Receptor-associated Protein Tyrosine Kinases Research Articles

Innate immune regulation by STAT-mediated transcriptional mechanisms.

The term innate immunity typically refers to a quick but non-specific host defense response against invading pathogens. The innate immune system comprises particular immune cell populations, epithelial barriers, and numerous secretory mediators including cytokines, chemokines, and defense peptides. Innate immune cells are also now recognized to play important contributing roles in cancer and pathological inflammatory conditions. Innate immunity relies on rapid signal transduction elicited upon pathogen recognition via pattern recognition receptors (PRRs) and cell:cell communication conducted by soluble mediators, including cytokines. A majority of cytokines involved in innate immune signaling use a molecular cascade encompassing receptor-associated Jak protein tyrosine kinases and STAT (signal transducer and activator of transcription) transcriptional regulators. Here, we focus on roles for STAT proteins in three major innate immune subsets: neutrophils, macrophages, and dendritic cells (DCs). While knowledge in this area is only now emerging, understanding the molecular regulation of these cell types is necessary for developing new approaches to treat human disorders such as inflammatory conditions, autoimmunity, and cancer.

Activation of mTORC1 Signaling Pathway in AIDS-Related Lymphomas

Using immunohistochemistry with antibodies against the phosphoserine residues in both S6rp and 4E binding protein 1, we identified the activation of the mammalian target of rapamycin (mTORC)1 pathway in 29 cases of AIDS-related lymphoma. These cases represented a diverse spectrum of histological types of non-Hodgkin lymphoma (24 cases) and classic Hodgkin lymphoma (five cases). mTORC1 was also activated in the hyperplastic but not involuted follicles of HIV-associated lymphadenopathy in eight cases, supporting the notion that mTORC1 activation is a common feature of transformed lymphocytes irrespective of either their reactive or malignant phenotype. We also found that in B-cell lines that represent diffuse large B-cell lymphoma, Burkitt lymphoma, Epstein-Barr virus-infected lymphocytes, and human herpesvirus 8-positive primary effusion lymphoma, inhibitors of Syk, MEK, and, seemingly, phosphoinositide 3 kinases suppressed mTORC1 activation, in particular when these inhibitors were used in combination. These findings indicate that AIDS-related lymphoma and other histologically similar types of lymphomas that are derived from transformed B lymphocytes may display clinical responses to inhibitors that directly target mTORC1 or, possibly, upstream activators of the mTORC1 pathway.

Redox Regulation of Interleukin-4 Signaling

The physiologic control of cytokine receptor activation is primarily mediated by reciprocal activation of receptor-associated protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Here, we show that immediately after ligand-dependent activation, interleukin (IL)-4 receptor generated reactive oxygen species (ROS) via phosphatidylinositol 3-kinase-dependent activation of NAD(P)H oxidase (NOX)1 and NOX5L. ROS, in turn, promoted IL-4 receptor activation by oxidatively inactivating PTP1B that physically associated with and deactivated IL-4 receptor. However, ROS were not required for the initiation of IL-4 receptor activation. ROS generated by other cytokine receptors, including those for erythropoietin, tumor necrosis factor-alpha, or IL-3, also promoted IL-4 signaling. These data indicate that inactivation of receptor-associated PTP activity by cytokine-generated ROS is a physiologic mechanism for the amplification of cytokine receptor activation in both cis and trans, revealing a role for ROS in cytokine crosstalk.

