Protein tyrosine kinase Syk is involved in Thy-1 signaling in rat basophilic leukemia cells

Thy-1, a glycosyl-phosphatidylinositol-anchored surface glycoprotein, has been shown to possess transmembrane signaling capacity. In rat mast cells and rat basophilic leukemia cells (RBL) aggregation of surface Thy-1 with antibodies triggers a series of intracellular events, resembling those induced by aggregation of the high-affinity receptor for IgE (Fc epsilonRI), including tyrosine phosphorylation of multiple proteins and release of secretory components. Unlike the Fc epsilonRI-mediated activation, where both the membrane-associated protein tyrosine kinase (PTK) Lyn and the cytoplasmic PTK Syk are responsible for initiating the signaling cascade, only Lyn has been implicated in Thy-1-mediated activation in RBL cells. Here we report that Syk is also rapidly tyrosine phosphorylated upon Thy-1 cross-linking. Increased Syk tyrosine phosphorylation is observed only in cells in which extensive aggregation of Thy-1 is induced by two layers of cross-linking reagents. RBL-derived mutant cells deficient in the expression of surface Thy-1 and transfectants re-expressing surface Thy-1 were used to exclude the possibility that Syk activation reflects an interaction of the cross-linking reagents with surface molecules other than Thy-1. As Fc epsilonRI gamma subunits are well known to promote activation of Syk and its recruitment to membrane complexes, we also investigated the role of these subunits in Thy-1-mediated Syk activation, using RBL-derived mutant cells deficient in the expression of Fc epsilonRI gamma subunits and their revertants. Consistent with the lack of Fc epsilonRI expression, no IgE-induced response could be elicited, while Thy-1-inducible Syk phosphorylation was preserved. Our results suggest that Syk might be one of the kinases responsible for signal propagation upon Thy-1 cross-linking in a Fc epsilonRI-independent pathway.     

The identification and characterization of umbilical cord blood-derived human basophils

Cross-linking allergen-specific immunoglobin E on human peripheral blood basophils results in the release of histamine and other inflammatory mediators that initiate allergy and asthma. The signaling pathways leading from IgE binding to mediator release have not been well established, mainly due to the difficulty in obtaining adequate numbers of highly purified basophils. It was the goal of this study to easily obtain Fc epsilonRI-positive human basophils in high yield and purity for studies of signal transduction pathways. We describe an in vitro culture system in which pulsing normal human cord blood leukocytes with interleukin-3 (IL-3) for 3-4 h followed by incubation in medium with fetal bovine serum generates a cell population that is predominately Fc epsilonRI positive between 14 and 28 days of culture. These cells resemble peripheral blood basophils when examined by light and electron microscopy. Like normal blood basophils, they express the integrins, CD11b, CD18, CD29, and CD49d. A majority of the IL-3-pulsed cells also express a marker recognized by the basophil-specific antibody, 2D7. Fc epsilonRI cross-linking results in a time and dose-dependent release of histamine. Fc epsilonRI cross-linking also stimulates protein-tyrosine phosphorylation, thought to be the first event leading to the IgE-mediated activation of peripheral blood basophils. These studies establish cord blood as an accessible source from which basophil-like cells can be developed to examine Fc epsilonRI-mediated 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.     

Early and late events in Fc epsilon RI signal transduction in human cultured mast cells

Protein tyrosine phosphorylation and other biochemical events have been shown to occur after cross-linking of Fc epsilonRI in rodent mast cells. To investigate the mechanism of Fc epsilonRI signal transduction in human mast cells, we used human cultured mast cells (HCMC) generated from cord blood cells in the presence of recombinant human stem cell factor and IL-6. We found that on cross-linking of Fc epsilonRI: 1) HCMC released histamine; 2) rapid tyrosine phosphorylation of multiple cellular substrates, including Syk, HS1, c-Cbl, ERK-1, and ERK-2, was observed; 3) intracellular Ca2+ and inositol phosphate production were increased within the first minute after Fc epsilonRI cross-linking; and 4) genistein, a tyrosine kinase inhibitor, inhibited both protein tyrosine phosphorylation and histamine release in a dose-dependent manner. These results were consistent with previous studies in rodent mast cells. In contrast, no tyrosine phosphorylation of phospholipase C gamma1 and Btk (Bruton's tyrosine kinase) were observed in our experimental conditions. These results suggest that the greater part of the early and late signaling events in HCMC is similar to those obtained with rodent mast cells and indicated that the requirement of tyrosine phosphorylation in the activation process of each of the signaling molecules might be different in HCMC and rodent mast cells. Our finding indicates that HCMC may be useful for analysis of Fc epsilonRI-mediated signal transduction in human mast 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.     

