柳 茂

In this study, we have identified a novel mitochondrial ubiquitin ligase, designated MITOL, which is localized in the mitochondrial outer membrane. MITOL possesses a Plant Homeo-Domain (PHD) motif responsible for E3 ubiquitin ligase activity and predicted four-transmembrane domains. MITOL displayed a rapid degradation by auto-ubiquitination activity in a PHD-dependent manner. HeLa cells stably expressing a MITOL mutant lacking ubiquitin ligase activity or MITOL-deficient cells by small interfering RNA showed an aberrant mitochondrial morphology such as fragmentation, suggesting the enhancement of mitochondrial fission by MITOL dysfunction. Indeed, a dominant-negative expression of Drp1 mutant blocked mitochondrial fragmentation induced by MITOL depletion. We found that MITOL associated with and ubiquitinated mitochondrial fission protein hFis1 and Drp1. Pulse-chase experiment showed that MITOL overexpression increased turnover of these fission proteins. In addition, overexpression phenotype of hFis1 could be reverted by MITOL co-overexpression. Our finding indicates that MITOL plays a critical role in mitochondrial dynamics through the control of mitochondrial fission proteins.

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG-polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin-proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. We have previously identified a unique member of this gene family, CRMP-associated molecule CRAM (CRMP-5), which is phylogenetically divergent from the other four CRMPs. In this study, we have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAM in the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. We propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.

Tamalin is a scaffold protein that forms a multiple protein assembly including metabotropic glutamate receptors (mGluRs) and several postsynaptic and protein-trafficking scaffold proteins in distinct mode of protein-protein association. In the present investigation, we report that tamalin possesses a typical immunoreceptor tyrosine-based activation motif (ITAM), which enables Syk kinase to be recruited and phosphorylated by the Src family kinases. Coimmunoprecipitation analysis of rat brain membrane fractions showed that tamalin is present in a multimolecular protein assembly comprising not only mGluR1 but also c-Src, Fyn, and a protein phosphatase, SHP-2. The protein association of both tamalin and c-Src, as determined by truncation analysis of mGluR1 in COS-7 cells, occurred at the carboxyl-terminal tail of mGluR1. Mutation analysis of tyrosine with phenylalanine in COS-7 cells revealed that paired tyrosines at the ITAM sequence of tamalin are phosphorylated preferentially by c-Src and Fyn, and this phosphorylation can recruit Syk kinase and enables it to be phosphorylated by the Src family kinases. The phosphorylated tyrosines at the ITAM sequence of tamalin were highly susceptible to dephosphorylation by protein-tyrosine phosphatases in COS-7 cells. Importantly, tamalin was endogenously phosphorylated and associated with Syk in retinoic acid-treated P19 embryonal carcinoma cells that undergo neuron-like differentiation. The present investigation demonstrates that tamalin is a novel signaling molecule that possesses a PDZ domain and a PDZ binding motif and mediates Syk signaling in an ITAM-based fashion.

We have previously demonstrated that Fes/Fps (Fes) tyrosine kinase is involved in Semaphorin3A-mediated signaling. Here we report a role for Fes tyrosine kinase in microtubule dynamics. A fibrous formation of Fes was observed in a kinase-dependent manner, which associated with microtubules and functionally correlated with microtubule bundling. Microtubule regeneration assays revealed that Fes aggregates colocalized with gamma-tubulin at microtubule nucleation sites in a Fes/CIP4 homology (FCH) domain-dependent manner and that expression of FCH domain-deleted Fes mutants blocked normal centrosome formation. In support of these observations, mouse embryonic fibroblasts derived from Fes-deficient mice displayed an aberrant structure of nucleation and centrosome with unbundling and disoriented filaments of microtubules. Our findings suggest that Fes plays a critical role in microtubule dynamics including microtubule nucleation and bundling through its FCH domain.

Background: Collapsin response mediator proteins (CRMPs) and CRAM belong to the unc-33 gene family which is implicated in axon guidance and outgrowth during neural development. However, their exact roles remain largely unknown. To understand the molecular basis of CRMP/CRAM function, we have undertaken to identify CRMP/CRAM interacting proteins. Results: We, have identified a novel mitochondrial septin (M-septin) as one of the CRMP/CRAM interacting proteins from the developing rat brain. M-septin is a major, alternatively spliced variant of the H5 gene in developing mouse brain and its expression is up-regulated during the neuronal differentiation of embryonal carcinoma P19 cells. In COS-7 cells, M-septin is specifically localized to mitochondria whereas H5 is diffusely distributed to the perinuclear cytoplasm and plasma membranes. In contrast to H5, M-septin induces the mitochondrial translocation of CRAM but not CRMP2. Finally, M-Septin is found to be transiently translocated to mitochondria before the induction of the neurites and then dissociates from the mitochondria after neurite extension in P19 cells. Conclusions: Our results suggest that M-septin has a role which is distinct from H5, and together with CRMP/CRAM, may play an important role in the neuronal-differentiation and axon guidance through the control of mitochondrial function.

