Ligand-stimulated tyrosine phosphorylation of the IL-2 receptor beta chain and receptor-associated proteins.

Interleukin-2 (IL-2) stimulates the rapid phosphorylation on tyrosine of several specific cellular proteins. However, the high-affinity human IL-2 receptor, composed of an alpha (p55) and beta (p70/75) subunit, does not contain a cytoplasmic tyrosine kinase domain. In this study, we investigated the identities of the proteins phosphorylated on tyrosine in response to IL-2 stimulation to examine possible pathways of signal transduction. By the use of immunoblotting with anti-phosphotyrosine antibodies, we demonstrate that IL-2 augments tyrosine phosphorylation of the IL-2 receptor beta chain in human cell lines expressing either high-affinity (alpha/beta) receptors or only the beta chain. In IL-2-dependent mouse T cell lines, a 100,000-Da protein was phosphorylated on tyrosine in response to IL-2 and is proposed to be the mouse IL-2 receptor beta chain. Two other cellular proteins, pp55 and pp105 in human or pp55 and pp115 in mouse cell lines, were phosphorylated on tyrosine in response to IL-2 and coimmunoprecipitated with the high-affinity IL-2 receptor after chemical crosslinking of IL-2-stimulated cells. Thus, the IL-2 receptor may associate with additional subunits or with cellular proteins involved in signal transduction.     

Stimulation of the antigen and interleukin-2 receptors on T lymphocytes activates distinct tyrosine protein kinases

The T cell antigen receptor complex (TCR) and the interleukin 2 (IL-2) receptor are responsible for signal transduction that results in T lymphocyte activation and proliferation. Stimulation of either the TCR or the IL-2 receptor induces an increase in tyrosine phosphorylation of several cellular proteins indicating that signal transduction by both of these receptors involves the activation of a tyrosine protein kinase. Although the tyrosine protein kinases activated by these receptors have not yet been characterized the receptors themselves are known not to contain a tyrosine protein kinase domain. To determine if these receptors are coupled to the activation of similar or distinct tyrosine protein kinases we examined the patterns and kinetics of tyrosine phosphorylation induced by stimulation of these receptors on a cloned cell line. Hut 78.3 cells co-express the TCR and the p75 IL-2 receptor. These cells were stimulated with either OKT3 antibodies, specific for the TCR, or with IL-2. Signal transduction by these receptors was found to increase the tyrosine phosphorylation of a set of proteins unique to each stimulus. The kinetics of the tyrosine phosphorylation induced by OKT3 antibodies also differed from that induced by IL-2. The OKT3-dependent tyrosine phosphorylation reached maximal levels within 2.5 min and began to decline by 5 min after stimulation. In contrast, the IL-2-induced tyrosine phosphorylation did not achieve maximal levels until 15 min after the addition of IL-2 and the proteins remained phosphorylated even after 60 min of incubation. In addition the tyrosine phosphorylations induced by OKT3 and IL-2 were not affected by prior stimulation with the other agent. These results demonstrate that the TCR and IL-2 receptor are coupled to different signal transduction pathways responsible for the independent activation of distinct tyrosine protein kinases.     

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.     

Why are multiple chains required for the interleukin 2 receptor?

The interleukin 2 receptor (IL-2R) is composed of at least two proteins, that is, a 55 kDa L chain (p55, α chain) and a 75 kDa H chain (p75, β chain). The high-affinity binding of IL-2 results in the formation of the ternary complex consisting of IL-2, the L chain and the H chain. Kinetic studies on the IL-2 binding to the high-affinity IL-2R have shown that the association of IL-2 to the L chain is the first step of the ternary complex formation and that expression of a larger number of L chains accelerates the association of IL-2 to the high-affinity IL-2R in agreement with the stepwise binding/affinity conversion model. This conclusion was supported by experiments using several monoclonal antibodies directed to either H or L chain and murine T cell lines which was transfected by the human L chain cDNA. Temperature-sensitive IL-2 binding to the high-affinity receptor is also consistent with the above conclusion. Signal transduction by the IL-2R appears to involve the activation of tyrosine protein kinase. IL-2 signal transduction seems to require the H chain and another yet unidentified molecule, which might have the kinase activity.     

Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.     

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.     

Phorbol 12-myristate 13-acetate inhibits granulocyte-macrophage colony stimulating factor-induced protein tyrosine phosphorylation in a human factor-dependent hematopoietic cell line.

