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.     

Critical role for hematopoietic cell kinase (Hck)-mediated phosphorylation of Gab1 and Gab2 docking proteins in interleukin 6-induced proliferation and survival of multiple myeloma cells

Interleukin-6 (LI-6) is a known growth and survival factor in multiple myeloma via activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling cascade. In this report we show that Grb2-associated binder (Gab) family adapter proteins Gab1 and Gab2 are expressed by multiple myeloma cells; and that interleukin-6 induces their tyrosine phosphorylation and association with downstream signaling molecules. We further demonstrate that these events are Src family tyrosine kinase-dependent and specifically identify the role of hematopoietic cell kinase (Hck) as a new Gab family adapter protein kinase. Conversely, inhibition of Src family tyrosine kinases by the pyrazolopyrimidine PP2, as in kinase-inactive Hck mutants, significantly reduces IL-6-triggered activation of extracellular signal-regulated kinase and AKT-1, leading to significant reduction of multiple myeloma cell proliferation and survival. Taken together, these results delineate a key role for Hck-mediated phosphorylation of Gab1 and Gab2 docking proteins in IL-6-induced proliferation and survival of multiple myeloma cells and identify tyrosine kinases and downstream adapter proteins as potential new therapeutic targets in multiple myeloma.     

CD40 signaling of monocyte inflammatory cytokine synthesis through an ERK1/2-dependent pathway. A target of interleukin (il)-4 and il-10 anti-inflammatory action

Ligation of CD40 on monocytes through its interaction with CD40 ligand (CD154) present on activated T helper cells, results in activation of monocyte inflammatory cytokine synthesis and rescue of monocytes from apoptosis induced through serum deprivation. Both of these consequences of CD40 stimulation have been shown to be dependent on the induction of protein tyrosine kinase activity. CD40-mediated activation of protein tyrosine kinase activity and subsequent inflammatory cytokine production are abrogated by treatment of monocytes with the T helper type 2 cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10). In the current study we demonstrate that stimulation of monocytes through CD40 resulted in the phosphorylation and activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinases, whereas phosphorylation of mitogen-activated protein kinases family members p38 and c-Jun N-terminal kinase was not observed in response to this stimuli over the time course examined. PD98059, an inhibitor of the upstream activator of ERK1/2, the MAP/ERK kinase MEK1/2, suppressed IL-1beta and tumor necrosis factor-alpha production in a dose-dependent fashion. Pretreatment of monocytes with IL-4 and IL-10 inhibited CD40-mediated activation of ERK1/2 kinase activity when used individually, and are enhanced in effectiveness when used in combination. Together, the data demonstrate that CD40-mediated induction of IL-1beta and tumor necrosis factor-alpha synthesis is dependent on a MEK/ERK pathway which is obstructed by signals generated through the action of IL-4 and IL-10.     

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.     

E3 Ubiquitin Ligase c-cbl Inhibits Microglia Activation After Chronic Constriction Injury

E3 ubiquitin ligase c-Caritas B cell lymphoma (c-cbl) is associated with negative regulation of receptor tyrosine kinases, signal transduction of antigens and cytokine receptors, and immune response. However, the expression and function of c-cbl in the regulation of neuropathic pain after chronic constriction injury (CCI) are unknown. In rat CCI model, c-cbl inhibited the activation of spinal cord microglia and the release of pro-inflammatory factors including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6), which alleviated mechanical and heat pain through down-regulating extracellular signal-regulated kinase (ERK) pathway. Additionally, exogenous TNF-α inhibited c-cbl protein level vice versa. In the primary microglia transfected with c-cbl siRNA, when treated with TNF-α or TNF-α inhibitor, the corresponding secretion of IL-1β and IL-6 did not change. In summary, CCI down-regulated c-cbl expression and induced the activation of microglia, then activated microglia released inflammatory factors via ERK signaling to cause pain. Our data might supply a novel molecular target for the therapy of CCI-induced neuropathic pain.     

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.     

Beta2 integrin-dependent phosphorylation of protein-tyrosine kinase Pyk2 stimulated by tumor necrosis factor alpha and fMLP in human neutrophils adherent to fibrinogen.

Tumor necrosis factor alpha and fMLP can activate a broad range of cellular functions in neutrophils adherent to biological surfaces. These functions are mediated by integrins and involve the activation of tyrosine kinases. Here, we report that Pyk2, a member of the focal adhesion kinase family, was present in human neutrophils and was rapidly phosphorylated and activated following tumor necrosis factor alpha and fMLP stimulation in an adhesion-dependent manner. Tyrosine phosphorylation of Pyk2 was attenuated by beta2 integrin blocking with specific antibodies. The tyrosine phosphorylation of Pyk2 was downstream of protein kinases Lyn, Syk and protein kinase C and cytoskeletal organization. The activation of Pyk2 may play a role in adhesion/cytoskeleton-associated neutrophils function.     