Background. Helicobacter pylori CagA is injected into the host cell and tyrosine-phosphorylated. We examined tyrosine-phosphorylation sites of CagA, as well as the function of CagA proteins in vivo and in vitro. Methods. After proteolytic digestion of CagA with lysyl endopeptidase, CagA tyrosine-phosphorylation sites were determined using quadropolar time-of-flight (Q-TOF) mass spectrometry analysis. Specific anti-pY CagA polyclonal and anti-CagA monoclonal antibodies were used to examine gastric mucosal biopsy specimens from H. pylori infected patients. Results. Mass spectrometry identified five crucial tyrosine-phosphorylation sites of CagA at Tyr893, Tyr912, Tyr965, Tyr999, and Tyr1033 within the five repeated EPIYA sequences of H. pylori (NCTC11637)-infected AGS cells. CagA protein also had an immuno-receptor tyrosine-based activation motif (ITAM)-like amino acid sequences in the 3' region of the cagA , EPIYATI x(27) EIYATI, which closely resembled the ITAM. CagA proteins: (i) were localized to the 1% TritonX-100 resistant membrane fraction (lipid rafts); (ii) formed a cluster of phosphorylated CagA protein complexes; (iii) associated with tyrosine-phosphorylated GIT1/Cat1 (G protein-coupled receptor kinase-interactor 1/Cool-associated tyrosine-phosphorylated 1), substrate molecules of receptor type protein-tyrosine phosphatase (RPTPzeta/beta), which is the receptor of VacA; and (iv) were involved in a delay and negative regulation of VacA-induced signal. Furthermore, immunohistochemical staining of gastric mucosal biopsy specimens provided strong evidence that tyrosine-phosphorylated CagA is found together with CagA at the luminal surface of gastric foveola in vivo. Conclusion. These findings suggest an important role for CagA containing ITAM-like sequences in the pathogenesis of H. pylori- related disease.

Collapsin response mediator proteins (CRMPs)/TOAD64/Ulips/DRPs and CRAM have emerged as strong candidates for a role in semaphorin signaling. In this study we identified Fes/Fps (Fes) tyrosine kinase in the CRMP-CRAM complex and investigated whether Fes was involved in semaphorin3A (Sema3A) signaling. In COS-7 cells, the interaction between Fes and plexinA1 (PlexA1) and the tyrosine phosphorylation of PlexA1 by Fes were observed; however, these events were significantly attenuated by co-expression of neuropilin-1 (NP-1). Even with NP-1 co-expression, Sema3A was able to enhance the association of Fes with PlexA1 and Fes-mediated tyrosine phosphorylation of PlexA1, CRAM and CRMP2. Co-expression of Fes with PlexA1 exhibited COS-7 cell contraction activity, indicating that Fes can convert inactive PlexA1 to its active form, whereas combination of Fes/NP-1/PlexA1 or Fes kinase-negative mutants/PlexA1 did not alter cell morphology. Finally, Sema3A-induced growth cone collapse of dorsal root ganglion neurons was suppressed by expression of Fes kinase-negative mutants. Taken together, our findings suggest that Fes links Sema3A signals to CRMP-CRAM, and that NP-1 negatively regulates PlexA1 activation by Fes in resting condition.

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. In this study, we have investigated the role of Syk in p38 activation and the regulation of cell-cycle progression upon oxidative stress. In B cells, p38 is activated by hydrogen peroxide (H2O stimulation. Syk is required for p38 activation following stimulation with 10-100 muM H2O2 V but not with 1 mM H2O2. H2O2-induced p38 activation is abrogated in phospholipase C-gamma2 (PLC-gamma2)-deficient as well as Syk-deficient cells, suggesting that Syk activates p38 through PLC-gamma2 upon H2O2 stimulation. Although stimulation with 20-100 muM H2O2 induces cellular apoptosis in B cells, pretreatment with SB203580, a p38-specific inhibitor, has no effect on H O-2(2)-induced apoptosis. Flow cytometric analysis reveals that B cells exposed to 10-20 muM H2O2 exhibit cell-cycle profile of G2/M arrest, and pretreatment with SB203580 inhibits only a little H2O2-induced G2/M arrest. On the other hand, Syk-deficient cells show no induction of G2/M arrest following H2O2 stimulation. These findings indicate that Syk plays a role in the regulation of cell-cycle progression in G2/M phase via p38-dependent and -independent pathways after oxidative stress.

BLNK (B cell linker protein) represents a central linker protein that bridges the B cell receptor-associated kinases with a multitude of signaling pathways. In this study, we have investigated the role of BLNK in oxidative stress signaling in B cells. H2O2 treatment of B cells induced a rapid tyrosine phosphorylation of BLNK in a H2O2 dose-dependent manner, which was inhibited in Syk-deficient DT40 cells. Calcium mobilization in BLNK-deficient as well as Syk-deficient and phospholipase C (PLC)-gamma2-deficient cells after H2O2 treatment was completely abolished. These were derived from decreased inositol 1,4,5-trisphosphate generation through PLC-gamma2 in BLNK-deficient cells. Moreover, viability of BLNK-deficient as well as PLC-gamma2-deficient cells after exposure to low doses of H2O2 was dramatically enhanced compared with that of the wild-type cells. Furthermore, c-Jun N-terminal kinase activation following high doses of H2O2 Stimulation, but not low doses of H2O2 stimulation, was abrogated in BLNK-deficient as well as Syk-deficient cells. These findings have led to the suggestion that BLNK is required for coupling Syk to PLC-gamma2, thereby accelerating cell apoptosis in B cells exposed to low doses of H2O2.