The human myeloid cell line MO7 requires either granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin 3 (IL-3) for proliferation. We have previously shown that both GM-CSF and IL-3 transiently induce tyrosine phosphorylation of a number of proteins, including two cytosolic proteins, p93 and p70, which are maximally phosphorylated 5-15 min after addition of growth factor to factor-deprived cells. GM-CSF-induced proliferation of MO7 cells was found to be inhibited by two activators of protein kinase C, phorbol 12-myristate 13-acetate (PMA) and bryostatin-1. PMA did not affect surface expression or affinity of the GM-CSF receptor but significantly inhibited GM-CSF- or IL-3-induced tyrosine phosphorylation of p93 and p70. In contrast, PMA augmented GM-CSF-induced tyrosine phosphorylation of another protein, p42. Pretreatment of cells with sodium orthovanadate to inhibit protein tyrosine phosphatases (PTPase) partially reversed the inhibitory effects of PMA. These results suggest that one aspect of GM-CSF and IL-3 signal transduction, protein tyrosine phosphorylation, can be inhibited by a mechanism which does not involve receptor down-regulation, and may involve either receptor down-regulation, and may involve either inhibition of a receptor-activated tyrosine kinase, activation of a protein tyrosine phosphatase, or both. This mechanism could be important in exerting control of proliferation of some types of hematopoietic cells.     

A tyrosine kinase physically associates with the beta-subunit of the human IL-2 receptor

Cell surface expression of the high affinity IL-2R regulates, in part, the proliferative response occurring in Ag- or mitogen-activated T cells. The functional high affinity IL-2R is composed of at least two distinct ligand-binding components, IL-2R alpha (Tac, p55) and IL-2R beta (p70/75). The IL-2R beta polypeptide appears to be essential for growth signal transduction, whereas the IL-2R alpha protein participates in the regulation of receptor affinity. We have prepared and characterized two mAb, DU-1 and DU-2, that specifically react with IL-2R beta. In vitro kinase assays performed with DU-2 immunoprecipitates, but not anti-IL-2R alpha or control antibody immunoprecipitates, have revealed co-precipitation of a tyrosine kinase enzymatic activity that mediates phosphorylation of IL-2R beta. Because both IL-2R alpha and IL-2R beta lack tyrosine kinase enzymatic domains, these findings strongly suggest that noncovalent association of a tyrosine kinase with the high affinity IL-2R complex. Deletion mutants of the intracellular region of IL-2R beta, lacking either a previously described "critical domain" between amino acids 267 and 322 or the carboxyl-terminal 198 residues (IL-2R beta 88), lacked the ability to co-precipitate this tyrosine kinase activity, as measured by phosphorylation of IL-2R beta in vitro. Both of these mutants also failed to transduce growth-promoting signals in response to IL-2 in vivo. Analysis of the IL-2R beta 88 mutant receptor suggested that a second protein kinase mediating phosphorylation on serine and threonine residues physically interacts with the carboxyl terminus of IL-2R beta. This kinase may be necessary but, alone, appears to be insufficient to support a full IL-2-induced proliferative response. These studies highlight the physical association of protein kinases with the cytoplasmic domain of IL-2R beta and their likely role in IL-2-induced growth signaling mediated through the multimeric high affinity IL-2R complex.     

Two-dimensional analysis of interleukin 2-regulated tyrosine kinase activation mediated by the p70-75 beta subunit of the interleukin 2 receptor

The proliferation of activated T lymphocytes is dependent on the interaction of the polypeptide growth factor interleukin 2 (IL 2) with its heterodimeric receptor, which consists of a p55 alpha subunit and a p70-75 beta subunit. Previously, it was shown that IL 2 stimulates rapid serine phosphorylation of several membrane and cytysolic proteins. Here, using anti-phosphotyrosine antibodies to purify phosphotyrosyl proteins and two-dimensional gel analysis, we show that IL 2 stimulates rapid tyrosine phosphorylation of a variety of cellular proteins, including pp180, pp92, and pp42 in activated human T lymphocytes. In addition, we have examined IL 2-induced tyrosine phosphorylation in the human cell line YT2C2 which expresses mostly the beta subunit of the IL 2 receptor and the gibbon cell line MLA-144 which expresses only the beta subunit. In both of these cell lines, IL 2 induced tyrosine phosphorylation of the same proteins phosphorylated in normal human T lymphocytes in response to IL 2. We conclude that the beta subunit is sufficient to induce tyrosine phosphorylation of the normal cellular target substrates involved in signal transduction.     

