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PubMed1998

Signal decay through a reverse phosphorelay in the Arc two-component signal transduction system.

Escherichia coli senses and signals anoxic or low redox conditions in its growth environment by the Arc two-component system. Under those conditions, the tripartite sensor kinase ArcB undergoes autophosphorylation at the expense of ATP and subsequently transphosphorylates its cognate response regulator ArcA through a His --> Asp --> His --> Asp phosphorelay pathway. In this study we used various combinations of wild-type and mutant ArcB domains to analyze in vitro the pathway for signal decay. The results indicate that ArcA-P dephosphorylation does not occur by direct hydrolysis but by transfer of the phosphoryl group to the secondary transmitter and subsequently to the receiver domain of ArcB. This reverse phosphorelay involves both the conserved His-717 of the secondary transmitter domain and the conserved Asp-576 of the receiver domain of ArcB but not the conserved His-292 of its primary transmitter domain. This novel pathway for signal decay may generally apply to signal transduction systems with tripartite sensor kinases.

Bacterial Outer Membrane Proteins↗

PubMed1999

Cross-talk between janus kinase-signal transducer and activator of transcription (JAK-STAT) and peroxisome proliferator-activated receptor-alpha (PPARalpha) signaling pathways. Growth hormone inhibition of pparalpha transcriptional activity mediated by stat5b.

Hepatic peroxisome proliferation induced by structurally diverse non-genotoxic carcinogens is mediated by the nuclear receptor peroxisome proliferator-activated receptor (PPARalpha) and can be inhibited by growth hormone (GH). GH-stimulated Janus kinase-signal transducer and activator of transcription 5b (JAK2/STAT5b) signaling and the PPAR activation pathway were reconstituted in COS-1 cells to investigate the mechanism for this GH inhibitory effect. Activation of STAT5b signaling by either GH or prolactin inhibited, by up to 80-85%, ligand-induced, PPARalpha-dependent reporter gene transcription. GH failed to inhibit 15-deoxy-Delta12, 14-prostaglandin-J2-stimulated gene transcription mediated by an endogenous COS-1 PPAR-related receptor. GH inhibition of PPARalpha activity required GH receptor and STAT5b and was not observed using GH-activated STAT1 in place of STAT5b. GH inhibition was not blocked by the mitogen-activated protein kinase pathway inhibitor PD98059. STAT5b-PPARalpha protein-protein interactions could not be detected by anti-STAT5b supershift analysis of PPARalpha-DNA complexes. The GH inhibitory effect required the tyrosine phosphorylation site (Tyr-699) of STAT5b, an intact STAT5b DNA binding domain, and the presence of a COOH-terminal trans-activation domain. Moreover, GH inhibition was reversed by a COOH-terminal-truncated, dominant-negative STAT5b mutant. STAT5b must thus be nuclear and transcriptionally active to mediate GH inhibition of PPARalpha activity, suggesting an indirect inhibition mechanism, such as competition for an essential PPARalpha coactivator or STAT5b-dependent synthesis of a more proximal PPARalpha inhibitor. The cross-talk between STAT5b and PPARalpha signaling pathways established by these findings provides new insight into the mechanisms of hormonal and cytokine regulation of hepatic peroxisome proliferation.

Animals↗

PubMed2000

Bovine papillomavirus E5 protein induces the formation of signal transduction complexes containing dimeric activated platelet-derived growth factor beta receptor and associated signaling proteins.

The bovine papillomavirus E5 protein binds to the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in constitutive activation of the receptor and cell growth transformation. By subjecting extracts from E5-transformed or PDGF-treated cells to velocity sedimentation in sucrose gradients, activated PDGF beta receptor complexes were separated from monomeric, inactive receptor. Rapidly sedimenting activated complexes contained oligomeric (apparently dimeric), tyrosine-phosphorylated PDGF beta receptor, the E5 protein, and associated cellular signaling proteins including the p85 subunit of phosphoinositol 3'-kinase, phospholipase Cgamma, and Ras-GTPase activating protein. These signaling proteins made the major contribution to the increased sedimentation rate of the activated receptor complexes. Pairwise analysis of components of these complexes indicated that multiple signaling proteins and the E5 protein were simultaneously present in the activated complexes. Our results also showed that the E5 protein and PDGF activated only a small fraction of the total PDGF beta receptor, that not all receptor molecules associated with the E5 protein were tyrosine-phosphorylated, and that signaling proteins could bind to hemiphosphorylated receptor dimers. On the basis of these results, we propose a model for the assembly of multiprotein, activated PDGF beta receptor complexes in response to the E5 protein.

