Shigeru Yanagi

Deletion of SHIP or SHP-1 Reveals Two Distinct Pathways for Inhibitory Signaling

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 gammaRIIB 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 gammaRIIB.

Involvement of Fes/Fps tyrosine kinase in semaphorin3A signaling

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 Is Required for the Activation of Akt Survival Pathway in B Cells Exposed to Oxidative Stress

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 μm), 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.

Protein-tyrosine kinase Syk expressed in human nasal fibroblasts and its effect on RANTES production.

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.

IL-1 Induced Chemokine Production Through the Association of Syk with TNF Receptor-Associated Factor-6 in Nasal Fibroblast Lines

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-α-signaling. In the present study, we found that Syk played a different role in IL-1- and TNF-α-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-α 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.

Syk protein-tyrosine kinase is involved in neuron-like differentiation of embryonal carcinoma P19 cells

Syk has been implicated in activated immunoreceptors to downstream signaling events in hematopoietic cells. Here we 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 be 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.

Helicobacter pylori CagA Containing ITAM‐Like Sequences Localized to Lipid Rafts Negatively Regulates VacA‐Induced Signaling In Vivo

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.

Syk is required for p38 activation and G2/M arrest in B cells exposed to oxidative stress.

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 (H(2)O(2)) stimulation. Syk is required for p38 activation following stimulation with 10-100 microM H(2)O(2), but not with 1 mM H(2)O(2). H(2)O(2)-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 H(2)O(2) stimulation. Although stimulation with 20-100 microM H(2)O(2) induces cellular apoptosis in B cells, pretreatment with SB203580, a p38-specific inhibitor, has no effect on H(2)O(2)-induced apoptosis. Flow cytometric analysis reveals that B cells exposed to 10-20 microM H(2)O(2) exhibit cell-cycle profile of G2/M arrest, and pretreatment with SB203580 inhibits only a little H(2)O(2)-induced G2/M arrest. On the other hand, Syk-deficient cells show no induction of G2/M arrest following H(2)O(2) 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.

Cross-linking of the B cell receptor induces activation of phospholipase D through Syk, Btk and phospholipase C-γ2

Phospholipase D (PLD) has been proposed to play a key role in the signal transduction of cellular responses to various extracellular signals. Herein we 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)‐γ2‐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‐γ2‐deficient cells, BCR engagement failed to activate PLD. These results demonstrate that Syk, Btk and PLC‐γ2 are essential for BCR‐induced PLD activation.

Requirement of Syk-phospholipase C-gamma2 pathway for phorbol ester-induced phospholipase D activation in DT40 cells.

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.