Modulation of the activation of extracellular signal-regulated kinase (ERK) and the production of inflammatory mediators by ADP-ribosylation inhibitors

ADP-ribosylation is involved in nuclear factor kappaB (NF-kappaB)-dependent gene expression induced by lipopolysaccharide in murine macrophages. Here we have investigated the mechanism by which ADP-ribosylation inhibitors block signaling pathways induced in macrophages. In RAW264.7 macrophages the inducers of NF-kappaB activate the production of reactive oxygen species and three mitogen-activated protein kinases (MAPK), the extracellular signal regulated kinase (ERK), the c-jun N-terminal kinase/stress-activated protein kinase (JNK), and p38. We demonstrate that ADP-ribosylation inhibitors specifically inhibit ERK MAPK activation and reduce the release of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha), IL-6 and nitrite.     

Protein kinase C and ERK activation are required for TFF-peptide-stimulated bronchial epithelial cell migration and tumor necrosis factor-alpha-induced interleukin-6 (IL-6) and IL-8 secretion

TFF-peptides (formerly P-domain peptides, trefoil factors) are typical secretory products of many mucous epithelia and are aberrantly secreted during chronic inflammatory diseases. They are known to enhance the migration of intestinal, corneal, and bronchial epithelial cells. Using the human bronchial epithelial cell line BEAS-2B as a model, it is shown here for the first time that TFF-peptides are capable of modulating the inflammatory response in vitro by regulating tumor necrosis factor-alpha-induced secretion of interleukin (IL)-6 and IL-8. In contrast, TFF2 itself does not change IL-6 and IL-8 secretion but triggers sustained activation of the extracellular signal-regulated kinases (ERK1/2) as well as phosphorylation of c-Jun N-terminal kinase (JNK). A complex differential regulation of tumor necrosis factor-alpha-induced IL-6 and IL-8 secretion by TFF2 is observed that involves signaling via protein kinase C and ERK1/2. Furthermore, the motogenic effect of TFF2 on BEAS-2B cells is analyzed using a modified Boyden chamber assay. This migratory effect is shown to be dependent not only on protein kinase C and ERK1/2 but also on the activation of the Src family of tyrosine kinases. Taken together, the data presented indicate an important physiological role of TFF-peptides during inflammatory conditions of mucous epithelia.     

Acetylcholine muscarinic receptor regulation of the Ras/Raf/MAP kinase pathway

Acetylcholine muscarinic m1 receptors and m2 receptors are predominantly coupled to the heterotrimeric G proteins Gq, 11 and Gi, respectively. Stimulation of the m1 and m2 receptors in different cell types activate the Ras/Raf/MAP kinase pathway. The ability of the m1 receptor to activate the MAP kinase pathway is dependent on the isoforms of adenylyl cyclase expressed in specific cell types. Specific adenylyl cyclases respond to different signals, including calcium and protein kinase C, with increased cAMP synthesis resulting in protein kinase A activation. Stimulation of protein kinase A inhibits Raf and subsequent MAP kinase activation by G protein-coupled receptors and growth factor receptor tyrosine kinases. G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated response pathways.     

TRAF6 and Src kinase activity regulates Cot activation by IL-1

Cot is one of the MAP kinase kinase kinases that regulates the ERK1/ERK2 pathway under physiological conditions. Cot is activated by LPS, by inducing its dissociation from the inactive p105 NFkappaB-Cot complex in macrophages. Here, we show that IL-1 promotes a 10-fold increase in endogenous Cot activity and that Cot is the only MAP kinase kinase kinase that activates ERK1/ERK2 in response to this cytokine. Moreover, in cells where the expression of Cot is blocked, IL-1 fails to induce an increase in IL-8 and MIP-1betamRNA levels. The activation of Cot-MKK1-ERK1/ERK2 signalling pathway by IL-1 is dependent on the activity of the transducer protein TRAF6. Most important, IL-1-induced ERK1/ERK2 activation is inhibited by PP1, a known inhibitor of Src tyrosine kinases, but this tyrosine kinase activity is not required for IL-1 to activate other MAP kinases such as p38 and JNK. This Src kinases inhibitor does not block the dissociation and subsequently degradation of Cot in response to IL-1, indicating that other events besides Cot dissociation are required to activate Cot. All these data highlight the specific requirements for activation of the Cot-MKK1-ERK1/ERK2 pathway and provide evidence that Cot controls the functions of IL-1 that are mediated by ERK1/ERK2.     

Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade

The epidermal growth factor receptor (EGFR) and the non-receptor protein tyrosine kinases Src and Pyk2 have been implicated in linking a variety of G-protein-coupled receptors (GPCR) to the mitogen-activated protein (MAP) kinase signaling cascade. In this report we apply a genetic strategy using cells isolated from Src-, Pyk2-, or EGFR-deficient mice to explore the roles played by these protein tyrosine kinases in GPCR-induced activation of EGFR, Pyk2, and MAP kinase. We show that Src kinases are critical for activation of Pyk2 in response to GPCR-stimulation and that Pyk2 and Src are essential for GPCR-induced tyrosine phosphorylation of EGFR. By contrast, Pyk2, Src, and EGFR are dispensable for GPCR-induced activation of MAP kinase. Moreover, GPCR-induced MAP kinase activation is normal in fibroblasts deficient in both Src and Pyk2 (Src-/-Pyk2-/- cells) as well as in fibroblasts deficient in all three Src kinases expressed in these cells (Src-/-Yes-/-Fyn-/- cells). Finally, experiments are presented demonstrating that, upon stimulation of GPCR, activated Pyk2 forms a complex with Src, which in turn phosphorylates EGFR directly. These experiments reveal a role for Src kinases in Pyk2 activation and a role for Pyk2 and Src in tyrosine phosphorylation of EGFR following GPCR stimulation. In addition, EGFR, Src family kinases, and Pyk2 are not required for linking GPCRs with the MAP kinase signaling cascade.     

CD40 signaling in human dendritic cells is initiated within membrane rafts

Despite CD40's role in stimulating dendritic cells (DCs) for efficient specific T-cell stimulation, its signal transduction components in DCs are still poorly documented. We show that CD40 receptors on human monocyte-derived DCs associate with sphingolipid- and cholesterol-rich plasma membrane microdomains, termed membrane rafts. Following engagement, CD40 utilizes membrane raft-associated Lyn Src family kinase, and possibly other raft-associated Src family kinases, to initiate tyrosine phosphorylation of intracellular substrates. CD40 engagement also leads to a membrane raft-restricted recruitment of tumor necrosis factor (TNF) receptor-associated factor (TRAF) 3 and, to a lesser extent, TRAF2, to CD40's cytoplasmic tail. Thus, the membrane raft structure plays an integral role in proximal events of CD40 signaling in DCs. We demonstrate that stimulation of Src family kinase within membrane rafts initiates a pathway implicating ERK activation, which leads to interleukin (IL)-1alpha/beta and IL-1Ra mRNA production and contributes to p38-dependent IL-12 mRNA production. These results provide the first evidence that membrane rafts play a critical role in initiation of CD40 signaling in DCs, and delineate the outcome of CD40-mediated pathways on cytokine production.     

Activation of phospholipase C-gamma 1 through transfected platelet-derived growth factor receptor enhances interleukin 2 production upon antigen stimulation in a T-cell line.

Phospholipase C-gamma 1 (PLC gamma 1) plays an important role in the signal transduction pathway by producing second messengers. However, the activation mechanism of PLC gamma 1 and the role of the phosphatidylinositol pathway for interleukin 2 (IL-2) production in T lymphocytes remain to be determined. To analyze the functional role of this pathway in T cells, we expressed an epidermal growth factor receptor (EGF) or platelet-derived growth factor (PDGF) receptor (EGF-R or PDGF-R), both of which are known to directly activate PLC gamma 1 in fibroblasts, into a murine T-cell hybridoma. Both receptors were expressed on the cell surface and caused tyrosine phosphorylation of multiple substrates, including the receptor itself, upon ligand binding. While EGF stimulation did not either cause phosphorylation of PLC gamma 1 or induce Ca2+ mobilization in the EGF-R transfectant in this system, PDGF treatment induced tyrosine phosphorylation of PLC gamma 1 and Ca2+ mobilization in the PDGF-R transfectant. Stimulation through PDGF-R enhanced IL-2 production upon antigen stimulation of the transfectants, although PDGF treatment alone did not induce IL-2 production. These results suggest that activation of the phosphatidylinositol pathway affects the downstream pathway to IL-2 production but is not sufficient to produce IL-2 and that cooperation with signals from tyrosine kinase cascades is required for IL-2 production.