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.     

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.     

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 tea infusions of various varieties or different manufacturing types on inhibition of mouse mast cell activation

We investigated effects of various tea infusions on mast cell activation using mouse mast cells. Among various tea extracts, infusions from cultivar 'Benihomare' and Taiwan lineage strongly inhibited histamine release after Fc epsilon RI cross-linking. Among three types of tea (from cultivar 'Benihomare'), extract from oolong tea or black tea inhibited histamine release more strongly than green tea extract. Furthermore, 'Benihomare' oolong tea extract suppressed tyrosine phosphorylation of cellular proteins after Fc epsilon RI cross-linking, but polyvinyl polypyrrolidone treatment of the extract to remove phenolic compounds, weakened the suppressive effect.     

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.     

Bruton's tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity

Mast cells derived from Bruton's tyrosine kinase (Btk)-defective xid or btk null mice showed greater expansion in culture containing interleukin-3 (IL-3) than those from wild-type (wt) mice. Although the proliferative response to IL-3 was not significantly different between the wt and xid mast cells, xid and btk null mast cells died by apoptosis more slowly than their wt counterparts upon IL-3 deprivation. Consistent with these findings, the apoptosis-linked c-Jun N-terminal kinase/stress-activated protein kinase (JNK) activity was compromised in these btk-mutated cells upon Fc(epsilon)RI crosslinking or upon stimulation with IL-3 or with stem cell factor. p38 activity was less severely, but significantly, affected by btk mutation, whereas extracellular signal-regulated kinases were not affected by the same mutation. Btk-mediated regulation of apoptosis and JNK activity was confirmed by reconstitution of btk null mutant mast cells with the wt btk cDNA. Furthermore, growth factor withdrawal induced the activation and sustained activity of JNK in wt mast cells, while JNK activity was consistently lower in btk-mutated mast cells. These results support the notion that Btk regulates apoptosis through the JNK activation.     

CGP 41251, a novel protein kinase inhibitor with in vitro selectivity for protein kinase C, strongly inhibits immunological activation of human skin mast cells and human basophils

The process of high-affinity IgE receptor (Fc epsilon RI)-mediated signal transduction in human basophils and mast cells is accompanied by activation of protein kinase C (PKC). The present study investigated the effects of a novel protein kinase inhibitor with in vitro selectivity for PKC (CGP 41251) in comparison with the potent but non-selective PKC inhibitor staurosporine on the activation of human peripheral basophilic leukocytes and enzymatically isolated human skin mast cells. CGP 41251 exerted strong concentration-dependent inhibitory effects on Fc epsilon RI-mediated histamine release from both cell populations. In addition, the IgE-mediated generation of arachidonic acid metabolites (leukotriene C4/D4 and prostaglandin E2) from human basophils was also significantly inhibited by this compound. Its action was not significantly different from the action of staurosporine. Direct activation of cellular PKC by the phorbol ester 12-o-tetradecanoyl-phorbol-13-acetate and subsequent histamine release from basophils was also inhibited by both compounds. CGP 41251 did not suppress N-formyl-met-leu-phe- or A23187-induced activation of basophils, whereas A23187-induced mediator release from human skin mast cells was inhibited in a concentration-dependent fashion. We conclude that an increase of in vitro selectivity for PKC does not significantly enhance inhibitory effects on immunological activation of histamine-containing cells. Moreover, nonimmunological pathways of signal transduction in basophils and mast cells appear to be mediated by distinct biochemical events.     

