Junyi Ding

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.

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.

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.

Role of BLNK in Oxidative Stress Signaling in B Cells

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.

A deficiency in Syk enhances ceramide-induced apoptosis in DT40 lymphoma B cells

Syk deficiency significantly enhanced ceramide‐induced apoptosis. Ectopic expression of wild‐type or kinase‐inactive Syk rendered Syk‐negative cells resistant to ceramide‐induced apoptosis. Furthermore, ceramide could not activate Syk, indicating that Syk protected DT40 cells from ceramide‐induced apoptosis, via a mechanism independent of its activity. In addition, a deficiency in Lyn also resulted in the cells becoming susceptible to ceramide‐induced apoptosis. However, no difference of Ara‐C‐induced apoptosis between wild‐type and mutant cells was observed. c‐Jun N‐terminal kinases appeared not to be important in mediating the enhanced apoptosis, as they were still activated in mutant cells following ceramide treatment.