Bovine papillomavirus 1↗

PubMed2000

Divergent signaling pathways requiring discrete calcium signals mediate concurrent activation of two mitogen-activated protein kinases by gonadotropin-releasing hormone.

Receptors coupled to heterotrimeric G proteins are linked to activation of mitogen-activated protein kinases (MAPKs) via receptor- and cell-specific mechanisms. We have demonstrated recently that gonadotropin-releasing hormone (GnRH) receptor occupancy results in activation of extracellular signal-regulated kinase (ERK) through a mechanism requiring calcium influx through L-type calcium channels in alphaT3-1 cells and primary rat gonadotropes. Further studies were undertaken to explore the signaling mechanisms by which the GnRH receptor is coupled to activation of another member of the MAPK family, c-Jun N-terminal kinase (JNK). GnRH induces activation of the JNK cascade in a dose-, time-, and receptor-dependent manner in clonal alphaT3-1 cells and primary rat pituitary gonadotrophs. Coexpression of dominant negative Cdc42 and kinase-defective p21-activated kinase 1 and MAPK kinase 7 with JNK and ERK indicated that specific activation of JNK by GnRH appears to involve these signaling molecules. Unlike ERK activation, GnRH-stimulated JNK activity does not require activation of protein kinase C and is not blocked after chelation of extracellular calcium with EGTA. GnRH-induced JNK activity was reduced after treatment with the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), whereas activation of ERK was not affected. Chelation of intracellular calcium also reduced GnRH-induced activation of JNK in rat pituitary cells in primary culture. GnRH-induced induction and activation of the JNK target c-Jun was inhibited after chelation of intracellular calcium, whereas induction of c-Fos, a known target of ERK, was unaffected. Therefore, although activation of ERK by GnRH requires a specific influx of calcium through L-type calcium channels, JNK activation is independent of extracellular calcium but sensitive to chelation of intracellular calcium. Our results provide novel evidence that GnRH activates two MAPK superfamily members via strikingly divergent signaling pathways with differential sensitivity to activation of protein kinase C and mobilization of discrete pools of calcium.

Animals↗

PubMed2000

Negative regulation of growth hormone receptor/JAK2 signaling by signal regulatory protein alpha.

Signal regulatory proteins (SIRPs) are receptor-like transmembrane proteins, the majority of which contain a cytoplasmic proline-rich region and four cytoplasmic tyrosines that, when phosphorylated, bind SH2 domain-containing protein tyrosine phosphatases (SHP). We demonstrated previously that growth hormone (GH) induces tyrosyl phosphorylation of SIRPalpha and association of SIRPalpha with SHP-2. The GH-activated tyrosine kinase JAK2 associates with and tyrosyl-phosphorylates SIRPalpha1. Here we show that JAK2-SIRPalpha1 association does not require phosphotyrosines in SIRPalpha1 or JAK2 or the proline-rich region of SIRPalpha1. However, when the C-terminal 30 amino acids of SIRPalpha1 containing the proline-rich region and tyrosine 495 are deleted, tyrosyl phosphorylation of SIRPalpha1 by JAK2 and association of SHP-2 with SIRPalpha1 are reduced. GH-dependent tyrosyl phosphorylation of JAK2 is reduced when wild-type SIRPalpha1 compared with SIRPalpha1 lacking the four cytoplasmic tyrosines (SIRP 4YF) is expressed in cells, suggesting that SIRPalpha1 negatively regulates GHR/JAK2 signaling. Consistent with reduced JAK2 activity, overexpression of wild-type SIRPalpha1 but not SIRP 4YF reduces GH-induced phosphorylation of ERKs 1 and 2, STAT3, and STAT5B. These results suggest that SIRPalpha1 is a negative regulator of GH signaling and that the ability of SIRPalpha1 mutants to negatively regulate GHR-JAK2 signaling correlates with their ability to bind SHP-2.