Identification and Functional Analysis of Phosphorylated Tyrosine Residues within EphA2 Receptor Tyrosine Kinase

EphA2 is a member of the Eph family of receptor tyrosine kinases. EphA2 mediates cell-cell communication and plays critical roles in a number of physiological and pathologic responses. We have previously shown that EphA2 is a key regulator of tumor angiogenesis and that tyrosine phosphorylation regulates EphA2 signaling. To understand the role of EphA2 phosphorylation, we have mapped phosphorylated tyrosines within the intracellular region of EphA2 by a combination of mass spectrometry analysis and phosphopeptide mapping using two-dimensional chromatography in conjunction with site-directed mutagenesis. The function of these phosphorylated tyrosine residues was assessed by mutational analysis using EphA2-null endothelial cells reconstituted with EphA2 tyrosine-to-phenylalanine or tyrosine-to-glutamic acid substitution mutants. Phosphorylated Tyr(587) and Tyr(593) bind to Vav2 and Vav3 guanine nucleotide exchange factors, whereas Tyr(P)(734) binds to the p85 regulatory subunit of phosphatidylinositol 3-kinase. Mutations that uncouple EphA2 with Vav guanine nucleotide exchange factors or p85 are defective in Rac1 activation and cell migration. Finally, EphA2 mutations in the juxtamembrane region (Y587F, Y593F, Y587E/Y593E), kinase domain (Y734F), or SAM domain (Y929F) inhibited ephrin-A1-induced vascular assembly. In addition, EphA2-null endothelial cells reconstituted with these mutants were unable to incorporate into tumor vasculature, suggesting a critical role of these phosphorylation tyrosine residues in transducing EphA2 signaling in vascular endothelial cells during tumor angiogenesis.

Stats: Multifaceted Regulators of Transcription

The high-affinity binding interactions between interferons (IFNs) and their cognate cell surface receptors lead to the activation of receptor-associated Janus protein tyrosine kinases (Jaks) and subsequent phosphorylation and activation of a group of transcription factors, the signal transducers and activators of transcription (Stats). Upon IFN-induced activation, these Stat proteins form homodimeric and heterodimeric complexes that translocate to the nucleus and bind specific elements within the promoters of IFN-stimulated genes (ISGs). In addition to the well-studied IFN-induced ISG factor 3 (ISGF3) and Stat1:1 complexes, IFNs induce the formation of a number of other Stat-containing complexes, including Stat3:3 and Stat5:5 homodimers, as well as Stat2:1 and Stat5:CrkL heterodimers, that also mediate gene transcription. Moreover, emerging evidence suggests that particular amino acid residues within the individual Stat proteins contribute to different aspects of Stat function. These residues modulate the transcriptional activation potential of Stat-containing complexes and thereby influence the expression of ISGs. Indeed, the Stat proteins function in a multifaceted manner to regulate the expression of proteins that mediate IFN responses.

Volumetric evaluation of cerebral cortex of patients with cerebral vascular disease at magnetic resonance imaging

With the purification and cloning of the interferon gamma (IFN-γ) receptor chains the mechanism of IFN-γ action and the resultant signal transduction events were delineated in remarkable detail. The interferon gamma (IFN-γ) receptor complex consists of two chains: IFN-γR1, the ligand-binding chain, and IFN-γR2, the accessory chain. Binding of IFN-γ causes oligomerization of the two IFN-γ receptor subunits, IFN-γR1 and IFN-γR2, which initiates the signal transduction events: activation of Jakl and Jak2 receptor associated protein tyrosine kinases, phosphorylation of the IFN-γR1 intracellular domain on Tyr440 followed by phosphorylation and activation of Stat1α, the latent transcriptional factor. With all these steps established, the IFN-γ receptor complex has provided the basic model for understanding the receptors for other members of the family of class II cytokine receptors.

Metabolic switches of T-cell activation and apoptosis.