Mice deficient in the Syk tyrosine kinase showed severe petechiae in utero and died shortly after birth. The mechanism of this bleeding, however, remains unknown. Here it is shown that this bleeding is caused by morphologic defects of Syk-deficient endothelial cells during embryogenesis. Immunoblot and reverse transcriptase-polymerase chain reaction Northern blot analysis indicated that Syk is expressed in several endothelial cell lines. Immunocytochemical analysis also confirmed that Syk is expressed in the normal embryonic endothelial cells and is absent in Syk-deficient mice. Furthermore, electron microscopic analysis of Syk-deficient mice revealed an abnormal morphogenesis and a decreased number of endothelial cells. The results indicate a critical role for Syk in endothelial cell function and in maintaining vascular integrity in vivo. (C) 2001 by The American Society of Hematology.

The effect of adhesion via CD43 (leukosialin, sialophorin) on cell proliferation and phosphorylation signaling were examined in a growth factor-dependent hematopoietic progenitor cell line, TF-1. TF-1 cells promptly resulted in death after withdrawal of growth factors. However, the viable cell number increased when TF-1 cells were cultured on anti-CD43 monoclonal antibody-coated plates. In this case, sustained activation of protein tyrosine kinase Syk and extracellular signal-regulated kinase (Erk) 1/2 were detected. Overexpression of exogenous Syk on TF-1 cells by the adenovirus vector system induced enhancement of the cell proliferation accompanied with enhancement of the Erk activation by a dominant-positive effect. The signal transducer and activator of transcription (STAT) 5 seemed not to be associated with the CD43-mediated cell proliferation. These results indicated that adhesion via CD43 induces the proliferation of TF-1 cells in the absence of growth factors in part by Syk-dependent Erk 1/2 signaling. (C) 2001 Academic Press.

Syk is a protein-tyrosine kinase that is widely expressed in haematopoietic cells and involved in coupling activated immunoreceptors to downstream signaling. On the other hand, Syk-deficient mice showed severe petechiae in utero and died shortly after birth. Recently we have shown the expression of Syk in endothelial cells and morphological defects of these cells in embryonic Syk-deficient mice. Here we report that both proliferation and migration of human umbilical vein endothelial cells were severely impaired by adenovirus-mediated expression of Syk dominant negative mutants. Furthermore, a close relationship between Syk kinase activity and extracellular signal-regulated kinase activation was suggested. Our results indicate that Syk plays a critical role in endothelial cell functions, including morphogenesis, cell growth, migration, and survival, and contributes to maintaining vascular integrity in vivo. (C) 2001 Academic Press.

The fibroblasts stimulated by cytokines released the chemokine and recruited the infiltrating cells, including eosinophils, that play a key role in the pathogenesis of airway disease. We established the human fibroblast lines showing high Syk expression and the lines showing low Syk expression from pieces of nasal polyp. IL-1 induces the interaction of TNFR-associated factor (TRAF) 6 with IL-1R-associated kinase, which is rapidly recruited to the IL-1R after IL-1 induction, whereas TRAF2 participates in TNF-alpha -signaling. In the present study, we found that Syk played a different role in IL-1- and TNF-alpha -induced chemokine production through a signaling complex involving Syk and TRAF6. Overexpression of wild-type Syk by gene transfer enhanced RANTES production from nasal fibroblasts stimulated with IL-1. The decrease of Syk expression by the administration of Syk antisense inhibited RANTES production in response to IL-1. However, the change of Syk expression did not affect RANTES production by TNF-alpha stimulation. We concluded that Syk is required for the IL-1-induced chemokine production through the association with TRAF-6 in fibroblasts of nasal polyps.

Background: Treatment of many cell types with phorbol esters stimulates phospholipase D (PLD) activity implying regulation of the enzyme by protein kinase C. Studies of the effects of several protein-tyrosine kinase (PTK) inhibitors have suggested that PTK(s) play some roles in the phorbol ester-induced PLD activation, but it remains unclear how and which PTK(s) is involved in this pathway. In this study, we investigated the roles of Syk and other PTKs for the phorbol esters, 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced PLD activation in K562 and DT40 cells. Results: TPA-induced PLD activation was remarkably reduced in both Syk dominant negative mutant K562 cells and Syk deficient DT40 B cells. Mutational analysis further indicated that two major autophosphorylation sites (Tyr-518 and Tyr-519) of Syk are critical for PLD activation. Similarly, TPA-induced PLD activation was reduced in Btk deficient cells, but unaffected in Lyn deficient cells. Finally, in cells deficient in the PLC-gamma2, one of the phosphorylated substrates regulated by Syk and Btk, TPA-induced PLD activation, as well as phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis was remarkably reduced. Conclusions: We demonstrated that the Syk, Btk and PLC-gamma2 pathways are required for TPA-induced PLD activation in DT40 cells.

