Hyunjung Ha

P2X7 Nucleotide Receptor Mediation of Membrane Pore Formation and Superoxide Generation in Human Promyelocytes and Neutrophils

The P2X7 receptor, which induces cation channel opening imparting significant permeability to Ca2+ and pore formation with changes in the plasma membrane potential, has been known to be rather restrictedly expressed in cells of the macrophage lineage including dendrites, mature macrophages, and microglial cells. However, we show here that the P2X7 receptor is also expressed in cells of granulocytic lineage such as HL-60 promyelocytes, granulocytic differentiated cells, and neutrophils. Exposure of these cells to 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP) triggered intracellular Ca2+ rise through the mediation of phospholipase C-independent and suramin-sensitive pathways. BzATP also induced depolarization of the plasma membrane in the absence of extracellular Ca2+, whereas it hyperpolarized the cells in the presence of external Ca2+, probably in part through the activation of Ca2+-activated K+ channels. However, the hyperpolarization phenomenon was markedly attenuated in differentiated HL-60 cells and neutrophils. RT-PCR and Northern blot analysis revealed the presence of P2X7 receptors on both HL-60 and neutrophil-like cells. This was further confirmed by pore formation through which the uptake of Lucifer yellow and YO-PRO1 occurred on BzATP treatment. BzATP stimulated in a concentration-dependent manner the production of superoxide in differentiated HL-60 cells via a pathway partially dependent on extracellular Ca2+. Moreover, in human neutrophils, BzATP was a more effective inducer of superoxide generation than PMA. Taken together, this is a first demonstration of the expression of P2X7 receptors on neutrophils, which shows that the receptor is functionally involved in the defense mechanism by activation of the respiratory burst pathway.

Role of phosphatidylinositol 3-kinases in chemotaxis in Dictyostelium.

Experiments in several cell types revealed that local accumulation of phosphatidylinositol 3,4,5-triphosphate mediates the ability of cells to migrate during gradient sensing. We took a systematic approach to characterize the functions of the six putative Class I phosphatidylinositol 3-kinases (PI3K1–6) in Dictyostelium by creating a series of gene knockouts. These studies revealed that PI3K1–PI3K3 are the major PI3Ks for chemoattractant-mediated phosphatidylinositol 3,4,5-triphosphate production. We studied chemotaxis of the pi3k1/2/3 triple knock-out strain (pi3k1/2/3 null cells) to cAMP under two distinct experimental conditions, an exponential gradient emitted from a micropipette and a shallow, linear gradient in a Dunn chamber, using four cAMP concentrations ranging over a factor of 10,000. Under all conditions tested pi3k1/2/3 null cells moved slower and had less polarity than wild-type cells. pi3k1/2/3 null cells moved toward a chemoattractant emitted by a micropipette, although persistence was lower than that of wild-type or pi3k1/2 null cells. In shallow linear gradients, pi3k1/2 null cells had greater directionality defects, especially at lower chemoattractant concentrations. Our studies suggest that although PI3K is not essential for directional movement under some chemoattractant conditions, it is a key component of the directional sensing pathway and plays a critical role in linear chemoattractant gradients, especially at low chemoattractant concentrations. The relative importance of PI3K in chemotaxis is also dependent on the developmental stage of the cells. Our data suggest that the output of other signaling pathways suffices to mediate directional sensing when cells perceive a strong signal, but PI3K signaling is crucial for detecting weaker signals.

Critical Role of Cysteine Residue 81 of Macrophage Migration Inhibitory Factor (MIF) in MIF-induced Inhibition of p53 Activity

Macrophage migration inhibitory factor (MIF) is a potent modulator of the p53 signaling pathway, but the molecular mechanisms of the effect of MIF on p53 function have so far remained unclear. Here we show that MIF physically interacts with the p53 tumor suppressor in vitro and in vivo. This association was significantly reduced by a C81S mutation but not C57S or C60S mutations, suggesting that Cys81 is essential for the in vivo association between MIF and p53. This association also depended on Cys242 (and, to some extent, on Cys238) within the central DNA binding domain of p53. Ectopic expression of MIF, but not MIF(C81S), inhibited p53-mediated transcriptional activation in a dose-dependent manner. Conversely, knockdown of endogenous MIF stimulated p53-mediated transcription. MIF inhibited p53-induced apoptosis and cell cycle arrest, whereas the MIF(C81S) mutant, which is unable to physically associate with p53, had no effect. Consistent with these findings, confocal microscopy showed that MIF prevented p53 translocation from the cytoplasm to the nucleus. We also demonstrated that MIF suppresses p53 activity by stabilizing the physical association between p53 and Mdm2. These results suggest that MIF physically associates with p53 and negatively regulates p53 function.