The signaling networks that mediate activation, proliferation, or programmed cell death of T lymphocytes are dependent on complex redox and metabolic pathways. T lymphocytes are primarily activated through the T-cell receptor and co-stimulatory molecules. Although activation results in lymphokine production, proliferation, and clonal expansion, it also increases susceptibility to apoptosis upon crosslinking of cell-surface death receptors or exposure to toxic metabolites. Activation signals are transmitted by receptor-associated protein tyrosine kinases and phosphatases through calcium mobilization to a secondary cascade of kinases, which in turn activate transcription factors initiating cell proliferation and cytokine production. Initiation and activity of cell death-mediating proteases are redox-sensitive and dependent on energy provided by ATP. Mitochondria play crucial roles in providing ATP for T-cell activation through the electron transport chain and oxidative phosphorylation. The mitochondrial transmembrane potential (DeltaPsi(m)) plays a decisive role not only by driving ATP synthesis, but also by controlling reactive oxygen species production and release of cell death-inducing factors. DeltaPsi(m) and reactive oxygen species levels are regulated by the supply of reducing equivalents, glutathione and thioredoxin, as well as NADPH generated in the pentose phosphate pathway. This article identifies redox and metabolic checkpoints controlling activation and survival of T lymphocytes.

Erythropoiesis in the absence of janus-kinase 2: BCR-ABL induces red cell formation in JAK2−/− hematopoietic progenitors

AbstractThe receptor-associated protein tyrosine kinase janus-kinase 2 (JAK2) is essential for normal red cell development and for erythropoietin receptor (EpoR) signaling. JAK2−/− embryos are severely deficient in erythropoiesis and die at an early stage of development from fetal anemia. The binding of erythropoietin (Epo) to the EpoR triggers the activation of JAK2, the phosphorylation of the EpoR, and the initiation of the EpoR signaling cascade. In addition to Epo binding to its receptor, signaling pathways downstream of the EpoR can also be stimulated by the BCR-ABL oncoprotein. This study explored whether JAK2 is required for BCR-ABL–mediated stimulation of erythropoiesis. Here, it is shown that JAK2 is constitutively tyrosine phosphorylated in cultured and primary erythroid cells expressing BCR-ABL. However, BCR-ABL effectively supports normal erythroid proliferation, differentiation, and maturation in JAK2-deficient fetal liver cells. Using mutants of BCR-ABL, this study shows that certain signaling pathways activated by BCR-ABL segments distinct from its tyrosine kinase domain are essential for rescue of erythropoiesis in JAK2−/− progenitors. The consequences of these multiple signaling pathways for normal erythroid development are discussed.

MEK and ERK Activation in Ras-Disabled RBL-2H3 Mast Cells and Novel Roles for Geranylgeranylated and Farnesylated Proteins in FcεRI-Mediated Signaling

AbstractCross-linking the high affinity IgE receptor FcεRI of basophils and mast cells activates receptor-associated protein-tyrosine kinases and stimulates a signaling cascade leading to secretion, ruffling, spreading, and cytokine production. Previous evidence that the pan-prenylation inhibitor lovastatin blocks Ag-stimulated Ca2+ influx, secretion, and membrane/cytoskeletal responses implicated isoprenylated proteins in the FcεRI-coupled signaling cascade but could not distinguish between contributions of C15 (farnesylated) and C20 (geranylgeranylated) species. Here we establish concentrations of lovastatin and the farnesyl-specific inhibitor BZA-5B that inhibit the farnesylation and Ag-induced activation of Ras species in RBL-2H3 cells (H-Ras, K-RasA, and K-RasB). These inhibitors have little effect on tyrosine kinase activation, which initiates FcεRI signaling. Although Ras is disabled, only lovastatin substantially blocks Raf-1 activation, and neither inhibitor affects mitogen-activated protein kinase kinase/extracellular signal regulated kinase kinase (MEK) or ERK1/ERK2 activation. Thus, the pathway to FcεRI-mediated MEK/ERK and ERK activation can apparently bypass Ras and Raf-1. Predictably, only lovastatin inhibits Ag-induced ruffling, spreading, and secretion, previously linked to geranylgeranylated Rho and Rab family members. Additionally, only lovastatin inhibits phospholipase Cγ-mediated inositol (1,4,5) trisphosphate production, sustained Ca2+ influx, and Ca2+-dependent IL-4 production, suggesting novel roles for geranylgeranylated (lovastatin-sensitive, BZA-5B-insensitive) proteins in FcεRI signal propagation. Remarkably, BZA-5B concentrations too low to inactivate Ras reduce the lag time to Ag-induced Ca2+ stores release and enhance secretion. These results link a non-Ras farnesylated protein(s) to the negative regulation of Ca2+ release from intracellular stores and secretion. We identified no clear role for Ras in FcεRI-coupled signaling but suggest its involvement in mast cell growth regulation based on the inhibition of cell proliferation by both BZA-5B and lovastatin.