Syk has been implicated in activated immunoreceptors to downstream signaling events in hematopoietic cells. Here rye report that Syk is expressed in neuron-like cells and involved in neuron-like differentiation of embryonal carcinoma P19 cells. Immunoblot, RT-PCR, and Northern analysis indicated that Syk is expressed in mouse brain, PC12 and P19 cells. In addition, Syk was found to he tyrosine phosphorylated during neuron-like differentiation of P19 cells, Furthermore, adenovirus-mediated overexpression of Syk induced supernumerary neurite formation and extracellular signal-regulated kinase (ERK) activation in P19 cells. These results suggest that Syk plays an important role in signaling steps leading to ERK activation in P19 cells. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Fibroblasts, a rich source of chemokines, interact with eosinophils and play a key role in the pathogenesis of airway disease. RANTES is produced by fibroblasts to attract and activate eosinophils, LPS is known to induce RANTES and cause protein tyrosine phosphorylation, Nonreceptor protein tyrosine kinase Syk is widely expressed and an important role in intracellular signal transduction in hemopoietic cells. In the present study, we examined whether Syk was expressed in a number of primary human nasal polyp tissue-derived fibroblast lines and whether it played some role in cellular function. Syk proteins were expressed in human nasal fibroblasts, but the expression level varied, There were positive correlations between the level of Syk expression and RANTES production induced by LPS, Overexpression of wild-type Syk by gene transfer enhanced RANTES production from nasal fibroblasts stimulated with LPS, The decrease of Syk expression by the administration of Syk antisense inhibited RANTES production. These results suggest that Syk expression affects RANTES production in fibroblasts of nasal polyps.

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. Here we report that Syk is required for the activation of the phosphatidylinositol (PI) 3-kinase-Akt survival pathway in B cells exposed to oxidative stress. Phosphorylation and activation of the serine-threonine kinase Akt were markedly increased in B cells treated with H2O2. In Syk-deficient DT40 cells treated with low doses of H2O2 (10-100 mu M), Akt activation was considerably reduced. Pretreatment with wortmannin, a PI 3-kinase-specific inhibitor, completely blocked the Syk-dependent Akt activation. Following stimulation by low doses of H2O2, a significant increase in PI 3-kinase activity was found in wild-type but not in Syk-deficient cells. These findings suggest that PI 3-kinase mediates Syk-dependent Akt activation pathway, Furthermore, viability of Syk-deficient cells, after exposure to H2O2, was dramatically decreased and caspase-9 activity was greatly increased compared with that of the wild-type cells. These results suggest that Syk is essential for the Akt survival pathway in B cells and enhances cellular resistance to oxidative stress-induced apoptosis.

Four members of collapsin response mediator proteins (CRMPs) are thought to be involved in the semaphorin-induced growth cone collapse during neural development. Here we report the identification of a novel CRMPS associated protein, designated CRAM for CRMP3-associated molecule, that belongs to the unc-33 gene family. The deduced amino acid sequence reveals that the CRAM gene encodes a protein of 563 amino acids, shows 57% identity with dihydropyrimidinase, and shows 50-51% identity with CRMPs. CRAM appears to form a large complex composed of CRMPS and other unidentified proteins in vivo. Indeed, CRAM physically associates with CRMPS when co-expressed in COS-7 cells. The expression of CRAM is brain-specific, is high in fetal and neonatal rat brain, and decreases to very low levels in adult brain. Moreover, CRAM expression is up-regulated during neuronal differentiation of embryonal carcinoma P19 and PC12 cells. Finally, immunoprecipitation analysis of rat brain extracts shows that CRAM is co-immunoprecipitated with proteins that contain protein-tyrosine kinase activity, Taken together, our results suggest that CRAM, which interacts with CRMP3 and protein-tyrosine kinase(s), is a new member of an emerging family of molecules that potentially mediate signals involved in the guidance and outgrowth of axons.

Syk plays a crucial role in the transduction of oxidative stress signaling:. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H2O2 treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma 2 phosphorylation, inositol 1,4,5-triphosphate (IP3) generation, Ca2+ release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma 2 was markedly induced in oxidative stress. the enhanced phosphorylation of mSH2(N) Syk and PLC-gamma 2, however did not link to Ca2+ mobilization from intracellular pools and IP3 generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.

Syk protein-tyrosine kinase (PTK) has been implicated in a variety of hematopoietic cell responses including immunoreceptor signaling. However, so far, there has been no evidence of the expression of Syk or Syk-related PTK in non-hematopoietic tissues. In this study, we have purified from blood cell-depleted rat liver a 72-kDa cytoplasmic PTK which shows cross-reactivity with anti-Syk antibody, Partial amino acid sequence analysis revealed that this 72-kDa PTK is identical to Syk, Immunohistochemical and RT-PCR analyses demonstrated that Syk is expressed in human hepatocytes and two rat liver-derived cell lines, JTC-27 and RLC-16. Furthermore, Syk is significantly tyrosine-phosphorylated in response to angiotensin II in JTC-27 cells, and angiotensin II-induced MAP kinase activation is blocked by the treatment of cells with a Syk-selective inhibitor piceatannol. These results suggest that Syk plays an important role in signaling events of hepatocytes, such as signaling steps leading to MAP kinase activation by G-protein-coupled receptors, This is the first report of the expression of Syk in non-hematopoietic tissue.

Phospholipase D (PLD) has been proposed to play a key role in the signal transduction of cellular responses to various extracellular signals. Herein me provide biochemical and genetic evidence that cross-linking of the B cell receptor (BCR) induces rapid activation of PLD through a Syk-, Btk- and phospholipase C (PLC)-gamma 2-dependent pathway in DT40 cells. Activation of PLD upon BCR engagement is completely blocked in Syk- or Btk-deficient cells, but unaffected in Lyn-deficient cells. Furthermore, in PLC- gamma 2-deficient cells, BCR engagement failed to activate PLD. These results demonstrate that Syk, Btk and PLC-gamma 2 are essential for BCR-induced PLD activation. (C) 1999 Federation of European Biochemical Societies.