NM23-H1 Tumor Suppressor and Its Interacting Partner STRAP Activate p53 Function *

p53 plays a critical role in a variety of growth inhibitory responses, including cell cycle arrest, differentiation, and apoptosis, and contributes to tumor suppression. Here we show that NM23-H1 and its binding partner STRAP (serine-threonine kinase receptor-associated protein) interact with p53 and potentiate p53 activity. Both NM23-H1 and STRAP directly interact with the central DNA binding domain within residues 113-290. The use of NM23-H1 and STRAP mutants revealed that Cys145 of NM23-H1 and Cys152 (or Cys270) of STRAP were responsible for p53 binding. Furthermore, Cys176 and Cys135 of p53 were required to bind NM23-H1 and STRAP, respectively. Ectopic expression of wild-type NM23-H1 and STRAP, but not NM23-H1(C145S) and STRAP(C152S/C270S), positively regulated p53-mediated transcription in a dose-dependent manner. Knockdown of endogenous NM23-H1 or STRAP produced an opposite trend and inhibited the p53-mediated transcription. Similarly, NM23-H1 and STRAP stimulated p53-induced apoptosis and growth inhibition, whereas the NM23-H1(C145S) and STRAP(C152S/C270S) mutants had no effect. We also demonstrated that p53 activation by NM23-H1 and STRAP was mediated by removing Mdm2, a negative regulator of p53, from the p53-Mdm2 complex. These results suggest that NM23-H1 and its interacting partner STRAP physically interact with p53 and positively regulate its functions, including p53-induced apoptosis and cell cycle arrest.

Inhibition of acetylcholine-mediated effects by borneol

We previously reported that the aqueous extract from a medicinal plant Dryobalanops aromatica specifically inhibits the nicotinic acetylcholine receptor (nAChR) (Oh et al. Pharmacol Res 2000;42(6):559-64). Here, the effect of borneol, the main constituent of D. aromatica, on nAChR activity was investigated in bovine adrenal chromaffin cells. Borneol inhibited a nAChR agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP)-induced calcium increase with a half maximal inhibitory concentration (IC(50)) of 56+/-9 microM. In contrast, borneol did not affect the calcium increases induced by high K+, veratridine, and bradykinin. The sodium increase induced by DMPP was also inhibited by borneol with similar potency (49+/-12 microM), suggesting that the activity of nAChRs is inhibited by borneol. Borneol inhibited DMPP-induced secretion of [3H]norepinephrine with an IC(50) of 70+/-12 microM. Carbon-fiber amperometry also confirmed the inhibition of DMPP-induced exocytosis by borneol in single chromaffin cells. [3H]nicotine binding, however, was not affected by borneol. The inhibitory effect by borneol is more potent than the effect by lidocaine, a commonly used local anesthetic. The data suggest that borneol specifically inhibits the nAChR-mediated effects in a noncompetitive way.

Regulation of p53 by activated protein kinase C-δ during nitric oxide-induced dopaminergic cell death

Selective cell death of dopaminergic neurons in the substantia nigra is the major cause of Parkinson disease. Current evidence suggests that this cell death could be mediated by nitric oxide by-products such as nitrate and peroxynitrite. Because protein kinase C (PKC)-δ is implicated in apoptosis of various cell types, we studied its roles and activation mechanisms in nitric oxide (NO)-induced apoptosis of SN4741 dopaminergic cells. When cells were treated with sodium nitroprusside (SNP), a NO donor, endogenous PKC-δ was nitrated and activated. Immunoprecipitation revealed that p53 co-immunoprecipitated with PKC-δ and was phosphorylated at the 15th serine residue in SNP-treated cells. An in vitro kinase assay revealed that p53 was directly phosphorylated by SNP-activated PKC-δ. The p53 Ser-15 phosphorylation was suppressed in SNP-treated cells when the NO-mediated activation of PKC-δ was inhibited by rottlerin or (-)-epigallocatechin gallate. Within 3 h of p53 phosphorylation, its protein levels increased because of decreased ubiquitin-dependent proteosomal proteolysis, whereas the protein levels of MDM2, ubiquitin-protein isopeptide ligase, were down-regulated in a p53 phosphorylation-dependent fashion. Taken together, these results demonstrate that nitration-mediated activation of PKC-δ induces the phosphorylation of the Ser-15 residue in p53, which increases its protein stability, thereby contributing to the nitric oxide-mediated apoptosis-like cell death pathway. These findings may be expanded to provide new insight into the cellular mechanisms of Parkinson disease.