Role of insulin-like growth factor-I in esophageal mucosal healing processes

The current study examines the stimulation of healing processes and signal transduction that is mediated by insulin-like growth factor-I (IGF-I) in an ex vivo esophageal explant model when using tyrphostin inhibition of receptor tyrosine kinase. The explant model provides a 3-dimensional cellular environment of multiple interacting cells isolated from the neural and vascular supply. Tyrphostins previously characterized for their interactions with epithelial growth factor (EGF) receptor-associated protein tyrosine kinases were tested for their potential effects on IGF-I growth-promoting activity. Explants of rabbit esophagus were incubated in media with or without IGF-I. Tyrphostins 1, 23, 25, 46, 47, 51, and 63 were added. We assessed DNA synthesis by tritiated thymidine incorporation. Outgrowth from the edge of the primary mucosa of the explant was evaluated on histologic sections, and cell proliferation was confirmed with immunohistology. IGF-I increased the incorporation of tritiated thymidine by 50% to 100%. Tyrphostins 23 and 47 eliminated IGF-I-induced proliferation in a dose-dependent manner. Tyrphostins 25, 46, and 51—along with negative controls tyrphostin 1 and tyrphostin 63—were ineffective, inasmuch as IGF-I-stimulated growth remained unchanged in their presence. Proliferative activity demonstrated by PCNA staining was confined to new mucosa. Two of 5 tyrphostins originally developed as EGF receptor protein tyrosine kinase inhibitors were effective in inhibiting the actions of exogenous IGF-I. We conclude that IGF-I stimulation may play an important role in repair processes in the esophagus and that this stimulation can be inhibited by using specific tyrphostins.

Activation of p21ras/MAPK Signal Transduction Molecules Decreases with Age in Mitogen-Stimulated T Cells from Rats

Signal transduction is ubiquitously involved in the initiation of physiological signals that lead to growth and proliferation of cells. The signaling cascade mediated by the mitogen-activated protein kinase (MAPK) is considered essential for T cell growth and function. Therefore, it was of interest to determine the influence of age on the induction of MAPK in mitogen-activated T cells. T cells from young (4–6 months) and old (24–26 months) rats responded to concanavalin A (Con A) stimulation by increasing MAPK, c-jun amino terminal kinase (JNK), and p21rasactivities. The time course of induction of MAPK/JNK and p21rasactivities was similar in T cells isolated from young and old rats. The induction of JNK activity did not change significantly with age; however, the induction of MAPK and p21rasactivities was significantly less (50 to 65%) in T cells from old rats than in T cells from young rats. Although the relative protein levels of p42 and p44 MAPK did not change with age, the proportion of the phosphorylated p44 MAPK decreased with age. In addition, it was found that thein vitrokinase activities of the T cell receptor-associated protein tyrosine kinase Lck (p56Lck) and ZAP-70 but not Fyn (p59Fyn) were lower in T cells from old rats than in T cells from young rats. The decline in activities of these signaling molecules with age was not associated with changes in their corresponding protein levels. Thus, our results demonstrate that aging alters the activation of the signal transduction cascade that leads to T cell activation.