ZAP-70 is another member of Syk family tyrosine kinases which plays an essential role in growth, differentiation, and function of T lymphocytes. In this study, we report the specific expression of a 66 kDa tyrosine kinase that is specifically cross-reacted with anti-ZAP-70 antibodies in the developing neurons. By immunoblot and immunoprecipitation assay using various anti-ZAP-70 antibodies, a 66 kDa tyrosine kinase was detected in lysates from rat brain. During the development of rat brain, expression levels of this 66 kDa tyrosine kinase were highest around 3 weeks after birth and decreased thereafter in the adult. In addition, immunoblot analysis demonstrated that this 66 kDa tyrosine kinase was expressed almost solely in the nervous system. These results suggest that this ZAP-70-related tyrosine kinase may play an important role in growth and differentiation in the developing neurons. Our observations will provide the clue to approach the regulatory system common to neurogenesis and immune response. (C) 1998 Academic Press.

Two signaling molecules have been implicated in the modulation of immune receptor activation by inhibitory coreceptors: an inositol polyphosphate 5'-phosphatase, SHIP, and a tyrosine phosphatase, SHP-1. To address the necessity, interaction, or redundancy of these signaling molecules, we have generated SHP-1-or SHIP-deficient B cell lines and determined their ability to mediate inhibitory signaling. Two distinct classes of inhibitory responses are defined, mediated by the selective recruitment of SHP-1 or SHIP. The Fc gamma RIIB class of inhibitory signaling is dependent on SHIP and not SHP-1; conversely, the KIR class requires SHP-1 and not SHIP. The consequence of this selective recruitment by inhibitory receptor engagement is seen in BCR-triggered apoptosis. SHP-1-mediated inhibitory signaling blocks apoptosis, while SHIP recruitment attenuates a proapoptotic signal initiated by Fc gamma RIIB.

Thrombin and epinephrine in combination exert synergistic effects on platelet activation. On the other hand, tyrosine phosphorylation and activation of tyrosine kinases including Syk have been shown to play a critical role in the induction of platelet responses to thrombin stimulation. This study investigated the role of tyrosine phosphorylation and Syk activation in the synergistic mechanisms between thrombin and epinephrine. Although epinephrine alone (4 mu M) slightly induced protein-tyrosine phosphorylation and Syk activation, the presence of epinephrine caused a shift to the left in the dose-dependence of thrombin (0.01-0.5 U/ml)-induced tyrosine phosphorylation and Syk activation, as well as platelet aggregation. Phenoxybenzamine, an alpha-adrenoceptor antagonist, canceled this potentiation by epinephrine. Since platelets dominantly express alpha(2)-adrenoceptor, this result indicates that epinephrine acts through the occupancy of alpha(2)-adrenoceptor. Furthermore, pretreatment with a tyrosine kinase inhibitor, genistein, or a cAMP-elevating agent, prostacyclin (PGI(2)), significantly reduced these synergistic effects of epinephrine. Taken together, our results suggested that the potentiation by epinephrine may be mediated via enhancement of tyrosine phosphorylation and Syk activation, in part through a decrease of intracellular cAMP levels.

CD45 is a tyrosine phosphatase that is required for normal B cell receptor (BCR)-mediated signaling. It has been shown that Src-family tyrosine kinases such as Lyn could be a potential substrate for CD45. In vitro studies indicate that activities of Src family tyrosine kinases are regulated by tyrosine phosphorylation; C-terminal phosphorylation is inhibitory, and autophosphorylation is stimulatory. We report here that both autophosphorylation and C-terminal negative regulatory tyrosines of Lyn were hyperphosphorylated in CD45-deficient DT40 B cells, In this mutant cell, BCR-induced protein-tyrosine phosphorylation and calcium mobilization were severely compromised, as seen in Lyn-deficient cells. Consistent with this observation, Lyn activation upon receptor ligation was profoundly decreased in CD45-deficient cells. Taken together, our results suggest that dephosphorylation of tyrosine residues at both autophosphorylation and negative regulatory sites is mediated by CD45 in vivo, and that dephosphorylation of C-terminal tyrosine is a prerequisite for participation of Lyn in BCR signaling.

Platelet glycoprotein (GP) VI is a so-far uncharacterized 62-kDa membrane protein, whose deficiency results in selective impairment in collagen-induced platelet aggregation, Our group previously reported a human polyclonal antibody (anti-p62 IgG) that induces activation of normal, but not of GPVI-deficient, platelets in an Fc-independent manner. The F(ab')(2) fragments of this antibody (F(ab')(2)-anti-p62) stimulated tyrosine phosphorylation of numerous proteins, which was not prevented even in the presence of cAMP-increasing agents such as prostacyclin. Pretreatment of platelets with the protein-tyrosine kinase (PTK) inhibitor tyrphostin A47 completely abolished F(ab')(2)-anti-p62-induced platelet aggregation in parallel with dose-dependent inhibition of protein-tyrosine phosphorylation, indicating an essential requirement of PTK activity for generating GPVI-mediated signaling. We found that two cytosolic PTKs, c-Src and Syk, became rapidly activated in response to F(ab')(2)-anti-p62 in a way insensitive to elevation of cAMP, In contrast, in the presence of prostacyclin, F(ab')(2)-anti-p62 did not stimulate tyrosine phosphorylation of the focal adhesion kinase, cAMP-insensitive activation of c-Src and Syk was also observed in collagen-but not thrombin-stimulated platelets, Moreover, either F(ab')(2)-anti-p62 or collagen stimulated cAMP-insensitive tyrosine phosphorylation of phospholipase C-gamma 2. These results indicate that the receptor-mediated activation of several PTKs in platelets is regulated through a cAMP-sensitive or -insensitive mechanism depending on the nature of each stimulus, and also suggest that GPVI engagement is coupled to cAMP-insensitive activation of c-Src and Syk accompanied by tyrosine phosphorylation of numerous substrates including phospholipase C-gamma 2 in a manner similar to collagen stimulation.