3T3 Cells↗

PubMed2001

Glutathione S-transferase mu modulates the stress-activated signals by suppressing apoptosis signal-regulating kinase 1.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that can activate the c-Jun N-terminal kinase and the p38 signaling pathways. It plays a critical role in cytokine- and stress-induced apoptosis. To further characterize the mechanism of the regulation of the ASK1 signal, we searched for ASK1-interacting proteins employing the yeast two-hybrid method. The yeast two-hybrid assay indicated that mouse glutathione S-transferase Mu 1-1 (mGSTM1-1), an enzyme involved in the metabolism of drugs and xenobiotics, interacted with ASK1. We subsequently confirmed that mGSTM1-1 physically associated with ASK1 both in vivo and in vitro. The in vitro binding assay indicated that the C-terminal portion of mGSTM1-1 and the N-terminal region of ASK1 were crucial for binding one another. Furthermore, mGSTM1-1 suppressed stress-stimulated ASK1 activity in cultured cells. mGSTM1-1 also blocked ASK1 oligomerization. The ASK1 inhibition by mGSTM1-1 occurred independently of the glutathione-conjugating activity of mGSTM1-1. Moreover, mGSTM1-1 repressed ASK1-dependent apoptotic cell death. Taken together, our findings suggest that mGSTM1-1 functions as an endogenous inhibitor of ASK1. This highlights a novel function for mGSTM1-1 insofar as mGSTM1-1 may modulate stress-mediated signals by repressing ASK1, and this activity occurs independently of its well-known catalytic activity in intracellular glutathione metabolism.

Animals↗

PubMed2001

Signaling through the Smad pathway by insulin-like growth factor-binding protein-3 in breast cancer cells. Relationship to transforming growth factor-beta 1 signaling.

We previously demonstrated in T47D cells transfected to express the transforming growth factor-beta receptor type II (TGF-betaRII) that insulin-like growth factor binding protein-3 (IGFBP-3) could stimulate Smad2 and Smad3 phosphorylation, potentiate TGF-beta1-stimulated Smad phosphorylation, and cooperate with exogenous TGF-beta1 in cell growth inhibition (Fanayan, S., Firth, S. M., Butt, A. J., and Baxter, R. C. (2000) J. Biol. Chem. 275, 39146-39151). This study further explores IGFBP-3 signaling through the Smad pathway. Like TGF-beta1, natural and recombinant IGFBP-3 stimulated the time- and dose-dependent phosphorylation of TGF-betaR1 as well as Smad2 and Smad3. This effect required the presence of TGF-betaRII. IGFBP-3 mutated in carboxyl-terminal nuclear localization signal residues retained activity in TGF-betaR1 and Smad phosphorylation, whereas IGFBP-5 was inactive. Immunoneutralization of endogenous TGF-beta1 suggested that TGF-beta1 was not essential for IGFBP-3 stimulation of this pathway, but it increased the effect of IGFBP-3. IGFBP-3, like TGF-beta1, elicited a rapid decline in immunodetectable Smad4 and Smad4.Smad2 complexes. IGFBP-3 and nuclear localization signal mutant IGFBP-3 stimulated the activation of the plasminogen activator inhibitor-1 promoter but was not additive with TGF-beta, suggesting that this end point is not a direct marker of the IGFBP-3 effect on cell proliferation. This study defines a signaling pathway for IGFBP-3 from a cell surface receptor to nuclear transcriptional activity, requiring TGF-betaRII but not dependent on the nuclear translocation of IGFBP-3. The precise mechanism by which IGFBP-3 interacts with the TGF-beta receptor system remains to be established.

Activin Receptors, Type I↗

PubMed2002

Modulation of Smad2-mediated signaling by extracellular signal-regulated kinase.

Components of the transforming growth factor-beta and mitogen-activated protein kinase pathways interact in controlling cell growth and differentiation. We show that phosphorylation of Smad2, a mediator of the activin/transforming growth factor-beta signal, by activated extracellular signal-regulated kinase 1 (ERK1) increases the amount of Smad2 protein and leads to enhanced transcriptional activity. Epidermal growth factor increased phosphorylation of Smad2 in COS7 cells, and Smad2-dependent transcription in a mink lung epithelial cell line, L17, was enhanced by co-transfection of a constitutively active MEK1. In addition, transfection of Smad2 mutants lacking ERK sites resulted in reduced transcription, whereas mutants that mimicked ERK phosphorylation stimulated transcription. The amount of Smad2 protein was increased by transfection with a constitutively active MEK1 and reduced by co-transfection with the ERK phosphatase, HVH2. The elevation of Smad2 protein levels was because of increased half-life and resulted in increased complex formation with Smad4. A site of ERK-dependent phosphorylation on Smad2 was located to Thr(8), a site that overlaps with the calmodulin binding region. We show that calmodulin inhibits Smad2 phosphorylation by ERK1, and overexpressing calmodulin, or stimulating calmodulin activity with ionomycin, reduces Smad2 levels. These findings suggest that the ERK pathway positively regulates Smad2 signaling by phosphorylating Smad2 and that negative regulation of Smad2 signaling by calmodulin is achieved in part by inhibiting this phosphorylation.