Increased Enzymatic Activity of the T-Cell Antigen Receptor-Associated Fyn Protein Tyrosine Kinase in Asymptomatic Patients Infected With the Human Immunodeficiency Virus

AbstractThe immune system of patients infected with human immunodeficiency virus (HIV) is in a state of chronic activation; however, the nature of HIV-related immune activation is unknown. As normal T-cell activation involves early tyrosine phosphorylation induced by the T-cell antigen receptor-associated src-family protein tyrosine kinase p59fyn(T) (Fyn), we examined a potential role for this kinase in HIV-related immune dysfunction. We determined the relative specific kinase activity of Fyn in lysates of peripheral blood mononuclear cells from 47 normal control individuals tested negative for HIV-1 and -2, human T-cell lymphotropic virus Type I, hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis; 14 asymptomatic HIV-infected patients having near-normal CD4+ T-cell counts (350 to 980 CD4+ cells/μL); 4 patients with symptomatic acquired immunodeficiency syndrome (AIDS) (<30 CD4+ cells/μL); 13 patients having chronic infection with HBV (6 patients) or HCV (7 patients); and 6 patients with systemic lupus erythematosis (SLE). All patients with asymptomatic HIV disease were shown to have a profound increase (mean increase of 19-fold; range threefold to 56-fold increase; p = 1.33 × 10−9) in the relative specific kinase activity of Fyn compared to uninfected controls or patients with hepatitis or SLE. In contrast, patients with AIDS had an Fyn-specific kinase activity that was much less affected (mean increase of threefold; range onefold to sevenfold increase; p = 1.30 × 10−5). It was further shown that HIV infection affects the Fyn-specific kinase activity in CD8+-enriched cells, suggesting abnormal Fyn activity in both CD8+ as well as CD4+ T lymphocytes. Initial results implicate a role for the CSK protein tyrosine kinase as responsible for the abnormal Fyn kinase activity observed in HIV-infected patients. These data indicate early and chronic activation of Fyn as a unique HIV-related effect that has the potential to be diagnostic for early HIV infection and/or may serve as a prognostic indicator for advancement to full-blown AIDS. More importantly, sustained activation of the protein tyrosine kinase associated with T-cell antigen receptor function may result in, or contribute to, the immunopathogenic effects associated with HIV infection.

The interferon gamma (IFN-γ) receptor: a paradigm for the multichain cytokine receptor

With the purification and cloning of the interferon gamma (IFN-γ) receptor chains the mechanism of IFN-γ action and the resultant signal transduction events were delineated in remarkable detail. The interferon gamma (IFN-γ) receptor complex consists of two chains: IFN-γR1, the ligand-binding chain, and IFN-γR2, the accessory chain. Binding of IFN-γ causes oligomerization of the two IFN-γ receptor subunits, IFN-γR1 and IFN-γR2, which initiates the signal transduction events: activation of Jakl and Jak2 receptor associated protein tyrosine kinases, phosphorylation of the IFN-γR1 intracellular domain on Tyr 440 followed by phosphorylation and activation of Stat1α, the latent transcriptional factor. With all these steps established, the IFN-γ receptor complex has provided the basic model for understanding the receptors for other members of the family of class II cytokine receptors.

Syk is required for BCR-mediated activation of p90Rsk, but not p70S6k, via a mitogen-activated protein kinase-independent pathway in B cells.

The tyrosine kinases Syk and Lyn are activated in B lymphocytes following antibody induced cross-linking of the B cell receptor for antigen (BCR). It has been suggested that activation of Syk is dependent on Lyn. We tested this hypothesis by comparing the phosphorylation and activation of several downstream effector molecules in parental DT40, DT40Syk- and DT40Lyn- B cells. The phosphorylation and activation of p90Rsk was ablated in Syk-deficient B cells but unaffected in Lyn-deficient B cells while the phosphorylation/activation of Ras GTPase activating protein (Ras GAP) and mitogen activated protein (MAP) kinase required both Syk and Lyn. Thus, these data indicate that Syk can be activated in the absence of Lyn after BCR cross-linking and results in the activation of p90Rsk via a MAP kinase-independent pathway in DT40Lyn- cells. We also demonstrated that BCR mediates the activation of p70S6k. However, activation of p70S6k in DT40Syk- and DT40Lyn- cells was comparable with that observed in parental cells. Thus, either Syk or Lyn may be sufficient for activation of p70S6k, or activation of p70S6k occurs independently of both Syk and Lyn. The kinase activity of Syk was required for the phosphorylation/activation of each of these downstream effector molecules but only the phosphorylation of Ras GAP was affected in cells expressing a mutant of Syk in which tyrosines 525 and 526 were substituted to phenlyalanines.