Radiation-induced biochemical events that mediate the intracellular signal transduction leading to cell apoptosis are largely unknown, Limited evidence suggests the possible involvement of one or more protein-tyrosine kinases (PTKs) in radiation-induced cellular responses, including apoptosis, However, so far, a PTK(s) responsible for the radiation-induced tyrosine phosphorylation of cellular substrates has not been identified and the role of the PTK(s) in the radiation-induced apoptosis remains unclear. To examine the roles of Syk and Lyn in radiation-induced signal transduction and radiation-induced apoptosis, we analyzed Syk-deficient or Lyn-deficient DT40 B cells along with wild-type cells following radiation, When DT40 B cells were exposed to radiation, the activity of Syk kinase dramatically increased and reached a maximum with 0.25 Grays (Gy) (15 s), and then decreased, whereas Lyn kinase activity increased and reached a maximum with a dose of 1.00 Gy (1 min), However, an apparent difference was not observed in radiation-induced apoptosis among wild-type, Syk-deficient, and Lyn-deficient DT40 B cells, These results indicate that Syk and Lyn kinases are involved in radiation-induced signal transduction, with different kinetics, In addition, our results revealed that functional inactivation of Syk or Lyn alone is not sufficient to prevent radiation-induced apoptosis, Thus, it is suggested that the activation of Syk or Lyn kinase alone may be sufficient to mediate the radiation-induced apoptosis in DT40 B cells, or both kinases may not be required for this biological process.

NNKY 1-19, anti-CD9 monoclonal antibody (MoAb), induced protein tyrosine phosphorylation of 125-, 97-, 75-, 64-, and 40-kDa proteins in human platelets, whereas F(ab')(2) fragments of NNKY 1-19 did not, suggesting that the stimulation of Fc gamma II receptors is required for the induction of protein tyrosine phosphorylation. Tyrosine-phosphorylated proteins of 97 and 125 kDa were associated with aggregation, while NNKY 1-19-induced protein tyrosine phosphorylation was completely inhibited by prostaglandin I-2 (PGI(2)). The activity of p72(syk) was assessed in immunoprecipitation kinase assays to determine at which step the signal transduction pathway leading to protein tyrosine phosphorylation was suspended. NNKY 1-19 induced a rapid and transient increase in the p72(syk)-associated tyrosine kinase activity that peaked at 10 s and subsided to the original level a min after stimulation. Coinciding with this time course, p60(c-src) transiently associated with p72(syk). In platelets preexposed to GRGDS peptides or PGI(2), NNKY 1-19 also increased the p72(syk)-associated tyrosine kinase activity and led to the association of p60(c-src) with p72(syk), However, in contrast to the control without any inhibitor, the elevated tyrosine kinase activity and the associated state of the two tyrosine kinases persisted as long as 5 min after stimulation, F(ab')(2) fragments of NNKY 1-19 induced changes similar to those observed with the effects of GRGDS peptides or PGI(2) treatment on intact IgG NNKY 1-19 stimulation. F(ab')(2) fragments of another CD9 MoAb, PMA2, had effects on p72(syk) essentially similar to those of NNKY 1-19. These findings suggest that the binding of anti-CDS MoAb to CD9 on the platelet membrane per se induces an increase in the p72(syk)-associated tyrosine kinase activity but that Fc gamma II receptor-mediated signal(s) is required for the full activation of platelets and the appearance of tyrosine-phosphorylated proteins. The elevated intracellular cAMP level induced by PGI(2) acts at a step distal to the activation of p72(syk) and inhibited the signal transduction pathway leading to protein tyrosine phosphorylation and aggregation. p72(syk) activation occurs in the absence of aggregation, but aggregation appears to reduce the elevated p72(syk) activity induced by anti-CDS MoAb.

Thrombin stimulation induces a dramatic increase in the activity of p72(syk) in platelets. We have found that activated p72(syk), which is phosphorylated on tyrosine residue(s), translocates from the Triton X-100-soluble fraction to the Triton X-100-insoluble, cytoskeleton-rich fraction after thrombin stimulation. In addition, the redistribution of p72(syk) from the 100,000 x g Triton X-soluble fraction and the membrane skeleton was found to correlate with an increased level of p72(syk) in the cytoskeleton. Furthermore, the early phase of p72(syk) translocation (within 60 s) was significantly inhibited with cytochalasin D, whereas the late phase of p72(syk) translocation (after 90 s) was completely inhibited with RGDS tetrapeptide treatment. These results suggest that translocation of the activated p72(syk) to the cytoskeleton correlates with different phases of the platelet activation process through actin polymerization and glyco protein IIb/IIIa-fibrinogen-mediated aggregation of platelets and, hence, may have a regulatory role in tyrosine phosphorylation of platelets.

