CBL-GRB2 interaction in myeloid immunoreceptor tyrosine activation motif signaling

In this study, we provide the first evidence for role of the CBL adapter protein interaction in Fc gammaRI receptor signal transduction. We study the Fc gammaRI receptor, an immunoreceptor tyrosine activation motif (ITAM)-linked signaling pathway, using IFN-gamma-differentiated U937 myeloid cells, termed U937IF cells. CBL is constitutively associated with both GRB2 and the ITAM-containing receptor subunit, Fc gammaRIgamma of Fc gammaRI, providing direct evidence that CBL functions in myeloid ITAM signaling. Fc gammaRI cross-linking of U937IF cells induces the tyrosine phosphorylation of CBL that is associated with an altered CBL-GRB2 interaction. Both GRB2-SH3 and SH2 domains bind CBL in resting cell lysates; upon Fc gammaRI stimulation, phosphorylated CBL binds exclusively to the GRB2-SH2 domain. Glutathione-S-transferase fusion protein data demonstrate that the constitutive interaction of CBL with GRB2 and CRKL is mediated via two discrete regions of the CBL C terminus. The proximal C terminus (residues 461-670) binds to GRB2 constitutively, and under conditions of receptor activation binds to the tyrosine-phosphorylated SHC adapter molecule. The distal C terminus of CBL (residues 671-906) binds the CRKL adapter protein. The data demonstrate that the CBL-GRB2 and GRB2-SOS protein complexes are distinct and mutually exclusive in U937IF cells, supporting a model by which the CBL-GRB2 and GRB2-SOS complexes function in separate pathways for myeloid Fc gammaRI signaling.     

Differential interaction of Crkl with Cbl or C3G, Hef-1, and gamma subunit immunoreceptor tyrosine-based activation motif in signaling of myeloid high affinity Fc receptor for IgG (Fc gamma RI)

Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following Fc gamma RI aggregation in U937IF cells. Fc gamma RI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing gamma subunit of Fc gamma RI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and Fc gamma RI gamma after Fc gamma RI activation and the constitutive association between C3G and Crkl provide the first evidence that a Fc gamma RI gamma-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.     

Intracluster restriction of Fc receptor gamma-chain tyrosine phosphorylation subverted by a protein-tyrosine phosphatase inhibitor

This study shows that aggregation of U937 cell high affinity IgG Fc receptor (Fc gamma RI) results in the transient tyrosine phosphorylation of Fc gamma RI gamma-chain but not the phosphorylation of gamma-chains associated with nonaggregated IgA Fc receptors (Fc alpha R) on the same cells. Thus, normally, tyrosine phosphorylation of gamma-chains is limited to FcR in aggregates. In contrast, aggregation of Fc gamma RI in the presence of vanadate induced the sustained tyrosine phosphorylation of Fc gamma RI gamma-chains and the rapid and extensive phosphorylation of nonaggregated Fc alpha R gamma-chains and low affinity IgG Fc receptors (Fc gamma RII). This global phosphorylation of motifs on nonaggregated FcR was also detected upon aggregation of Fc alpha R or Fc gamma RII, which induced the phosphorylation of nonaggregated Fc gamma RI gamma-chains. Vanadate prevented dephosphorylation of proteins and increased kinase activity in stimulated cells. Evidence failed to support alternative explanations such as acquisition of phospho-gamma through subunit exchange or a coalescence of nonaggregated with aggregated FcR. It is likely, therefore, that activated kinases interacted with nonaggregated FcR in stimulated cells. Pervanadate induced the tyrosine phosphorylation of gamma-chains in the absence of FcR cross-linking, indicating that the kinases could be activated by phosphatase inhibition and could react with nonaggregated substrates. We conclude that under normal conditions there is a vanadate-sensitive mechanism that prevents tyrosine phosphorylation of nonaggregated FcR gamma-chain motifs in activated cells, restricting their phosphorylation to aggregates.     