Animals↗

PubMed2002

A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.

Animals↗

PubMed2005

Protein kinase D specifically mediates apoptosis signal-regulating kinase 1-JNK signaling induced by H2O2 but not tumor necrosis factor.

Although both tumor necrosis factor (TNF) and H2O2 induce activation of c-Jun N-terminal kinase (JNK) kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. In this study, we first examined a variety of pharmacological inhibitors on TNF and H2O2-induced JNK activation. Go6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H2O2-induced JNK activation. However, Go6976 and calphostin, which can inhibit protein kinase C as well as protein kinase D (PKD), blocked H2O2- but not TNF-induced JNK activation, suggesting that PKD may be specifically involved in H2O2-induced JNK activation. Consistently, H2O2, but not TNF, induced phosphorylation of PKD and translocation of PKD from endothelial cell membrane to cytoplasm where it associates with the JNK upstream activator, apoptosis signal-regulating kinase 1 (ASK1). The association is mediated through the pleckstrin homology domain of PKD and the C-terminal domain of ASK1. Inhibition of PKD by Go6976 or by small interfering RNA of PKD blocked H2O2-induced ASK1-JNK activation and endothelial cell apoptosis. Interestingly, H2O2 induced 14-3-3 binding to PKD via the phospho-Ser-205/208 and phospho-Ser-219/223 and H2O2-induced 14-3-3 binding of PKD was specifically blocked by Go6976 but not by Go6983. More significantly, the 14-3-3-binding defective forms of PKD failed to associate with ASK1 and to activate JNK signaling, highlighting the importance of 14-3-3 binding of PKD in H2O2-induced activation of ASK1-JNK cascade. Thus, our data have identified PKD as a critical mediator in H2O2- but not TNF-induced ASK1-JNK signaling.

14-3-3 Proteins↗

PubMed2005

"Host tissue damage" signal ATP promotes non-directional migration and negatively regulates toll-like receptor signaling in human monocytes.

The activation of Toll-like receptors (TLRs) by lipopolysaccharide or other ligands evokes a proinflammatory immune response, which is not only capable of clearing invading pathogens but can also inflict damage to host tissues. It is therefore important to prevent an overshoot of the TLR-induced response where necessary, and here we show that extracellular ATP is capable of doing this in human monocytes. Using reverse transcription-PCR, we showed that monocytes express P2Y(1), P2Y(2), P2Y(4), P2Y(11), and P2Y(13) receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca(2+) signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca(2+), which corresponded to internal Ca(2+) release followed by activation of store-operated Ca(2+) entry. The evoked Ca(2+) signals stimulated Ca(2+)-activated K(+) channels, producing transient membrane hyperpolarization. In addition, ATP promoted cytoskeleton reorganization and cell migration; however, unlike chemoattractants, the migration was non-directional and further analysis showed that ATP did not activate Akt, essential for sensing gradients. When TLR2, TLR4, or TLR2/6 were stimulated with their respective ligands, ATPgammaS profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-alpha and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATPgammaS) strongly increased cAMP levels, and, moreover, the TLR-response was inhibited by forskolin, whereas UTP neither increased cAMP nor inhibited the TLR-response. Thus, our data suggest that ATP promotes non-directional migration and, importantly, acts as a "host tissue damage" signal via the G(s) protein-coupled P2Y(11) receptor and increased cAMP to negatively regulate TLR signaling.

Adenosine Triphosphate↗

PubMed2006

IRF-8/interferon (IFN) consensus sequence-binding protein is involved in Toll-like receptor (TLR) signaling and contributes to the cross-talk between TLR and IFN-gamma signaling pathways.

