Reactivation of oxidized PTP1B and PTEN by thioredoxin 1.

The transient inactivation of protein phosphatases contributes to the efficiency and temporal control of kinase-dependent signal transduction. In particular, members of the protein tyrosine phosphatase family are known to undergo reversible oxidation of their active site cysteine. The thiol oxidation step requires activation of colocalized NADPH oxidases and is mediated by locally produced reactive oxygen species, in particular H2 O2 . How oxidized phosphatases are returned to the reduced active state is less well studied. Both major thiol reductive systems, the thioredoxin and the glutathione systems, have been implicated in the reactivation of phosphatases. Here, we show that the protein tyrosine phosphatase PTP1B and the dual-specificity phosphatase PTEN are preferentially reactivated by the thioredoxin system. We show that inducible depletion of thioredoxin 1(TRX1) slows PTEN reactivation in intact living cells. Finally, using a mechanism-based trapping approach, we demonstrate direct thiol disulphide exchange between the active sites of thioredoxin and either phosphatase. The application of thioredoxin trapping mutants represents a complementary approach to direct assays of PTP oxidation in elucidating the significance of redox regulation of PTP function in the control of cell signaling. STRUCTURED DIGITAL ABSTRACT: TRX1 physically interacts with PTP1B by anti tag coimmunoprecipitation (1, 2).

The SH2-domain of SHIP1 interacts with the SHIP1 C-terminus: impact on SHIP1/Ig-α interaction.

The SH2-containing inositol 5'-phosphatase, SHIP1, negatively regulates signal transduction from the B cell antigen receptor (BCR). The mode of coupling between SHIP1 and the BCR has not been elucidated so far. In comparison to wild-type cells, B cells expressing a mutant IgD- or IgM-BCR containing a C-terminally truncated Ig-α respond to pervanadate stimulation with markedly reduced tyrosine phosphorylation of SHIP1 and augmented activation of protein kinase B. This indicates that SHIP1 is capable of interacting with the C-terminus of Ig-α. Employing a system of fluorescence resonance energy transfer in S2 cells, we can clearly demonstrate interaction between the SH2-domain of SHIP1 and Ig-α. Furthermore, a fluorescently labeled SH2-domain of SHIP1 translocates to the plasma membrane in an Ig-α-dependent manner. Interestingly, whereas the SHIP1 SH2-domain can be pulled-down with phospho-peptides corresponding to the immunoreceptor tyrosine-based activation motif (ITAM) of Ig-α from detergent lysates, no interaction between full-length SHIP1 and the phosphorylated Ig-α ITAM can be observed. Further studies show that the SH2-domain of SHIP1 can bind to the C-terminus of the SHIP1 molecule, most probably by inter- as well as intra-molecular means, and that this interaction regulates the association between different forms of SHIP1 and Ig-α.

In situ kinetic trapping reveals a fingerprint of reversible protein thiol oxidation in the mitochondrial matrix.

Reactive oxygen species (ROS) are released at the mitochondrial inner membrane by the electron transport chain (ETC). Increasing evidence suggests that mitochondrial H2O2 acts as a signaling molecule and participates in the (feedback) regulation of mitochondrial activity and turnover. It seems likely that key mitochondrial components contain redox-sensitive thiols that help to adapt protein function to changes in electron flow. However, the identity of most redox-regulated mitochondrial proteins remains to be defined. Thioredoxin 2 (Trx2) is the major protein-thiol-reducing oxidoreductase in the mitochondrial matrix. We used in situ mechanism-based kinetic trapping to identify disulfide-exchange interactions of Trx2 within functional mitochondria of intact cells. Mass spectrometry successfully identified known and suspected Trx2 target proteins and, in addition, revealed a set of new candidate target proteins. Our results suggest that the mitochondrial protein biosynthesis machinery is a major target of ETC-derived ROS. In particular, we identified mitochondrial methionyl-tRNA synthetase (mtMetRS) as one of the most prominent Trx2 target proteins. We show that an increase in ETC-derived oxidants leads to an increase in mtMetRS oxidation in intact cells. In conclusion, we find that in situ kinetic trapping provides starting points for future functional studies of intramitochondrial redox regulation.

A cysteine-rich receptor-like kinase NCRK and a pathogen-induced protein kinase RBK1 are Rop GTPase interactors.

In plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two-hybrid screening we have found three previously uncharacterized receptor-like protein kinases to be Rop GTPase-interacting molecules: a cysteine-rich receptor kinase, named NCRK, and two receptor-like cytosolic kinases from the Arabidopsis RLCK-VIb family, named RBK1 and RBK2. Uniquely for Rho-family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP-bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro, suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co-localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single-copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK-VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2. We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross-talk between Rop GTPases and specific receptor-like kinases through direct molecular interaction.

Identification of redox-active cell-surface proteins by mechanism-based kinetic trapping.

A number of thiol-dependent oxidoreductases are released from cells and act on the cell surface. Correspondingly, several cell-surface processes appear to depend on catalyzed thiol-disulfide exchange, including integrin activation and the fusion of viral particles with the host membrane. Tumor cells frequently increase the abundance of secreted and cell-surface forms of particular oxidoreductases, and evidence suggests that oxidoreductases released from tumor cells promote growth and contribute to the remodeling of the cellular microenvironment. Few cell-surface or membrane proteins that are targeted by extracellular redox enzymes have been identified. One major reason for this slow progress is the highly transient nature of thiol-disulfide exchange, making its detection by conventional techniques difficult or impossible. Here we describe the application of an activity-based proteomics approach, also known as "mechanism-based kinetic trapping," to identify individual cell-surface target proteins that engage in disulfide exchange with thiol-dependent oxidoreductases. Although we have applied this approach to thioredoxin-1, it should also be applicable to other members of the thioredoxin superfamily whose activity is based on the CXXC active-site motif.

Selective redox regulation of cytokine receptor signaling by extracellular thioredoxin-1.

The thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of Trx1 require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular Trx1 redox activity to cellular responses have not been identified so far. Using a mechanism-based kinetic trapping technique to identify disulfide exchange interactions on the intact surface of living lymphocytes, we found that Trx1 catalytically interacts with a single principal target protein. This target protein was identified as the tumor necrosis factor receptor superfamily member 8 (TNFRSF8/CD30). We demonstrate that the redox interaction is highly specific for both Trx1 and CD30 and that the redox state of CD30 determines its ability to engage the cognate ligand and transduce signals. Furthermore, we confirm that Trx1 affects CD30-dependent changes in lymphocyte effector function. Thus, we conclude that receptor-ligand signaling interactions can be selectively regulated by an extracellular redox catalyst.

Insulin and insulin-like growth factor-1 promote mast cell survival via activation of the phosphatidylinositol-3-kinase pathway.

OBJECTIVE: Mast cells (MCs) play central roles for the onset and development of immediate-type and inflammatory allergic reactions. Since the inverse relationship between atopic disorders and diabetes mellitus has been observed in animals and humans, we investigated the effects of insulin (Ins) on MC signaling and biological function. METHODS: In bone marrow-derived MCs (BMMCs) from wild-type as well as SHIP-deficient mice Ins as well as insulin-like growth factor-1 (IGF-I)-triggered intracellular signaling events and MC effector functions were studied. RESULTS: We found that the addition of either Ins or IGF-1 to BMMCs triggers the phosphorylation of protein kinase B (PKB) and p38 kinase but not extracellular signal-regulated kinase (Erk). We also found that Ins/IGF-1 stimulates the tyrosine phosphorylation of SHIP1 and, in keeping with this, Ins/IGF-1-induced PKB phosphorylation is higher in SHIP1-/- BMMCs and is inhibited in SHIP+/+ as well as SHIP1-/- BMMCs with inhibitors of phosphatidylinositol-3-kinase (PI3K). Ins/IGF-1, like antigen (Ag), also stimulates the Rac-dependent activation of PAK as well as the production of hydrogen peroxide (H2O2). To elucidate the role of Ins and IGF-1 in MC biology, we studied their effects on Ag-mediated degranulation and MC survival. Although both only slightly enhanced Ag-mediated degranulation, they significantly promoted MC survival in the absence of IL-3 in a PI3K-dependent manner. CONCLUSION: The promotion of BMMC survival by induction of Ins/IGF-1 signaling may, in part, be responsible for the inverse correlation observed between atopic disorders and diabetes mellitus.