The CBL-related protein CBLB participates in FLT3 and interleukin-7 receptor signal transduction in pro-B cells

The FLT3 receptor tyrosine kinase and its ligand, FL, play an important role in early hematopoietic development. We have found that CBLB, a recently characterized molecule closely related to the CBL protooncogene product, is phosphorylated on tyrosine(s) following FL treatment of JEA2 human pro-B cells and THP1 monocytic cells. Treatment of JEA2 cells with interleukin (IL)-7 induces CBLB phosphorylation as well. FL and IL-7, respectively, induce and increase association of tyrosine-phosphorylated SHC and the p85 subunit of phosphatidylinositol 3'-kinase with CBLB. In these cells, CBLB constitutively binds the GRB2 adaptor predominantly through its N-terminal SH3 domain, to form a complex that is distinct from the GRB2.CBL and GRB2.SOS1 complexes. Together with the fact that CBLB is consistently found in blast cells from acute leukemias and in peripheral blood mononuclear cells, this suggests that CBLB has a role in tyrosine kinase-regulated signaling pathways in many hematolymphoid cells.     

Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction

Engagement of immunoreceptors in hemopoietic cells leads to activation of Src family tyrosine kinases as well as Syk or ZAP-70. Current models propose that Src family kinases are critical in immune response signal transduction through their role in phosphorylation of tyrosine residues within immunoreceptor tyrosine activation motifs (ITAMs; which recruit the SH2 domains of Syk or ZAP-70) and by direct phosphorylation of Syk and ZAP-70. Several lines of evidence suggest that Syk may not show the same dependence on activation by Src family kinases as ZAP-70. In this report, we used COS cells transiently transfected with components of the Fc epsilon RI complex (Lyn, Syk, and a chimeric CD8 receptor containing the cytoplasmic domain of the gamma subunit of Fc epsilon RI (CD8-gamma)) to examine the regulation of Syk activity. Syk was activated and phosphorylated in COS cells cotransfected with Lyn; however, in cells expressing CD8-gamma, activation of Syk and phosphorylation of CD8-gamma did not require coexpression of Lyn. Additional experiments indicate that gamma phosphorylation is dependent on Syk kinase activity and is independent of endogenous COS cell kinases. In parallel experiments, ZAP-70 was not activated by cotransfection with CD8-gamma, nor was CD8-gamma phosphorylated when coexpressed with ZAP-70 alone. Taken together, these studies indicate that Syk can be distinguished from ZAP-70 in its ability to be activated by coexpression with an ITAM-containing receptor without coexpression of a Src family kinase, and that Syk is capable of phosphorylating ITAM tyrosines under certain experimental conditions.     

A molecular switch changes the signalling pathway used by the Fc gamma RI antibody receptor to mobilise calcium

BACKGROUND: Leukocytes express Fc gamma receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody-directed cellular cytotoxicity. The nature of the myeloid response to Fc gamma receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability. RESULTS: We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fc gamma receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor Fc gamma RI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the gamma chain, an Fc gamma RI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of Fc gamma RI resulted in the Fc gamma RIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated. CONCLUSIONS: The switch in Fc gamma RI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by Fc gamma RI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.     

Cell growth inhibition by a novel vitamin K is associated with induction of protein tyrosine phosphorylation

We have shown that a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), potently inhibits cell growth and suggested that the analog exerts its effects mainly via sulfhydryl arylation rather than redox cycling. Since protein-tyrosine phosphatases (PTPases), which have pivotal roles in many cellular functions, have a critical cysteine in their active site, we have proposed PTPases as likely targets for Cpd 5. To test this hypothesis, we examined the effects of Cpd 5 on protein tyrosine phosphorylation of cellular proteins and on the activity of PTPases. We found that Cpd 5 rapidly induced protein tyrosine phosphorylation in a human hepatocellular carcinoma cell line (Hep3B) at growth inhibitory doses, and the effect was blocked by thiols but not by non-thiol antioxidants or tyrosine kinase inhibitors. Cpd 5 inhibited PTPase activity, which was also significantly antagonized by reduced glutathione. Furthermore, the well studied PTPase inhibitor orthovanadate also induced protein tyrosine phosphorylation and growth inhibition in Hep3B cells. These results suggest that inhibition of cellular PTPases by sulfhydryl arylation and subsequent perturbation of protein tyrosine phosphorylation may be involved in the mechanisms of Cpd 5-induced cell growth inhibition.     