Fc epsilon RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling

Detergent-resistant plasma membrane structures, such as caveolae, have been implicated in signalling, transport, and vesicle trafficking functions. Using sucrose gradient ultracentrifugation, we have isolated low-density, Triton X-100-insoluble membrane domains from RBL-2H3 mucosal mast cells that contain several markers common to caveolae, including a src-family tyrosine kinase, p53/56lyn. Aggregation of Fc epsilon RI, the high-affinity IgE receptor, causes a significant increase in the amount of p53/56lyn associated with these low-density membrane domains. Under our standard conditions for lysis, IgE-Fc epsilon RI fractionates with the majority of the solubilized proteins, whereas aggregated receptor complexes are found at a higher density in the gradient. Stimulated translocation of p53/56lyn is accompanied by increased tyrosine phosphorylation of several proteins in the low-density membrane domains as well as enhanced in vitro tyrosine kinase activity toward these proteins and an exogenous substrate. With a lower detergent-to-cell ratio during lysis, significant Fc epsilon RI remains associated with these membrane domains, consistent with the ability to coimmunoprecipitate tyrosine kinase activity with Fc epsilon RI under similar lysis conditions [Pribluda, V. S., Pribluda, C. & Metzger, H. (1994) Proc. Natl. Acad. Sci. USA 91, 11246-11250]. These results indicate that specialized membrane domains may be directly involved in the coupling of receptor aggregation to the activation of signaling events.     

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.     

Modulation of MAP kinase signaling and growth characteristics by the overexpression of protein kinase C in NIH3T3 cells

This study was performed to examine effects of the overexpression of protein kinase C (PKC) isoforms (i.e., beta I, beta II, gamma, delta, eta, and zeta) on mitogen-activated protein (MAP) kinase (Erk-1 and -2) signaling and growth characteristics of NIH3T3 cells. Phorbol ester (PMA) activated endogenous and ectopically expressed PKC alpha, beta I, beta II, gamma, delta, epsilon, and eta. Overexpression of the examined PKC isoforms enhanced PMA-induced MAP kinase activation. Potentiation of MAP kinase activation was also observed upon stimulation of cells with platelet-derived growth factor (PDGF) although there was no indication for the activation PKC isoforms by PDGF. Inhibition of PKC blocked PMA- but not PDGF-induced MAP kinase activation. Thus, potentiation of PDGF-induced MAP kinase activation appears to be independent to PKC activity, while PMA-induced MAP kinase activation requires PKC activity. The ability of PKC isoforms to potentiate MAP kinase activation is not related to the growth characteristics of cells because individual PKC isoforms differentially regulated maximum density and proliferation of 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.     

In vivo induction of specific cytotoxic T lymphocytes in mice and rhesus macaques immunized with DNA vector encoding an HIV epitope fused with hepatitis B surface antigen

BACKGROUND: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to Ca2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production. METHODS AND RESULTS: The role of tyrosine kinases in human Fc epsilonRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. Fc epsilonRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to Fc epsilonRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion). CONCLUSIONS: Our data demonstrate the association of anti-IgE-stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of Fc epsilonRI-mediated signaling in human basophils.     

Effects of suramin-related and other clinically therapeutic polyanions on protein kinase C activity

The mechanism of the antineoplastic effects of suramin may involve interference with signal transduction, but in general is not well understood. We examined several polyanions to determine their effects on the kinase activity of the protein kinase C (PKC) beta1 and other PKC isoforms. Similar to suramin, a phosphorothioate oligodeoxynucleotide 28-mer homopolymer of cytidine (SdC28) inhibited the phosphatidylserine and Ca2+-dependent phosphorylation of an epidermal growth factor receptor octapeptide substrate. The inhibition by suramin was mixed competitive/noncompetitive with respect to ATP, but uncompetitive with respect to substrate. In contrast, the inhibition by SdC28 was competitive with respect to substrate (Ki = 5.4 microM) and not competitive with respect to ATP. The PKC alpha and beta1 isoforms were inhibited to the same extent with SdC28, while PKC epsilon was not inhibited. SdC28, in the absence of lipid cofactor, stimulated substrate phosphorylation, and in the absence of substrate induced PKC beta1 autophosphorylation. Similar behavior was seen with another polyanion, the polysulfated carbohydrate pentosan polysulfate (polyxylyl hydrogen sulfate). H4, a bis-naphthalene disulfonate tetraanion structurally related to suramin, also inhibited kinase activity but was not competitive with respect to ATP. Dianions closely related to H4 failed to inhibit PKC beta1, suggesting that multiple (>2) negative charges are required. The interactions of polyanions with PKC are complex, and are dependent on the molecular structure of the polyanion, the presence of cofactors, and the PKC isoform.     

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.     

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.     