Identification of a novel pathway important for proliferation and differentiation of primary erythroid progenitors.

Homodimerization of the erythropoietin (EPO) receptor (EPO-R) in response to EPO binding transiently activates the receptor-associated protein tyrosine kinase JAK2. Tyrosine phosphorylation of the EPO-R creates "docking sites" for SH2 domain(s) in signaling molecules such as the protein tyrosine phosphatases SH-PTP1 and SH-PTP2, phosphoinositide 3-kinase (PI3 kinase), and STAT5. However, little is known about the specific intracellular signals essential for proliferation and differentiation of erythroid progenitors. Here we show that an EPO-R containing only one cytosolic (phospho)tyrosine residue, Y479, induces a signal transduction pathway sufficient for proliferation and differentiation of fetal liver progenitors of erythroid colony-forming units from EPO-R(-/-) mice as well as for proliferation of cultured hematopoietic cells. This cascade involves sequential EPO-induced recruitment of PI3 kinase to the EPO-R and activation of mitogen-activated protein kinase activity, independent of the Shc/Grb2-adapter pathway and of STAT5. Protein kinase C epsilon may be one of the mediators connecting PI3 kinase with the mitogen-activated protein kinase signaling cascade. Our results identify a signaling cascade important in vivo for erythroid cell proliferation and differentiation.

Synthesis and biological activity of 5-[(2,5-dihydroxybenzyl)amino]salicylic acid analogs as inhibitors of EGF receptor-associated protein tyrosine kinase

The synthesis and biological activity of a series of 5-[(2,5-dihydroxybenzyl)amino]salicylic acid derivatives ( 3–6) as analogs of the active partial structure ( 2) of the potent EGF-R tyrosine kinase inhibitor lavendustin A ( 1) are described. Analogs with an electron-withdrawing group in place of the carboxyl group of 2 showed activity. The N-hexylsalicylamide analog 6b (IC 50=0.9 μM) was about four times more potent than 2.

Defective Activation of Mitogen-Activated Protein Kinase After Allogeneic Bone Marrow Transplantation

Allogeneic bone marrow transplant (BMT) recipients have increased susceptibility to infections for prolonged periods after phenotypic reconstitution of donor cells. This immunodeficiency status is characterized by multiple T-cell functional abnormalities. This study was designed to investigate several signaling pathways involved in T-cell activation during this period of immune deficiency. In initial experiments using equal numbers of CD3+ cells or highly purified T-cell subpopulations obtained from normal controls and BMT recipients, we confirmed that abnormal T-cell proliferation after CD3 cross-linking, phytohemagglutinin stimulation, or phorbol myristate acetate (PMA) stimulation of peripheral blood mononuclear cells from BMT recipients was due to a qualitative T-cell deficiency rather than to low numbers of circulating T cells. We next investigated the ability of the T-cell receptor/CD3 complex to transduce signals via receptor-associated protein tyrosine kinases. In all BMT recipients, CD3 cross-linking induced protein tyrosine phosphorylation of several proteins in a similar fashion to that seen in controls, including phosphorylation of a 21-kD protein that represents the zeta subunit of the receptor itself. Further investigation showed that CD3 cross-linking and PMA stimulation did not increase 42-44-kD mitogen-activated protein kinase (MAPK) activity. The failure of MAPK activation in BMT recipients occurred despite tyrosine phosphorylation of the 42-44-kD proteins, which, in normal controls, parallels enzyme activation. Our results indicate that T-cell immunodeficiency in BMT recipients is associated with a selective failure of MAPK activation, possibly related to abnormal posttranslational positive regulation of this enzyme.