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.     

Differential intrinsic enzymatic activity of Syk and Zap-70 protein-tyrosine kinases

Syk and Zap-70 are related protein-tyrosine kinases implicated in antigen and Fc receptor signaling. While Zap-70 is restricted to T-cells and natural killer cells, Syk accumulates in B-cells, mast cells, platelets, and immature T-cells. In addition, we found that an isoform of Syk (SykB), which carries a 23-amino acid deletion in the "linker" region, is prominently expressed in bone marrow. To better understand the relative impact of Syk, SykB, and Zap-70 on signal transduction, we compared their intrinsic enzymatic properties in transiently transfected COS-1 cells and in hemopoietic cells. Using modified versions of these enzymes bearing a common Myc epitope at the amino terminus, we determined that the ability of Syk and SykB to undergo autophosphorylation and to phosphorylate erythrocyte band 3 in immune complex kinase reactions was at least 100-fold greater than that of Zap-70. Similarly, Syk and SykB, but not Zap-70, caused prominent tyrosine phosphorylation of p120(c-)cbl in COS-1 cells. A similar pattern of activity was also noted for endogenous Syk and Zap-70 from hemopoietic cells. To understand the structural basis for these characteristics, we also created and analyzed a series of chimeras between Syk and Zap-70. These studies indicated that the catalytic domain of Syk and Zap-70, but not their SH2 domains, linker region or carboxyl-terminal tail, was responsible for their respective activity. Taken together, these data demonstrated that the intrinsic enzymatic activity of Syk and SykB is superior to that of Zap-70 and that such a distinction relates to structural variations in the catalytic domain.     

Measurement of 6-MV X-ray surface dose when topical agents are applied prior to external beam irradiation

The Syk protein-tyrosine kinase is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. After cross-linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues. We have previously reported that Syk expressed in COS cells is predominantly phosphorylated at both Tyr518 and Tyr519 at its putative autophosphorylation site. In this study, we have examined the role of each of these two residues for the catalytic activity of Syk in vitro and for the Syk-induced phosphorylation of cellular proteins in intact cells. Mutation of either residue had minor effects on the catalytic activity of Syk, and even the double mutant [F518, F519]Syk was about 60% as active as the wild-type enzyme. In intact cells, however, all three mutants consistently failed to induce the extensive tyrosine phosphorylation of cellular proteins typically observed with wild-type Syk. We have recently shown that the doubly phosphorylated Y518/Y519 site is also the site for association of Syk with the SH2 domain of the Lck kinase, which suggests that although phosphates at Y518/Y519 may enhance the catalytic activity of Syk, its interaction with Src family protein-tyrosine kinases is at least equally important for the induction of downstream substrate phosphorylation.     

Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa

The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, the tyrosine residues within this motif become phosphorylated. We prepared a recombinant fusion protein of the cytoplasmic tail of Fc(gamma)RIIa (containing the ITAM) with glutathione-S-Transferase (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk (immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP. This association was also observed in immunoprecipitates of Fc(gamma)RIIa from resting neutrophils, suggesting that Lyn might be the kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.     