Molecules in focus: diacylglycerol kinase

Recent observations suggest that diacylglycerol kinase (DGK) is one of the key enzymes involved in the regulation of signal transduction. It attenuates protein kinase C activity and cell cycle progression of T-lymphocytes, through controlling the intracellular levels of the second messengers, diacylglycerol and phosphatidic acid. To date, eight DGK isozymes containing characteristic zinc finger structures in common have been identified. Type I DGKs (alpha, beta and gamma) contain EF-hand motifs that contribute to the calcium-dependent activities of this type of DGK. A pleckstrin homology and/or an EPH C-terminal tail homology domains are found in type II isozymes (DGK delta and eta). DGK epsilon represents a third type of DGK that selectively phosphorylates arachidonate-containing diacylglycerol. DGK zeta (type IV) and DGK theta (type V) contain four tandem ankyrin repeats and a Ras-associating domain, respectively.     

The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta

The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca2+]i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGF's angiogenic effects.     

Phosphorylation events mediated by protein kinase C alpha and epsilon participate in regulation of tau steady-state levels and generation of certain "Alzheimer-like" phospho-epitopes

Hyperactivation of protein kinase C (PKC) in intact neuroblastoma cells by several methods increases site-specific tau phosphorylation as shown by increases in paired helical filament-I (PHF-I) and ALZ-50 but not AT-8 immunoreactivity. In the present study, the influence of PKC on tau metabolism was further examined by isoform-specific antisense oligonucleotide-mediated PKC downregulation in human SH-SY-5Y neuroblastoma cells and by generation of stably-transfected subclones expressing isoform-specific anti-PKC mRNA sequences. Downregulation of PKC epsilon by both of these methods reduced PHF-I and ALZ-50 immunoreactivity, suggesting that this PKC isoform, perhaps via downstream kinase cascades, regulated tau phosphorylation events that normally generate these epitopes. By contrast, downregulation of either PKC epsilon or PKC alpha reduced immunoreactivity towards the phosphate-independent anti-tau antibodies 5E2 and JM, suggesting that both of these isoforms participated in regulation of tau steady-state levels. Downregulation of PKC beta did not affect any of the above changes. The above roles were apparently unique for PKC epsilon and PKC alpha, since activation of multiple PKC isoforms by phorbol ester treatment and/or other calcium-dependent kinase(s) by ionophore-mediated calcium influx could not compensate for downregulation of PKC alpha or PKC epsilon in maintaining tau steady-state levels or PHF-I/ALZ-50 immunoreactivity, respectively. These findings suggest that hyperactivation of signal transduction pathways, including those regulated by PKC, could evoke changes in neuronal cells reminiscent of those seen in affected neurons in Alzheimer's disease.     

Src family tyrosine kinase Lyn binds several proteins including paxillin in rat basophilic leukemia cells

Aggregation of the high affinity IgE receptors on rat basophilic leukemia (RBL-2H3) cells results in protein tyrosine phosphorylation although the receptor has no intrinsic enzymatic activity. The Src related protein tyrosine kinase p53/56lyn present in RBL-2H3 cells could play a role in this reaction. Here we have isolated the cDNA for rat Lyn and found it to be very homologous at the amino acid level to both the human and mouse proteins. A bacterially expressed maltose binding protein-Lyn (MBP-Lyn) fusion protein was already tyrosine phosphorylated and had tyrosine kinase activity. In a filter-binding assay, MBP-Lyn fusion protein (at 0.1 microM) specifically bound to several proteins of RBL-2H3 cells. In lysates of IgE receptor-activated cells, there was increased binding of MBP-Lyn to 65, 72, 78 and 110 kDa tyrosine phosphorylated proteins. The 72, 78 and 110 kDa tyrosine phosphorylated proteins were precipitated by a fusion protein containing the Lyn Src Homology 2 (SH2) domain. The 72 kDa Lyn binding protein was different from p72syk. Furthermore, paxillin, a cytoskeletal protein, was identified as one of the Lyn binding proteins. Thus Fc epsilon RI mediated signal transduction in RBL-2H3 cells may result from the interaction of p53/56lyn with paxillin, pp72, pp110 and other proteins.