Protein tyrosine kinases in activation signal of human basophils through the immunoglobulin E receptor type l

Human basophils activated through high-affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) are involved in the late phase of the allergic reaction. To investigate the possible involvement of protein-tyrosine kinases in this activation we used human acute basophilic leukemia (ABL) cells in culture as well as a pure population of normal basophils in vitro-derived from human bone marrow precursor cells (HBMB). ABL cells were 50-80% basophils at various stages of maturation as assessed by staining, morphology, ultrastructure, and flow cytometry analysis, and only basophils in ABL cells expressed Fc epsilon RI. Aggregation of Fc epsilon RI by IgE and anti-IgE, IgE and antigen, or anti-Fc epsilon RI monoclonal antibodies on ABL cells or on HBMB, led to increased tyrosine phosphorylation of 120-, 100-, 80-, 72-, 50- to 65-, and 38-kDa substrates. Tyrosine phosphorylations in ABL cells were in basophils because 1) they were detected after a 5-s stimulation, 2) they were observed under conditions where mediator release is minimal, i.e., in the absence of extracellular calcium, 3) hapten addition during antigen stimulation resulted in almost total disappearance of tyrosine phosphorylations within 30 s. There was correlation between histamine release and tyrosine phosphorylation in anti-IgE dose-responses and in dose-responses of the tyrosine kinase inhibitor genistein. The tyrosine kinase p72syk was detected in the cells. Stimulation of ABL cells for 1 min resulted in extracellular calcium-independent tyrosine phosphorylation and activation of p72syk. Therefore, tyrosine kinases are involved in the early steps of human Fc epsilon RI signaling in basophils. Tyrosine kinases and their substrates could represent new potential therapeutic targets to prevent the development of the allergic reaction.     

Metabolic activation of halothane to neoantigens in C57Bl/10 mice: immunochemical studies

Previous studies have shown that protein-serine/threonine kinases and protein-tyrosine kinase(s) are activated by cross-linking of the high-affinity receptor for IgE, Fc epsilon RI, on mast cells and basophils. In vitro kinase assays (ISDR kinase assays) on cellular proteins immobilized on polyvinylidene difluoride membrane after denaturation and renaturation were employed to estimate the complexity of protein kinases expressed in mouse mast cells. The results demonstrated that a large number (more than 60) of both serine/threonine- and tyrosine-specific kinases are present in a mouse mast cell line, PT-18. Cross-linking of Fc epsilon RI-induced activation of a subset of both serine/threonine kinases and tyrosine kinases in PT-18 as well as bone marrow-derived mouse mast cells, as revealed by the ISDR kinase assay. Among them, MAP kinase (or ERK2) was shown to be tyrosine phosphorylated and activated transiently upon Fc epsilon RI cross-linking, suggesting its potential role in mast cell signal transduction.     

Endogenous hematopoietic reconstitution induced by human umbilical cord blood cells in immunocompromised mice: implications for adoptive therapy

Cross-linking the heterotrimeric (alpha beta gamma 2) IgE receptor, Fc epsilon RI, of mast cells activates two tyrosine kinases: Lyn, which phosphorylates beta and gamma subunit immunoreceptor tyrosine-based activation motifs, and Syk, which binds gamma-phospho-immunoreceptor tyrosine-based activation motifs and initiates cellular responses. We studied three Fc epsilon RI-dimerizing mAbs that maintain similar dispersed distributions over the surface of RBL-2H3 mast cells but elicit very different signaling responses. Specifically, mAb H10 receptor dimers induce very little inositol 1,4,5-trisphosphate synthesis, Ca2+ mobilization, secretion, spreading, ruffling, and actin plaque assembly, whereas dimers generated with the other anti-Fc epsilon RI mAbs induce responses that are only modestly lower than that to multivalent Ag. H10 receptor dimers activate Lyn and support Fc epsilon RI beta and gamma subunit phosphorylation but are poor Syk activators compared with Ag and the other anti-Fc epsilon RI mAbs. H10 receptor dimers have two other distinguishing features. First, they induce stable complexes between activated Lyn and receptor subunits. Second, the predominant Lyn-binding phospho-beta isoform found in mAb H10-treated cells is a less tyrosine phosphorylated, more electrophoretically mobile species than the predominant isoform in Ag-treated cells that does not coprecipitate with Lyn. These studies implicate Lyn dissociation from highly phosphorylated receptor subunits as a new regulatory step in the Fc epsilon RI signaling cascade required for Syk activation and signal progression.     

