Mei-yi Pu

Nitric Oxide Controls Src Kinase Activity through a Sulfhydryl Group Modification-mediated Tyr-527-independent and Tyr-416-linked Mechanism

c-Src kinase was activated when either murine NIH3T3 fibroblast cells or immunoprecipitated c-Src proteins were treated with nitric oxide generator,S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside. Nitric oxide (NO) scavenger hemoglobin and N2O3 scavenger homocysteine abolished the SNAP-mediated c-Src kinase activation. Phosphoamino acid analysis and peptide mapping of in vitro labeled phospho-c-Src proteins revealed that SNAP promoted the autophosphorylation at tyrosine, which preferentially took place at Tyr-416. Peptide mapping of in vivo labeled c-Src kinase excluded the involvement of phospho-Tyr-527 dephosphorylation in the SNAP-mediated activation mechanism. Correspondingly, protein-tyrosine phosphatase inhibitor Na3VO4 did not abolish the SNAP-mediated activation of Src kinase, and the constitutively activated v-Src kinase was also further up-regulated in activity by SNAP. SNAP, however, failed to up-regulate the kinase activity of Phe-416 mutant v-Src. 2-Mercaptoethanol or dithiothreitol, which should disrupt N2O3-mediated S-nitrosylation and subsequent formation of the S-S bond, abolished the up-regulated catalytic activity, and the activity was regained after re-exposing the enzyme to SNAP. Exposure of Src kinase to SNAP promoted both autophosphorylation and S-S bond-mediated aggregation of the kinase molecules, demonstrating a linkage between the two events. These results suggest that the NO/N2O3-provokedS-nitrosylation/S-S bond formation destabilizes the Src structure for Tyr-416 autophosphorylation-associated activation bypassing the Tyr-527-linked regulation.

Redox-linked ligand-independent cell surface triggering for extensive protein tyrosine phosphorylation

Exposure of lymphocytes to 0.2–2 mM HgCl2, a thiol‐reactive heavy metal, induced extensive tyrosine phosphorylation of multiple cellular proteins. The phosphorylation started as quickly as 5 s after exposure to HgCl2, and was irreversible. Another 3 thiol‐reactive chemicals also displayed similar, though less marked, actions, whereas dithiothreitol, a reducing agent, antagonized the HgCl2 action. The demonstrated new action of HgCl2 indispensably required membrane‐intact cells as a target. Whereas exposure of lymphocytes to > 0.2 mM HgCl2 caused rapid cell death, 0.01–0.1 mM HgCl2 affected the cells so as to accelerate their c‐fos transcription. These results suggest a novel redox‐linked mechanism of cell surface triggering of intracellular protein kinase activity, which is independent of receptor‐ligand interactions.

Cell type-oriented differential modulatory actions of saikosaponin-d on growth responses and DNA fragmentation of lymphocytes triggered by receptor-mediated and receptor-bypassed pathways

We examined the immunoregulatory action of saikosaponin-d (SSd), which was isolated from the root of Bupleurum talcatum L. and had a steroid-like structure, on murine thymocytes, and compared the action with that on spleen cells. Constitutive DNA synthesis or the growth response stimulated with anti-CD3mAb of thymocytes were down-regulated by 3 micrograms/ml SSd, whereas with spleen cells these were up-regulated by the same concentration of SSd. On the other hand, 3 micrograms/ml of SSd greatly up-regulated the growth response and interleukin 2 (IL-2)/interleukin 4 (IL-4) production induced through a receptor-bypassed pathway by calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA) in thymocytes, whereas it only slightly up-regulated them in spleen cells. Moreover, the same concentration of SSd inhibited DNA fragmentation in thymocytes induced by A23187 or PMA. These results suggest a unique cell type-dependent immuno-modulatory action of SSd.

Mercuric chloride mediates a protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase which is independent of the phosphorylation / dephosphorylation of a carboxyl terminal tyrosine

Little is known about the regulatory mechanism of c‐Src kinase in cells except the suggested regulation through phosphorylation and dephosphorylation of its carboxyl terminal tyrosine residue (Y527). We here demonstrated that exposure of NIH3T3 cells to mercuric chloride (HgCl2) induces both aggregation and activation of Src kinase protein through a redox‐linked mechanism. The aggregation of Src proteins was suggested to be induced by the sulfhydryl groups‐to‐Hg2+ reaction‐mediated polymerization of cell membrane proteins to which the Src proteins associate noncovalently. The possibility was ruled out that the aggregation occurred secondarily to the promotion of protein tyrosine phosphorylation. Further study revealed that the Src kinase was activated by HgCl2 at least in part independent of the known Csk kinase‐linked or Y527‐phosphorylation/dephosphorylation‐mediated control. Correspondingly, CNBr cleavage mapping of phosphopeptides for autophosphorylated c‐Src protein demonstrated selective promotion of phosphorylation at Y416 in HgCl2‐treated cells without obvious change in the phosphorylation level at Y527. These results suggest a unique protein sulfhydryl modification‐based pathway of signal transduction for activating Src kinase in NIH3T3 cells.