Toll-like receptor (TLR) and interferon-gamma (IFN-gamma) signaling pathways are important for both innate and adaptive immune responses. However, the cross-talk between these two signaling pathways is incompletely understood. Here we show that IFN-gamma and LPS synergistically induce the expression of proinflammatory factors, including interleukin-1 (IL-1), IL-6, IL-12, NO, and tumor necrosis factor-alpha (TNF-alpha). Comparable synergism was observed between IFN-gamma and peptidoglycan (PGN; a TLR2 ligand) and poly(I:C) (a TLR3 ligand) in the induction of IL-12 promoter activity. IFN-gamma enhanced lipopolysaccharide (LPS)-induced ERK and JNK phosphorylation but had no effect on LPS-induced NF-kappaB activation. Interestingly, we found that IRF-8-/- macrophages were impaired in the activation of LPS-induced ERK and JNK and the production of proinflammatory cytokines induced by LPS or IFN-gamma plus LPS. Retroviral transduction of IRF-8 into IRF-8-/- macrophages rescued ERK and JNK activation. Furthermore, co-immunoprecipitation experiments show that IRF-8 physically interacts with TRAF6 at a binding site between amino acid residues 356 and 305 of IRF-8. Transfection of IRF-8 enhanced TRAF6 ubiquitination, which is consistent with a physical interaction of IRF-8 with TRAF6. Taken together, the results suggest that the interaction of IRF-8 with TRAF6 modulates TLR signaling and may contribute to the cross-talk between IFN-gamma and TLR signal pathways.

Cell Line↗

PubMed1994

The COOH-terminal ends of internal signal and signal-anchor sequences are positioned differently in the ER translocase.

Signal peptides (SPs) target proteins to the secretory pathway and are cleaved from the nascent chain once the translocase in the ER has been engaged. Signal-anchor (SA) sequences also interact transiently with the ER translocase, but are not cleaved and move laterally out of the translocase to become permanent membrane anchors. One obvious difference between SP and SA sequences is the considerably longer hydrophobic regions (h regions) of the latter. To study the interaction between SP/SA sequences and the ER translocase, we have constructed signal sequences with poly-Leu h regions ranging in length from 8 to 29 residues and have characterized their locations within the translocase using both a new assay that measures the minimum number of amino acids needed to span the distance between the COOH-terminal end of the h region and the active site of the oligosaccharyl transferase enzyme and an assay where the efficiency of signal peptidase catalyzed cleavage is measured. Our results suggest that SP and SA sequences are positioned differently in the ER translocase.

Amino Acid Sequence↗

PubMed1990

Analysis of T cell signaling by class I MHC molecules: the cytoplasmic domain is not required for signal transduction.

The structural requirements for signal transduction by class I major histocompatibility complex (MHC) molecules were examined. Native or mutant HLA-A2 or HLA-B27 constructs lacking most of their cytoplasmic domains were co-transfected with pSV2neo into Jurkat cells. Transfection of either native or mutant constructs resulted in a comparable expression of the gene products. Stimulation of transfectants expressing either native or truncated A2 or B27 molecules with specific mAb evoked an increase in [Ca2+]i upon crosslinking. Moreover, crosslinking native or truncated A2 or B27 induced IL-2 production upon co-stimulation with phorbol myristate acetate. These results confirm that crosslinking class I MHC molecules transduces an activation signal to human T cells. Effective signaling was observed when all but four of the intracytoplasmic residues were deleted, indicating that signal transduction does not require this portion of the molecule.

Animals↗

PubMed1995

Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4.

Interleukin 12 (IL-12) initiates the differentiation of naive CD4+ T cells to T helper type 1 (Th1) cells critical for resistance to intracellular pathogens such as Leishmania major. To explore the basis of IL-12 action, we analyzed induction of nuclear factors in Th1 cells. IL-12 selectively induced nuclear DNA-binding complexes that contained Stat3 and Stat4, recently cloned members of the family of signal transducers and activators of transcription (STATs). While Stat3 participates in signaling for several other cytokines, Stat4 was not previously known to participate in the signaling pathway for any natural ligand. The selective activation of Stat4 provides a basis for unique actions of IL-12 on Th1 development. Thus, this study presents the first identification of the early events in IL-12 signaling in T cells and of ligand activation of Stat4.