Regulation of T-cell antigen receptor signalling by Syk tyrosine protein kinase

T-cell antigen receptor (TCR) signalling has been shown to involve two classes of tyrosine protein kinases: the Src-related kinases p56(lck) and p59(fyr), and the Zap-70/Syk family kinases. Lck and FynT are postulated to initiate TCR-triggered signal transduction by phosphorylating the CD3 and zeta subunits of the TCR complex. This modification permits the recruitment of Zap-70 and Syk, which are presumed to amplify the TCR-triggered signal, by phosphorylating additional intracellular proteins. While Zap-70 is expressed in all T cells, Syk is present in thymocytes and mature T-cell populations such as intraepithelial gammadelta T cells and naive alphabeta T cells. To better understand the role of Syk in these cells, its impact on the physiology of an antigen-specific T-cell line was tested. Our results showed that compared to Zap-70 alone, Syk was a strong positive regulator of antigen receptor-induced signals in BI-141 cells. Surprisingly, they indicated that, like Src family kinases, Syk augmented TCR-triggered tyrosine phosphorylation of CD3/zeta. Syk, but not Zap-70 alone, could also stimulate tyrosine phosphorylation of a zeta-bearing chimera in transiently transfected Cos-1 cells. Finally, evidence was provided that Syk has the capacity to directly phosphorylate a zeta-derived peptide in vitro. These findings suggested that Syk may have a unique role in T cells, as a consequence of its ability to efficiently phosphorylate multiple components of the TCR signalling cascade. Furthermore, they raised the possibility that Syk can regulate the initiation of TCR signalling, by promoting phosphorylation of the immunoreceptor tyrosine-based activation motifs of the TCR complex.     

Translocation of glucokinase in pancreatic beta-cells during acute and chronic hyperglycemia

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.     

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.     

Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells

Activation of the high affinity IgE receptor (Fc epsilon RI) of mast cells, a member of the antigen receptor family, leads to the release of allergic mediators, a critical event in the onset of immediate hypersensitivity. Stimulation of Fc epsilon RI results in the rapid association and activation of the Syk tyrosine kinase. Using Syk-deficient mast cells we show that they fail to degranulate, synthesize leukotrienes and secrete cytokines when stimulated through Fc epsilon RI, conclusively demonstrating an essential role for Syk in Fc epsilon RI signalling. Furthermore, our data strongly supports a model of Fc epsilon RI engagement leading to the sequential activation of the tyrosine kinases Lyn and then Syk. A similar mechanism is likely to apply to signal transduction through all members of the antigen receptor family.     

Syk, but not Lyn, recruitment to B cell antigen receptor and activation following stimulation of CD45- B cells

B cell Ag receptor (BCR) signaling occurs via tyrosine phosphorylation of CD79a and CD79b ITAMs, leading to recruitment and activation of Lyn and Syk tyrosine kinases and subsequent downstream events. CD45 expression is required for BCR triggering of certain of these downstream events, such as calcium mobilization and p21ras activation. However, the site in the BCR signaling cascade at which CD45 impinges is poorly defined. To address this question, we have studied CD45 function in the CD45-deficient (CD45-) and CD45-reconstituted (CD45+) J558L mu m3 plasmacytoma. In both CD45+ and CD45- cells, Ag stimulation led to CD79a and CD79b tyrosine phosphorylation as well as Syk tyrosine phosphorylation, recruitment to the receptors, and activation. In contrast to CD45+ cells, Lyn exhibited high basal tyrosine phosphorylation in the CD45- cells and was not further phosphorylated upon Ag stimulation. Mapping studies indicated that the observed constitutive phosphorylation of Lyn reflects phosphorylation of its C-terminal tyrosine, Y508, at high stoichiometry. Constitutively Y508-phosphorylated Lyn was neither recruited to the BCR nor activated upon Ag stimulation. Moreover, CD79a-ITAM phosphopeptides failed to bind Lyn from the CD45- cells. Thus, Y508 phosphorylation of Lyn occurs in the absence of cellular CD45 expression and appears to render the kinase unable to associate with the phosphorylated receptor complex via its Src homology 2 domain and to participate in signal propagation. Surprisingly, in view of previous findings implicating Src family kinases in ITAM phosphorylation, the data indicate that Ag-induced CD79a and CD79b tyrosine phosphorylation and Syk recruitment and activation can occur in the absence of CD45 expression and, hence, Src-family kinase activation.     