Direct evidence of involvement of glycosylphosphatidylinositol-anchored proteins in the heavy metal-mediated signal delivery into T lymphocytes

The biological significance of the action of glycosylphosphatidylinositol (GPI)‐anchored proteins in cell physiology and pathology when stimulated with their natural agonists is not known. Here we provide evidence that GPI‐anchored proteins play a crucial role in the recently defined heavy metal (HgCl2)‐triggered signal delivery to T lymphocytes. Thiol‐reactive HgCl2, a multi‐potent crosslinker of cell membrane proteins, induced heavy aggregation of Thy‐1, a representative GPI‐anchored protein, on murine thymocytes, and delivered a signal to induce heavy tyrosine phosphorylation of cellular proteins. This rather unusual signal delivery by HgCl2 is diminished by the pre‐treatment of cells with phosphatidylinositol‐specific phospholipase C, which partially cleaved GPI‐anchored proteins from the cell surface. Direct evidence for the involvement of GPI or GPI‐anchored proteins in the HgCl2‐mediated signaling is provided by the loss of signaling in a mutant thymoma cell line defective in the phosphatidylinositol glycan‐class A gene (PIG‐A), and its restoration in a transfectant with PIG‐A.

Oncogene-linked in situ immunotherapy of pre-B lymphoma arising in E mu/ret transgenic mice.

We attempted to induce anti-tumour immunity for rejecting pre-B lymphoma derived from E mu/ret transgenic mice (TGM). We established pre-B-lymphoma cell lines of C57BL/6 x Balb/c background (H-2b/d) into which H-2k alloantigen and C3H background were introduced (retL1-6 and retL6-6), and we inoculated BCF1 mice with these immunising tumour cells. After these tumours were rejected by alloantigen (H-2k/C3H background)-specific effector cells, the mice were challenged with the pre-B-lymphoma cell line derived from the original E mu/ret TGM (ret0-2). All non-immunised control mice died within 80 days, whereas half the immunised mice survived for over 300 days. The immunity was also effective against primary pre-B-lymphoma cells from E mu/ret TGM and the ret-driven melanoma cell line (MEL-ret), but not against the pre-B-lymphoma cell line from E mu/myc TGM. This immunity was at least in part mediated by cell-mediated cytotoxicity that was specific to the ret oncogene product or ret-regulated antigen. Next we immunised E mu/ret TGM by inoculating them with retL6-6 cells once every 2 weeks beginning at the age of 1 month. Interestingly, this immunisation enabled the TGM to survive longer than the non-immunised control group (P < 0.05). Moreover, 2 of 11 transgenic mice receiving such immunisation were free from both macroscopic and microscopic tumours at the time when all of the 12 non-immunised control TGM had died from their tumour. This provides a new model for oncogene-linked immunotherapy research.

T Cell maturation stage-linked heterogeneity of the glycosylphosphatidylinositol membrane anchor of Thy-1

We showed that some of Thy-1 molecules on murine thymocytes are resistant to phosphatidylinositol-specific phospholipase C (PI-PLC) derived from Bacillus thuringiensis. Both immature thymocytes with low CD3 expression and mature thymic T lymphocytes with high CD3 expression carried the PI-PLC-resistant Thy-1, and the PI-PLC-sensitivity of Thy-1 extensively varied among thymocyte subpopulations. In contrast, the same PI-PLC fully hydrolysed the anchor of Thy-1 on peripheral T lymphocytes. When the latter cells were activated with mitogen in vitro, however, some Thy-1 on them became resistant to PI-PLC. We then found that virtually all Thy-1 molecules on thymocytes became sensitive to PI-PLC when they were treated with hydroxylamine that should cleave ester-linked lipids. The result ruled out the possibility that the PI-PLC-resistant Thy-1 had a transmembranous peptide sequence, and suggested the presence of an additional fatty acyl group on the inositol ring of the Thy-1 anchor. In addition, the molecular size of the PI-PLC-resistant membrane-bound Thy-1 was only marginally larger than that of the PI-PLC-sensitive solubilized Thy-1 in detergent-partitioning SDS-PAGE analysis.

Delivery of an accessory signal for cell activation by exogenous phosphatidylinositol-specific phospholipase C.

Digestion of phosphatidylinositol (PI) or glycosylphosphatidylinositol (GPI) anchors of membrane proteins on the external cell surface with exogenous PI‐specific phospholipase C (PIPLC) fromBacillus thuringiensis was shown to transmit a signal into the thymocyte to modulate the TCR/CD3 complex‐induced signal delivery for cell activation. This was demonstrated for very early protein tyrosine phosphorylation, early c‐fos transcription and late DNA synthesis. For this effect preincubation of the cells with PIPLC was required, but there was no evidence of involvement of any soluble products released form the cell surface by PIPLC in the signaling, suggesting a crucial role of the membrane‐bound counterpart (diacylglycerol or diradylglycerol) of the PI/GPI hydrolysate. A possible role for this accessory signal in the microorganism‐linked control of the T cell receptor function is discussed.