NM23-H1 Tumor Suppressor Physically Interacts with Serine-Threonine Kinase Receptor-associated Protein, a Transforming Growth Factor-β (TGF-β) Receptor-interacting Protein, and Negatively Regulates TGF-β Signaling

NM23-H1 is a member of the NM23/NDP kinase gene family and a putative metastasis suppressor. Previously, a screen for NM23-H1-interacting proteins that could potentially modulate its activity identified serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor (TGF)-β receptor-interacting protein. Through the use of cysteine to serine amino acid substitution mutants of NM23-H1 (C4S, C109S, and C145S) and STRAP (C152S, C270S, and C152S/C270S), we demonstrated that the association between these two proteins is dependent on Cys145 of NM23-H1 and Cys152 and Cys270 of STRAP but did not appear to involve Cys4 and Cys109 of NM23-H1, suggesting that a disulfide linkage involving Cys145 of NM23-H1 and Cys152 or Cys270 of STRAP mediates complex formation. The interaction was dependent on the presence of dithiothreitol or β-mercaptoethanol but not H2O2. Ectopic expression of wild-type NM23-H1, but not NM23-H1(C145S), negatively regulated TGF-β signaling in a dose-dependent manner, enhanced stable association between the TGF-β receptor and Smad7, and prevented nuclear translocation of Smad3. Similarly, wild-type NM23-H1 inhibited TGF-β-induced apoptosis and growth inhibition, whereas NM23-H1(C145S) had no effect. Knockdown of NM23-H1 by small interfering RNA stimulated TGF-β signaling. Coexpression of wild-type STRAP, but not STRAP(C152S/C270S), significantly stimulated NM23-H1-induced growth of HaCaT cells. These results suggest that the direct interaction of NM23-H1 and STRAP is important for the regulation of TGF-β-dependent biological activity as well as NM23-H1 activity.

Regulation of Transforming Growth Factor-β Signaling and PDK1 Kinase Activity by Physical Interaction between PDK1 and Serine-Threonine Kinase Receptor-associated Protein

To gain more insights about the biological roles of PDK1, we have used the yeast two-hybrid system and in vivo binding assay to identify interacting molecules that associate with PDK1. As a result, serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor-β (TGF-β) receptor-interacting protein, was identified as an interacting partner of PDK1. STRAP was found to form in vivo complexes with PDK1 in intact cells. Mapping analysis revealed that this binding was only mediated by the catalytic domain of PDK1 and not by the pleckstrin homology domain. Insulin enhanced a physical association between PDK1 and STRAP in intact cells, but this insulin-induced association was prevented by wortmannin, a phosphatidylinositol 3-kinase inhibitor. In addition, the association between PDK1 and STRAP was decreased by TGF-β treatment. Analysis of the activities of the interacting proteins showed that PDK1 kinase activity was significantly increased by coexpression of STRAP, probably through the inhibition of the binding of 14-3-3, a negative regulator, to PDK1. Consistently, knockdown of the endogenous STRAP by the transfection of the small interfering RNA resulted in the decrease of PDK1 kinase activity. PDK1 also exhibited an inhibition of TGF-β signaling with STRAP by contributing to the stable association between TGF-β receptor and Smad7. Moreover, confocal microscopic study and immunostaining results demonstrated that PDK1 prevented the nuclear translocation of Smad3 in response to TGF-β. Knockdown of endogenous PDK1 with small interfering RNA has an opposite effect. Taken together, these results suggested that STRAP acts as an intermediate signaling molecule linking between the phosphatidylinositol 3-kinase/PDK1 and the TGF-β signaling pathways.

Cloning and expression of a cDNA encoding a novel protein serine/threonine kinase predominantly expressed in hematopoietic cells.

We have isolated a cDNA clone of a new member of protein serine/threonine kinases, MPK38, from a cDNA library constructed from the murine teratocarcinoma PCC4 cell line by the polymerase chain reaction. MPK38 was transcribed as an approx. 2.5 kb mRNA encoding for a protein of 643 amino acids. N-terminus of MPK38 contains the kinase catalytic domain which exhibits approximately 60% protein sequence identity with the SNF1 serine/threonine kinase family. The MPK38 cDNA directs the in vitro translation of two protein species of approx. 70 and approx. 50 kDa, which appear to result from an internal initiation of translation. MPK38 was predominantly expressed in thymus and spleen, but was not detectable in kidney, liver, and muscle in the adult tissues. In addition, MPK38 was apparently expressed in T lineage cells and a macrophage/monocyte cell, but was not detectable in a B cell line and an embryonic cell line. However, a low level of MPK38 transcript was detectable in a mast cell line after a longer exposure. Furthermore, MPK38 gene product showed the kinase activity which was assessed by immune complex kinase assay. Thus, MPK38 gene product seems to play an important role in signal transduction of certain lineages of hematopoietic cells.

Murine Protein Serine/Threonine Kinase 38 Activates Apoptosis Signal-regulating Kinase 1 via Thr838 Phosphorylation

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H2O2 or tumor necrosis factor α treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr838 phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H2O2-mediated apoptosis by enhancing the ASK1 activity through Thr838 phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.