which the signal initiated by GH binding to its cell-surface receptor is transduced. The GH receptor has been reported to be phosphorylated on tyrosine in 3T3-F442A cells, a cell line in which GH promotes differentiation and inhibits mitogen-stimulated growth; however, it is not known whether tyrosine phosphorylation plays a role in GH signal transduction. We report that GH treatment of 3T3-F442A cells resulted in the rapid tyrosine phosphorylation of at least four proteins. These included 42- (pp42) and 45-kDa (pp45) proteins immunologically related to ERK1 (extracellular signal-regulated kinase 1), a member of a family of serine/threonine protein kinases that are phosphorylated on tyrosine in response to mitogens. Prolonged phorbol ester pretreatment attenuated the tyrosine phosphorylation of pp42 and pp45 in platelet-derived growth factor-treated cells, but not in GH-treated cells. Maximal GH-stimulated tyrosine phosphorylation of pp42 and pp45 coincided with peak levels of a 42-kDa renaturable MBP kinase activity in lysates of GH-treated cells resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The observation that multiple cellular proteins are rapidly phosphorylated on tyrosine in response to physiological concentrations of GH suggests that tyrosine phosphorylation plays a role in GH signal transduction. Moreover, the stimulation of tyrosine phosphorylation of ERK-related proteins by GH suggests that mitogens and nonmitogens may employ common phosphotyrosyl proteins in the activation of ultimately distinct cellular programs.     

IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.     

Oncostatin M induces tyrosine phosphorylation in endothelial cells and activation of p62yes tyrosine kinase.

Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that has diverse biologic effects, including growth inhibition of tumor cells and induction of IL-6 expression in cultured human endothelial cells (HEC). HEC are highly responsive to oncostatin M and express high levels of oncostatin M receptors relative to other cell types. Oncostatin M has previously been found to bind a specific receptor of 150 to 160 kDa. We have found through the use of anti-phosphotyrosine immunoblotting that oncostatin M induces tyrosine phosphorylation in HEC. Anti-phosphotyrosine antibodies specifically immunoprecipitated labeled oncostatin M cross-linked to its receptor, demonstrating that the oncostatin M receptor is either directly phosphorylated on tyrosine after ligand binding or is tightly associated with a phosphotyrosyl protein in these cells. The tyrosine kinase inhibitor herbimycin A blocked the induction of IL-6 by oncostatin M in HEC. In addition, immune complex kinase assays showed that oncostatin M markedly increased the activity of the p62yes tyrosine kinase with a small increase in p59fyn but no increase in p56lyn tyrosine kinase activity in HEC. We conclude that oncostatin M utilizes a tyrosine phosphorylation signal transduction pathway in HEC involving the activation of the p62yes tyrosine kinase, and that this tyrosine phosphorylation pathway leads to the induction of IL-6 expression.     

IL-4 activates a distinct signal transduction cascade from IL-3 in factor-dependent myeloid cells.

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.