It has been demonstrated that activation of platelets by platelet-activating factor (PAF) results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. We report here that p(72syk) is a potential candidate for the protein-tyrosine phosphorylation following PAF stimulation in porcine platelets. Immunoprecipitation kinase assay revealed that PAF stimulation resulted in a rapid activation of p72(syk) which peaked at 10 s. The level of activation was found to be dose dependent and could be completely inhibited by the PAF receptor antagonist, CV3988. Phosphorylation at the tyrosine residues of p72(syk) coincided with activation of p72(syk). Pretreatment of platelets with aspirin and apyrase did not affect PAF induced activation of p72(syk). Furthermore, genistein, a potent protein-tyrosine-kinase inhibitor, diminished PAF-induced p72(syk) activation and Ca2+ mobilization as well as platelet aggregation. These results suggest that p72(syk) may play a critical role in PAF-induced aggregation, possibly through regulation of Ca2+ mobilization.

Previous studies have demonstrated that activation of platelets by collagen results in a dramatic increase in tyrosine phosphorylation of several cellular proteins, including pp125(FAK), through the interaction of collagen with integrin alpha(2) beta(1) (GP Ia-IIa). In this study, we report that p72(syk) is a potential candidate for the protein-tyrosine phosphorylation event following collagen stimulation in porcine platelets. Washed platelets were stimulated with collagen and the activation of p72(syk) was assessed in an immunoprecipitation kinase assay. The activity of p72(syk) increased within 1 min, reached a maximum at 5 min after stimulation by collagen, and the phosphorylation at tyrosine residues of p72(syk) in platelets also occurred in the same time course as the activation of p72(syk). Prior treatment of platelets with cytochalasin D to inhibit actin polymerization, or with aspirin and apyrase to inhibit the secondary reaction, or EGTA and the acetoxymethyl ester of 5,5'-dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid to chelate both extracellular and intracellular Ca2+, did not affect the activation of p72(syk) induced by collagen. Furthermore, herbimycin A, a potent protein tyrosine-kinase inhibitor, was capable of reducing collagen-evoked p72(syk) activation, Ca2+ mobilization and platelet aggregation. These results suggest that upon stimulation by collagen p72(syk) is physically activated by a process that is independent of the effects of Ca2+, ADP, and actin polymerization, and may participate in the regulation of Ca2+ mobilization mediated by collagen in platelets.

We previously demonstrated that thrombin-induced activation of p72(syk) was independent of intracellular Ca2+ elevation in platelets. However, our previous studies also demonstrated that activation of platelets by ionophore A23187 results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. In the present study, we investigated the effect of Ca2+ elevation on the activity of p72(syk). When washed porcine platelets were stimulated with ionophore A23187 and the activity of p72(syk) was assessed by means of an immunoprecipitation kinase assay, A23187 caused a time- and dose-dependent increase in the specific activity of p72(syk). In addition, pretreatment of platelets with both aspirin and ADP scavengers or chelation of extracellular Ca2+ by EGTA had no effect on the A23187-induced activation of p72(syk). These results indicate that A23187-induced activation of p72(syk) is independent of the formation of endoperoxide/thromboxage A(2), released ADP and extracellular Ca2+, suggesting the existence of a novel pathway for activation of p72(syk). Furthermore, evidence is presented which indicates a synergistic effect of A23187 and thrombin on the activation of p72(syk), and an inhibitory effect of pretreatment with phorbol 12-myristate 13-acetate, a protein kinase C activator, on the activation of p72(syk) induced by either A23187 or thrombin.

We have previously reported that a non-receptor-type protein-tyrosine kinase p72(syk), exists in both membrane and cytosolic fractions in porcine platelets and is activated after thrombin stimulation. To facilitate the understanding of the function of p72(syk), we have investigated the topological features, kinase activities and the interaction with another signal-transducing molecule, namely phosphatidylinositol 3-kinase, during platelet activation. Membrane and cytosolic fractions were separated from thrombin-treated porcine platelets, and the amount of p72(syk) was quantified by the immunoblot technique or the kinase activity of each fraction was determined by an immunoprecipitation kinase assay. After stimulation by thrombin, cytosolic p72(syk) rapidly translocated to the membrane fraction within 10 s and there was also a significant increase in the amount of p72(syk) in the cytoskeletal fraction. The autophosphorylation activity of membrane-associated p72(syk) significantly increased approximately tenfold and reached a maximum at 10 s; the activity subsequently decreased to almost the basal level within 120 s. For similar time courses, association of p72(syk) with phosphatidylinositol 3-kinase and tyrosine phosphorylation of p72(syk) were observed. These results suggest that translocation, activation, and association of p72(syk) with transducing molecules such as phosphatidylinositol 3-kinase, events which occur during platelet activation, may participate in early signal-transduction events.

In the previous study, we reported purification and characterization of cytosolic protein-tyrosine kinase, CPTK71, from bovine platelets (Nakamura, S., Yanagi, S. and Yamamura, H. (1988) Eur. J. Biochem. 174, 471-477). In the present study, we have investigated the relationship between CPTK71 and p72(syk) which was purified from cytosolic fraction of porcine spleen and sequenced from porcine cDNA library. The elution patterns of CPTK71 activity in each purification step closely corresponded with the pattern of immunoreactivity with p72(syk). Immunoprecipitation study revealed that specific antibodies against p72(syk) precipitated the activity of both phosphorylating [Val(5)]angiotensin II and autophosphorylation of highly purified CPTK71. In addition, the same result was obtained using another anti-p72(syk) antibody which recognized different amino acid sequence of 72(sky). From these results, we conclude that CPTK71 is identical or closely related to p72(syk). (C) 1994 Academic Press, Inc. .