Activation of protein-tyrosine kinase Pyk2 is downstream of Syk in FcepsilonRI signaling

Aggregation of the FcepsilonRI, a member of the immune receptor family, induces the activation of proteintyrosine kinases and results in tyrosine phosphorylation of proteins that are involved in downstream signaling pathways. Here we report that Pyk2, another member of the focal adhesion kinase family, was present in the RBL-2H3 mast cell line and was rapidly tyrosine-phosphorylated and activated after FcepsilonRI aggregation. Tyrosine phosphorylation of Pyk2 was also induced by the calcium ionophore A23187, by phorbol myristate acetate, or by stimulation of G-protein-coupled receptors. Adherence of cells to fibronectin dramatically enhanced the induced tyrosine phosphorylation of Pyk2. Although Src family kinases are activated by FcepsilonRI stimulation and tyrosine-phosphorylate the receptor subunits, the activation and tyrosine phosphorylation of Pyk2 were downstream of Syk. In contrast, tyrosine phosphorylation of Pyk2 by stimulation of G-protein-coupled receptors was independent of Syk. Therefore, the FcepsilonRI-induced tyrosine phosphorylation of Pyk2 is downstream of Syk and may play a role in cell secretion.     

T cell activation induced by novel gain-of-function mutants of Syk and ZAP-70

The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.     

Differential regulation of oxidative and osmotic stress induced Syk activation by both autophosphorylation and SH2 domains

Syk, a nonreceptor protein-tyrosine kinase, is activated by both oxidative and osmotic stress and plays different roles in the transduction of stress signals. In this study, the regulation of oxidative and osmotic stress induced Syk activation was investigated utilizing Syk-negative DT40 cells, expressing various Syk mutants. Phosphorylation of Y518Y519 was demonstrated to be required for both oxidative and osmotic stress induced Syk activation. Syk activation by these two types of stress stimuli was a combination of both autophosphorylation and the activities of additional tyrosine kinases. Oxidative stress induced Syk tyrosine phosphorylation was almost completely attributed to autophosphorylation, whereas other tyrosine kinases were largely responsible for osmotic stress induced Syk tyrosine phosphorylation. Moreover, the Src homology 2 (SH2) domains of Syk differentially regulated Syk activation. Both mSH2(N) Syk and mSH2(C) Syk, in which the phosphotyrosine-dependent binding motif within the SH2 domains contained point mutations, showed a significantly higher activity than that observed in wild-type Syk, following osmotic stress treatment. In comparison, in response to oxidative stress, only mSH2(N) Syk demonstrated a stronger activation than wild-type Syk. Therefore, differential activation and regulation of Syk may give an insight into the distinctive functions of Syk in oxidative and osmotic stress signaling.     

Syk and Fyn are required by mouse megakaryocytes for the rise in intracellular calcium induced by a collagen-related peptide

Stimulation of platelets by collagen leads to activation of a tyrosine kinase cascade resulting in secretion and aggregation. We have recently shown that this pathway involves rapid tyrosine phosphorylation of an Fc receptor gamma chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM), enabling interaction with the tandem SH2 domains of the tyrosine kinase Syk. Activation of Syk lies upstream of tyrosine phosphorylation of phospholipase Cgamma2. In the present study we sought to test directly the role of the ITAM/Syk interaction and the role of the Src-related kinases in collagen receptor signaling using mouse megakaryocytes. We demonstrate that the calcium-mobilizing action of a collagen-related peptide (CRP) is kinase-dependent, inhibited by the microinjection of the tandem SH2 domains of Syk and abolished in Syk-deficient mice. Furthermore, the CRP response is abolished by the Src family kinase inhibitor PP1 and inhibited in Fyn-deficient mice. In contrast, the calcium response to the G-protein-linked receptor agonist thrombin is not significantly altered under these conditions. These results provide direct evidence of the functional importance of Fyn and Syk in collagen receptor signaling and support the megakaryocyte as a model for the study of proteins involved in this pathway.