Animals↗

PubMed2000

Erythropoietin receptors that signal through Stat5 or Stat3 support fetal liver and adult erythropoiesis: lack of specificity of stat signals during red blood cell development.

Erythropoietin (Epo) is essential for formation of mature red blood cells (RBC). However, the function of Epo receptor (EpoR)-dependent signaling pathways in the regulation of erythropoiesis remains unclear. To determine whether specific Stat signals are required for RBC development, we changed the Stat signaling specificity of the EpoR. The wild-type EpoR activates only Stat5. Thus, we substituted the major Stat5 binding sites (residues 343 and 401) in the EpoR cytoplasmic region with the Stat3 binding/activation motif from gp130. We demonstrated that activated EpoRs containing a single substitution stimulate Stat5 and Stat3, whereas an EpoR with both substitutions stimulates Stat3 but not Stat5. We then determined the ability of these receptors to support fetal liver and adult erythropoiesis. Our results show that erythropoiesis is stimulated by EpoRs that activate Stat5, both Stat5 and Stat3, or Stat3 in place of Stat5. These findings demonstrate that the specificity of EpoR Stat signaling is not essential for RBC development.

Animals↗

PubMed2002

Hypochlorous acid activates tyrosine phosphorylation signal pathways leading to calcium signaling and TNFalpha production.

Hypochlorous acid is an important oxidizing agent produced by neutrophils to aid in defense against pathogens. Although hypochlorous acid is known to cause tissue damage due to its cytotoxicity, the effect of this oxidizing agent on signal transduction by cells of the immune system and its effects on their responses are not well understood. In this study, hypochlorous acid was found to induce cellular tyrosine phosphorylation in both T and B lymphocytes, activate the ZAP-70 tyrosine kinase, and induce cellular calcium signaling in a tyrosine kinase-dependent manner. These signaling events also occurred in T cell lines that did not express the T-cell receptor, indicating the ability of hypochlorous acid to bypass normal receptor control. Hypochlorous acid induced tumor necrosis factor-alpha production in peripheral blood mononuclear cells in a tyrosine kinase-dependent manner. These results suggest that hypochlorous acid may contribute to inflammatory responses by activating signal pathways in cells of the immune system.

Calcium Signaling↗

PubMed2000

Unraveling distinct intracellular signals that promote survival and proliferation: study of erythropoietin, stem cell factor, and constitutive signaling in leukemic cells.

This review summarizes selected recent studies of the intracellular signals that allow erythroid cells to survive and proliferate under the control of erythropoietin (EPO) and alteration in signals that contribute to EPO-independent survival and proliferation. The hypothesis explored is that the proliferation and survival signals are distinct and can be separately studied with the proper cell lines and growth factor stimulation. The anti- and pro-apoptotic proteins Bcl-XL and BAD are highly implicated in EPO-dependent survival of erythroid cells. Stat5 activity appears to be upstream of Bcl-XL expression such that pathologic, constitutive activation of Stat5 may be a common event in leukemic cells that become resistant to apoptosis by constitutive expression of Bcl-XL. Other signals apparently also control the expression of Bcl-XL, such as the expression of JunB which seem to be required to suppress Bcl-XL expression when EPO is withdrawn. Apoptosis may also be triggered by inactivation of Bcl-XL by BAD. Dephosphorylation of BAD as a result of withdrawal of survival factors converts prosurvival BAD to proapoptotic BAD. Phosphorylation of BAD at the serine 112 residue seems critical to promoting survival. Constitutive activation of a kinase that phosphorylates BAD serine 112 may, therefore, contribute to resistance to apoptosis in leukemic cells. We describe the resistance of erythroleukemic cells to apoptosis induced by EPO withdrawal apparently caused by constitutive BAD phosphorylation. The resistance to apoptosis in these cells is reversed by treatment with the PI3-kinase inhibitor, LY294002, suggesting that resistance to apoptosis in these cells likely results from constitutive P13-kinase that is an upstream activator of an S-112 BAD kinase. The MAP kinase cascade is apparently active in EPO-dependent and stem cell factor (SCF)-dependent proliferation but not survival. In addition, autocrine tumor necrosis factor-a! (TNF-alpha) may also be a proliferation factor not affecting survival. P13-kinase seems to be required for full EPO-dependent proliferation but is not required for EPO-dependent survival (but it can promote survival when activated).

Animals↗