We have succeeded in purifying p72(syk), a non-receptor-type protein-tyrosine kinase carrying high susceptibility to proteolysis [Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T, Nakamura, S. and Yamamura, H. (1991) J. Biol. Chem. 266, 15790-15796] from porcine spleen. The purification procedure involves a sequential column chromatography, following extraction with 0.5 M NaCl from spleen homogenate, on phosphocellulose, Sephacryl S-200, heparin-Sepharose CL-6B, Mono Q and Mono S. SDS/PAGE of the final purified sample revealed a 72-kDa protein band with about 95% purity and immunodepletion analysis showed immunological cross-reactivity with anti-p72(syk) antibody which does not recognize ZAP-70. It was purified approximately 3000-fold with an overall yield of 0.54% according to [Val5]angiotensin II phosphorylation activity and the specific activity of the final sample (30 nmol phosphate min(-1) mg protein(-1)) was relatively lower than that of the 40-kDa kinase, a catalytic fragment of p72(syk) which lacks two src homology regions 2 domains. The p72(syk) had an autophosphorylation activity that was performed by intramolecular catalysis accompanied by a phosphate exchange reaction, and could efficiently phosphorylate tubulin, myelin basic protein and H2B histone. Employing [Val5]angiotensin II as a substrate, the apparent K-m value for the peptide was 0.91 mM and that for ATP was 0.48 mu M. Mn2+, Mg2+ and Co2+ were effective divalent cations and optimum pH was around 8.0-8.5 for the expression of the activity. These results suggest that the purified p72(syk) may exist as a less active form compared with the 40-kDa kinase and that the part of p72(syk) containing two src homology region 2 domains may participate in the regulation of its activity though the enzymic character is quite similar to that of the 40-kDa kinase.

Thrombin dramatically activated p72(syk) in a time- and dose-dependent fashion in extracts of resting porcine platelets in the presence of EDTA. Separation analysis using Sephacryl S-300 column chromatography has demonstrated that p72(syk) may exist as large (complex) and small (monomer) forms in resting platelets, and activation of p72(syk) was only observed in the fraction of large form. Pretreatment with ATP scavenger, GDP beta S and protein phosphatase inhibitors had no effect on this activation. Furthermore, washed immune-precipitates of large form p72(syk) were also activated by thrombin or fibrinogen. These results suggest that p72(syk) may associate with thrombin receptor or other agonist receptors and there may be a novel activation mechanism of non-receptor type protein-tyrosine kinase, which does not require the modification by other protein kinases, protein phosphatases and GTP binding proteins. (C) 1994 Academic Press, Inc.

Activation of platelets by thrombin results in a dramatic increase in tyrosine phosphorylation on multiple cellular proteins (Ferrell, J. E., and Martin, G. S. (1988) Mol. Cell. Biol. 8, 3603-3610; Golden, A., and Brugge, J. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 901-905; Nakamura, S., and Yamamura, H. (1989) J. Biol. Chem. 264, 7089-7091). However, none of the responsible protein-tyrosine kinase has been reported so far. We report here that p72syk, one of the non-receptor-type protein-tyrosine kinases, is activated following thrombin stimulation in blood platelets. Washed porcine platelets were stimulated by thrombin, and the activation of p72syk was assessed in an immunoprecipitation kinase assay. The activity of p72syk increased within 5 s, reached a maximum at 10 s, and decreased to a basal level within 60 s after 0.5 unit/ml thrombin stimulation. The amount of immunoprecipitated p72syk was not altered throughout the time course. This activation was greatly enhanced in a dose-dependent manner and was completely canceled by the pretreatment of platelet suspension with hirudin, a specific antagonist of thrombin. In the Ca2+-depleted condition both extra- and intracellularly, the activation of p72syk was still persistent; in contrast, the deactivation process was completely abrogated even at 120 s after thrombin stimulation. In addition, the replenishment of Ca2+ resulted in a similar deactivation pattern as seen in the Ca2+-rich condition. Furthermore, this deactivation was also canceled by the pretreatment of platelets with W7, a calmodulin antagonist, as well as ML9, a myosin-light-chain kinase inhibitor. These results indicate that p72syk can be a responsible enzyme to the protein-tyrosine phosphorylation events following the platelet activation by thrombin and may be negatively regulated by Ca2+ in a calmodulin-dependent manner, inter alia myosin light-chain kinase, in thrombin-stimulated platelets.

We have investigated the involvement of protein-tyrosine kinases in thrombin-induced aggregation of human platelets, using ST638 and genistein which are known inhibitors of protein-tyrosine kinase. Preincubation of platelets with 50-mu-M of ST638 or 25-mu-g/ml of genistein completely blocked the platelet aggregation induced with 0.05 unit/ml of thrombin. The increase of protein-tyrosine phosphorylation bands (135-, 124-, 76-, 64-, and 60-kDa) induced with thrombin was also inhibited by these inhibitors in a dose-dependent manner. These inhibitors also blocked the platelet aggregation and protein-tyrosine phosphorylation induced with thrombin in aspirin-treated platelets. Increase of the intracellular Ca2+ concentration induced by thrombin was also inhibited by higher concentrations of genistein. These results suggest that the protein-tyrosine phosphorylation plays a certain role in platelet activation having some relation to the intracellular Ca2+ concentration.