Inhibition of cytokines and JAK-STAT activation by distinct signaling pathways.

An important component of cytokine regulation of cell growth and differentiation is rapid transcriptional activation of genes by the JAK-STAT (signal transducer and activator of transcription) signaling pathway. Ligation of cytokine receptors results in tyrosine phosphorylation and activation of receptor-associated Jak protein tyrosine kinases and cytoplasmic STAT transcription factors, which then translocate to the nucleus. We describe the interruption of cytokine triggered JAK-STAT signals by cAMP, the calcium ionophore ionomycin, and granulocyte/macrophage colony-stimulating factor. Jak1 kinase activity, interleukin 6-induced gene activation, Stat3 tyrosine phosphorylation, and DNA-binding were inhibited, as was activation of Jak1 and Stat1 by interferon gamma. The kinetics and requirement for new RNA and protein synthesis for inhibition of interleukin 6 by ionomycin and GM-CSF differed, but both agents increased the association of Jak1 with protein tyrosine phosphatase ID (SH2-containing phosphatase 2). Our results demonstrate that crosstalk with distinct signaling pathways can inhibit JAK-STAT signal transduction, and suggest approaches for modulating cytokine activity during immune responses and inflammatory processes.

Alteration of B-cell Antigen Receptor Signaling by CD19 Co-ligation.: A STUDY WITH BISPECIFIC ANTIBODIES

The activation of B-cell antigen receptor-associated protein tyrosine kinases is an early and crucial event in B-cell signaling. Apart from the B-cell antigen receptor (BCR), the B-cell-specific transmembrane glycoprotein CD19 has also been shown to directly activate intracellular signaling cascades. In addition, because CD19 and the BCR are associated on the surface of activated B-cells, it has been proposed that close approximation between these two entities is crucial for optimal B-cell triggering. To test this hypothesis, bispecific antibodies were generated that bind membrane IgM and CD19 simultaneously. Although CD19 bispecific antibodies strongly induced tyrosine phosphorylation, they were, in contrast to muF(ab)2 fragments, unable to induce a proliferative response. Detailed analysis of the early signaling events showed that compared with muF(ab)2 fragments CD19 bispecific antibodies potently raised the intracellular [Ca2+], which was correlated with an efficient tyrosine phosphorylation of syk. Strikingly, the assembly of Grb2 complexes that may couple the BCR to p21(ras) was clearly altered by the CD19 bispecific antibody. In addition to the reported Shc and 145-kDa phosphoproteins, a prominent 90-95-kDa phosphoprotein resembling CD19 was detected in the Grb2 complexes. Thus, studies with CD19 bispecific antibodies show that CD19 co-ligation both quantitatively and qualitatively alters BCR signaling.

The Effect of Purified Extract ofFagopyrum esculentum(Buckwheat) on Protein Kinases Involved in Signal Transduction Pathways

The effect of a purified extract of the flowering herb of Fagopyrum esculentum (buckwheat) on various protein kinases involved in signal transduction was examined. We observed that buckwheat contains red fluorescent compounds having photosensitizing properties. Spectrophotometric analysis of the extract has indicated structural similarity to hypericin. Dose- and light-dependent inhibition of various protein kinases was observed. The purified buckwheat extract strongly inhibited two receptor-associated protein tyrosine kinases (EGF-R and Ins-R) and a Ser/Thr kinase (PK-C) at an ng/ml concentration range. Selectivity was exhibited as a decreased sensitivity to cytosolic PTKs and protein kinase CK-2. The protein kinases are important components of the signal transduction pathway. Aberration of signal transduction is a hallmark of several proliferative diseases. Our results indicate that photosensitizing compounds in buckwheat are potential antiproliferative agents.