Effects of an antiallergic drug, pemirolast potassium on tyrosine phosphorylation and map kinase activation in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells

Aggregation of high affinity IgE Fc receptors (Fc epsilon RI) on RBL-2H3 cells results in tyrosine phosphorylation of 33-, 42-, 44-, 72-, 80-, 90-, 125-kDa proteins. The 42 and 44 kDa proteins were identified as mitogen-activated protein (MAP) kinases with immunoblotting of anti-MAP kinase antibody. The effects of an antiallergic drug, pemirolast potassium (TBX) on Ag-induced protein tyrosine phosphorylation and MAP kinase activation were investigated. When RBL-2H3 cells were stimulated with Ag in the presence of TBX, tyrosine phosphorylation of three proteins (33, 42 and 44 kDa) was inhibited concentration-dependently (0.1-10 micrograms/ml). Inhibition of Ag-induced tyrosine phosphorylation of 33 kDa protein, which could be a beta subunit of Fc epsilon RI, suggests that TBX may prevent the activation of Fc epsilon RI. TBX suppressed activation of MAP kinases (42 and 44 kDa) in response to Ag as well as phorbol myristate acetate (100 nM) or calcium ionophore A23187 (500 nM), implying that the drug acts on signal transduction component(s) between the second messengers and MAP kinases. However, TBX had no effects on protein tyrosine phosphorylation and MAP kinase activation in MC3T3-E1 osteoblastic cells. These results indicate that TBX may affect Fc epsilon RI and also may act as a step distal of Ca2+ mobilization and protein kinase C activation leading to MAP kinase activation in RBL-2H3 cells.     

Mapping a telomere using the translocation eT1(III;V) in Caenorhabditis elegans

TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase.     

Stimulation of Fc(alpha) receptors induces tyrosine phosphorylation of phospholipase C-gamma(1), phosphatidylinositol phosphate hydrolysis, and Ca2+ mobilization in rat and human mesangial cells

Although knowledge of IgA Fc receptor (Fc(alpha)R) structure and gene organization has progressed in the past few years, signal transduction pathways elicited by its activation have hardly been studied. Previously, we have demonstrated that mesangial cells (MC) possess Fc(alpha)R stimulation triggers several biologic responses. In this work, we studied the early biochemical signals triggered by Fc(alpha)R stimulation in MC. MC incubation with aggregated IgA (AIgA) elicited a dose-dependent increase in cytosolic Ca2+ ([Ca2+]i). The response was rapid and transient, and slowly fell to the original baseline. Ca2+ mobilization was dependent on the Fc region of the IgA, because Fc, but neither Fab fragment nor carbohydrates, inhibited the [Ca2+] rise. The initial induction of [Ca2+]i rise was due to Ca2+ mobilization from inositol trisphosphate (IP3)-sensitive intracellular stores, while sustained levels were maintained through extracellular Ca2+ influx. Stimulation of Fc(alpha)R also resulted in production of IP3, temporally correlated with Ca2+ mobilization. Protein tyrosine kinase inhibitors abolished [Ca2+]i rise, indicating that tyrosine phosphorylation of some substrates is required for Ca2+ mobilization. Stimulation through Fc(alpha)R gave rise to a marked increase in tyrosine phosphorylation of several proteins, including the 147-kDa band, similar in size to phospholipase C-gamma(1) (PLC-gamma(1)). Tyrosine phosphorylation of PLC-gamma(1) reached a maximum 30 s after stimulation, as determined by immunoprecipitation and Western blot. Collectively, these results indicate that the Fc(alpha)R signaling pathway in MC involves PLC-(gamma(1) activation, IP3 formation, and Ca2+ mobilization, and is linked to activation of tyrosine kinases.     