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.     

Five isoforms of the phosphatidylinositol 3-kinase regulatory subunit exhibit different associations with receptor tyrosine kinases and their tyrosine phosphorylations

There are five isoforms of the regulatory subunit for the heterodimeric type of phosphatidylinositol 3-kinase. These five regulatory subunit isoforms were overexpressed using an adenovirus transfection system, and their own tyrosine phosphorylations and associations with various tyrosine kinase receptors were investigated. When overexpressed in CHO-PDGFR cells, the associations of these regulatory subunit isoforms with the platelet-derived growth factor receptor were similar. However, when overexpressed in CHO-IR cells, p55gamma exhibited a significantly lower ability to bind with IRS-1 upon insulin stimulation, as compared with other regulatory subunit isoforms. Furthermore, p55alpha and p55gamma were found to be tyrosine-phosphorylated. Finally, interestingly, when overexpressed in CHO-EGFR cells or A431 cells and stimulated with epidermal growth factor (EGF), phosphorylated EGF receptor was detected in p85alpha, p85beta and p50alpha immunoprecipitates, but not in p55alpha and p55gamma immunoprecipitates. In addition, EGF-induced tyrosine phosphorylation was observed in p85alpha, p85beta, p55alpha and p55gamma, but not in p50alpha, immunoprecipitates. Thus, each regulatory subunit exhibits specific responses regarding both the association with tyrosine-phosphorylated substrates and its own tyrosine phosphorylation. These results suggest that each isoform possesses specific roles in signal transduction, based on its individual tyrosine kinase receptor.     

Interleukin 2-induced activation of Ras requires two domains of interleukin 2 receptor beta subunit, the essential region for growth stimulation and Lck-binding domain.

Interleukin 2 (IL-2) can stimulate the proliferation of various kinds of T-cell lines. The receptor for IL-2 is composed of at least two subunits (alpha and beta), of which beta subunit plays the major role in transducing growth signals into the cells. A nonreceptor-type tyrosine kinase, Lck, is associated with IL-2 receptor beta subunit, and the binding of IL-2 to its receptor induces the activation of Lck. On the other hand, it has been shown that stimulation of T-cells with IL-2 causes rapid activation of Ras protein. In this paper, we describe that both of the two regions in IL-2 receptor beta subunit, the indispensable region for the induction of cell growth (serine-rich region) and the binding region of Lck protein (acidic region), are required for the activation of Ras. These two regions are also required for tyrosine phosphorylation of an 85-kDa cellular protein (p85) and the accumulation of fos and jun mRNAs. This observation suggests also that the activation of a receptor-associated tyrosine kinase in response to IL-2-stimulation is primarily responsible for subsequent activation of the pathway through Ras to Fos and Jun.     

Interleukin 10 modulation of tumour necrosis factor receptors requires tyrosine kinases but not the PI 3-kinase/p70 S6 kinase pathway

We have previously shown that interleukin (IL-)10-induced proliferation of the murine mast cell line D36, was dependent upon the activation of PI 3-kinase and p70 S6 kinase. Conversely, we were able to show that this pathway was not involved in the signal transduction pathway mediating IL-10 inhibition of pro-inflammatory cytokine release from monocytes. We have extended these studies to investigate the induction of p75 tumour necrosis factor receptor (TNF-R) shedding, another anti-inflammatory property of IL-10. Using the inhibitors of PI 3-kinase (LY294002 and wortmannin) and an inhibitor of p70 S6 kinase activation (rapamycin), we were able to show that this anti-inflammatory effect of IL-10 was not mediated by the PI 3-kinase/p70 S6 kinase pathway, indicating that another signalling cascade(s) was involved. Further studies also investigated the role of tyrosine kinases in the response to IL-10. Two distinct tyrosine kinase inhibitors, herbimycin and genistein affected the expression of TNF-R in response to IL-10 but, surprisingly, with opposite effects. However, both compounds inhibited the activation of both PI 3-kinase and p70 S6 kinase, with a concomitant inhibition of IL-10-induced proliferation. We observed that whilst tyrosine kinase activity was involved in the regulation of TNF-R expression, IL-10-induced activation of JAK kinases was not sensitive to inhibition by the tyrosine kinase inhibitors. These data suggest that multiple unknown tyrosine kinases are mediating the IL-10-induced signal transduction pathways leading to the regulation of TNF-R expression and IL-10-induced proliferation.     