Monoclonal antibody to the type II Fc receptor for human IgG blocks potentiation of monocyte and neutrophil IgG-induced respiratory burst activation by aggregated C-reactive protein

The acute phase protein, CRP, when heat-aggregated (Agg-CRP), binds to human monocytes and neutrophils and potentiates the respiratory burst stimulated by heat-aggregated IgG (Agg-IgG). Earlier data from our laboratory and others have indicated that CRP binds to phagocytic cells at membrane sites associated with IgG Fc receptors. The present study utilized monoclonal antibodies (MAb) to determine whether the Agg-CRP potentiation of oxidative metabolism could be linked to activation through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Preincubation of monocytes with MAb 32.2, which recognizes an Fc gamma RI epitope distinct from its IgG binding site, had only a minimal (20%) inhibitory effect on Agg-IgG-induced luminol chemiluminescence (CL) and exerted no significant effect on its enhancement by Agg-CRP. MAb 10.1, which blocks IgG binding to Fc gamma RI, reduced Agg-IgG-induced monocyte CL by 40%, but did not alter the Agg-CRP-mediated enhancement. In contrast, exposure to MAb IV.3, which binds to Fc gamma RII on monocytes and neutrophils and blocks IgG binding to this receptor, resulted in a greater than 70%, inhibition of Agg-IgG-induced CL and also significantly suppressed the enhancement by Agg-CRP. MAb Leu-11b, which reacts with Fc gamma RIII on neutrophils, reduced Agg-IgG-induced CL by 70% but did not suppress the Agg-CRP potentiation. Preincubation of monocytes and neutrophils with anti-Leu-M1, anti-CR1, or anti-CR3 failed to block Agg-IgG-induced CL or its enhancement by Agg-CRP. Although the potentiating effect of Agg-CRP on Agg-IgG-elicited CL was blocked by MAb IV.3, this antibody failed to reduce binding of Agg-CRP to either monocytes or neutrophils. These results indicate that, although Agg-CRP does not bind to phagocytic cells at the IgG-binding determinant of Fc gamma RII, it alters Agg-IgG-induced cell activation through this receptor.     

Surface makers for mast cell subtypes: low affinity IgG receptors and gp49 family

Members of the Immunoglobulin Superfamily (Ig) present in the surface of rodent mast cells include the high affinity IgE receptor (Fc epsilon RI), the low affinity receptors for the Fc portion of IgG, the Fc gamma RII family and Fc gamma RIII as well as the recently cloned gp49 family that includes three members gp49A, gp49B1 and gp49B2. Fc epsilon RI and Fc gamma RIII are members of the multi-chain immune recognition receptor (MIRR) family since they possess a multimeric structure in which the signal transducing chains (gamma chains) contain six acids that conform the Antigen Homology Receptor 1 Motif (ARH1M), also present in the T cell receptor (TCR) transducing chains. gp49B1, gp49B2 and the FC gamma R family are monomeric chains that also contain the partial of full AHR1M motif in their cytoplasmic domain indicating the capability for signal transduction through tyrosine phosphorylation and the possibility of cell activation with mediator (s) or cytokine (s) release. Distribution of the Fc gamma R receptors and gp49 family members varies in the different stages of mast cell differentiation and maturation.     

Activation and interaction with protein kinase C of a cytoplasmic tyrosine kinase, Itk/Tsk/Emt, on Fc epsilon RI cross-linking on mast cells

Cross-linking of the high-affinity IgE receptor (Fc epsilon RI) on mast cells induces rapid phosphorylation on serine, threonine, and tyrosine residues and increases the enzymatic activity, of a Tec subfamily tyrosine kinase, Itk/Tsk/Emt (Emt). The pleckstrin homology domain of Emt at its amino-terminal interacts directly with multiple isoforms of protein kinase C (PKC) in vitro. In addition, a portion of Emt is physically associated with multiple isoforms of PKC in intact mast cells. PKC phosphorylates a bacterial fusion protein containing the pleckstrin homology domain of Emt in vitro. Coexpression of Emt in COS-7 cells with Ca(2+)-dependent PKC isoforms (alpha, beta I, or beta II) induces an enhancement in tyrosine phosphorylation of Emt. In vivo inhibition of PKC expression or activity attenuates tyrosine phosphorylation and enzymatic activity of Emt induced upon Fc epsilon RI cross-linking. These data collectively suggest that PKC phosphorylates Emt and activates its autophosphorylating activity. Alternatively, PKC could activate another tyrosine kinase that phosphorylates Emt, or PKC-mediated phosphorylation of Emt may render it a target for another tyrosine kinase. In any case, PKC appears to play a major role in the activation of Emt induced upon Fc epsilon RI cross-linking.