Hck is a key regulator of gene expression in alternatively activated human monocytes

IL-13 is a Th2 cytokine that promotes alternative activation (M2 polarization) in primary human monocytes. Our studies have characterized the functional IL-13 receptor complex and the downstream signaling events in response to IL-13 stimulation in alternatively activated monocytes/macrophages. In this report, we present evidence that IL-13 induces the activation of a Src family tyrosine kinase, which is required for IL-13 induction of M2 gene expression, including 15-lipoxygenase (15-LO). Our data show that Src kinase activity regulates IL-13-induced p38 MAPK tyrosine phosphorylation via the upstream kinases MKK3 or MKK6. Our findings also reveal that the IL-13 receptor-associated tyrosine kinase Jak2 is required for the activation of both Src kinase as well as p38 MAPK. Further, we found that Src tyrosine kinase-mediated activation of p38 MAPK is required for Stat1 and Stat3 serine 727 phosphorylation in alternatively activated monocytes/macrophages. Additional studies identify Hck as the specific Src family member, stimulated by IL-13 and involved in regulating both p38 MAPK activation and p38 MAPK-mediated 15-LO expression. Finally we show that the Hck regulates the expression of other alternative state (M2)-specific genes (Mannose receptor, MAO-A, and CD36) and therefore conclude that Hck acts as a key regulator controlling gene expression in alternatively activated monocytes/macrophages.     

Interleukin-2 (IL-2) induces tyrosine kinase-dependent translocation of active raf-1 from the IL-2 receptor into the cytosol.

Stimulation of the interleukin-2 (IL-2) receptor results in phosphorylation and activation of cytosolic Raf-1 serine/threonine kinase. Herein, we report that enzymatically active Raf-1 is physically associated with the IL-2 receptor beta chain (p75) in T-cell blasts. Following stimulation with IL-2, Raf-1 dissociates from the IL-2 receptor complex and translocates to the cytosol. Genistein, a protein tyrosine kinase inhibitor, prevents the dissociation of enzymatically active Raf-1 from the ligand-stimulated IL-2 receptor complex. These data favor a model of IL-2 receptor activation in which an IL-2-activated protein tyrosine kinase phosphorylates the IL-2 receptor and/or receptor-bound Raf-1. Following tyrosine phosphorylation, enzymatically active Raf-1 dissociates from the IL-2 receptor and translocates into the cytosol.     

Reconstitution of a functional IL-2 receptor by the beta-chain cDNA. A newly acquired receptor transduces negative signal

The high-affinity IL-2R results from the noncovalent association between at least two subunits; alpha (p55) and beta (p70), both of which are capable of binding IL-2 with a low and intermediate affinity, respectively. Although the alpha-chain itself has been shown to be nonfunctional, suggestions have been made that the beta-chain mediates an IL-2 signal. To directly study the role of the beta-chain in the signal transduction, we transfected with the cDNA encoding the IL-2R beta-chain a human T lymphotropic virus-I-transformed T cell line, MT-1 originally expressing low-affinity alpha-chain alone, and established a stable transformant (designated MT-beta 7) which expressed both alpha- and beta-chains simultaneously. We showed 1) MT-beta 7 manifested the high-affinity IL-2 binding, which was completely disrupted by the anti-beta chain mAb (Mik-beta 1), 2) the 125I-IL-2 crosslinking patterns of MT-beta 7 were indistinguishable from those of cells expressing the native high-affinity IL-2R, 3) MT-beta 7, but not parental MT-1, internalized the bound IL-2 and responded to IL-2 with a negative signal, i.e., inhibition of the de novo DNA synthesis. These results clearly demonstrate that the beta-chain not only participates in forming the high-affinity IL-2R with the alpha-chain but also is directly involved in the IL-2 signal transduction.     

The p75 neurotrophin receptor enhances TrkA signalling by binding to Shc and augmenting its phosphorylation

Nerve growth factor (NGF) is an important neuronal survival factor, especially during development. Optimal sensitivity of the survival response to NGF requires the presence of TrkA and the p75 neurotrophin receptor, p75(NTR). Signalling pathways used by TrkA are well established, but the mechanisms by which p75(NTR) enhances NGF signalling remain far from clear. A prevalent view is that p75(NTR) and TrkA combine to form a high-affinity receptor, but definitive evidence for this is still lacking. We therefore investigated the possibility that p75(NTR) and TrkA interact via their signal transduction pathways. Using antisense techniques to down-regulate p75(NTR) and TrkA, we found that p75(NTR) specifically enhanced phosphorylation of the 46- and 52-kDa isoforms of Shc during nerve growth factor-induced TrkA activation. p75(NTR) did not enhance tyrosine phosphorylation of other TrkA substrates. Serine phosphorylation of Akt, downstream of Shc activation, was also p75(NTR)-dependent. We consistently detected co-immunoprecipitation of p75(NTR) and Shc. These data indicate that p75(NTR) interacts with Shc physically, via a binding interaction, and functionally, by assisting its phosphorylation. Whilst providing evidence that p75(NTR) augments TrkA signal transduction, these results do not preclude the presence of a p75(NTR)-TrkA high-affinity NGF receptor.     

Human IL-3 induction of c-jun in normal monocytes is independent of tyrosine kinase and involves protein kinase C.

We have used normal human monocytes as a model system to begin elucidating the signal transduction mechanism associated with the IL-3R. Normal human monocytes deprived of human serum and CSF become quiescent in vitro. Stimulation of these cells with rIL-3 induces expression of the c-jun protooncogene, as detected by Northern blotting of total monocyte RNA. This protooncogene is also induced in these cells by phorbol ester through direct stimulation of protein kinase C. Concentrations of the protein kinase C inhibitor I-(5-isoquindinyl-sulfonyl)-2 methylpiperazine (H-7) between 30 and 100 microM (5-20 x Ki) inhibit this induction by phorbol ester. The same concentration-range of H-7 completely inhibited the induction of c-jun by human IL-3. A structural analog of H-7 designated HA-1004 preferentially inhibits cyclic nucleotide-dependent protein kinase rather that protein kinase C. HA-1004 at 5 to 20 x Ki did not inhibit IL-3-induced c-jun mRNA accumulation. Further 30 microM genistein that is an effective inhibitor of cellular tyrosine kinases did not inhibit IL-3-induced c-jun expression. Immunoprecipitation of lysates from [32P]orthophosphate labeled cells with antiphosphotyrosine polyclonal antibody showed that IL-3-stimulated phosphorylation of a 70-kDa protein and a 110-kDa protein on tyrosine, and that these protein phosphorylations were completely inhibited by 30 microM genistein. As further confirmation that IL-3 is stimulating protein kinase C in human monocytes we have found that IL-3 stimulates phosphorylation of the unique protein kinase C substrate myristoylated alanine-rich C kinase substrate in these cells. It is therefore likely that the interaction of IL-3 with its receptor generates diacylglycerol and stimulates the Ca2+/phospholipid-dependent protein kinase C.     

Costimulation of resting B lymphocytes alters the IL－4－activated IRS2 signaling pathway in a STAT6 independent manner：implications for cell survival and proliferation

IL-4 is an important B cell survival and growth factor. IL-4 induced the tyrosine phosphorylation of IRS2 in resting B lymphocytes and in LPS- or CD40L-activated blasts. Phosphorylated IRS2 coprecipitated with the p85 subunit of PI 3' kinase in both resting and activated cells. By contrast, association of phosphorylated IRS2 with GRB2 was not detected in resting B cells after IL-4 treatment although both proteins were expressed. However, IL-4 induced association of IRS2 with GRB2 in B cell blasts. The pattern of IL-4-induced recruitment of p85 and GRB2 to IRS2 observed in B cells derived from STAT6 null mice was identical to that observed for normal mice. While IL-4 alone does not induce activation of MEK, a MEK1 inhibitor suppressed the IL-4-induced proliferative response of LPS-activated B cell blasts. These results demonstrate that costimulation of splenic B cells alters IL-4-induced signal transduction independent of STAT6 leading to proliferation. Furthermore, proliferation induced by IL-4 in LPS-activated blasts is dependent upon the MAP kinase pathway.