Biomedical subjects

Gary L Johnson

Publications and source records attributed to Gary L Johnson.

At least 37 records · Page 2Linked to original sources

PubMed2003

The in vitro production and characterization of neutrophils from embryonic stem cells.

An embryonic stem (ES) cell/OP9 coculture system for the effective production of functional neutrophils is described. A 3-step differentiation strategy was developed that uses liquid culture, enabling reliable and abundant production of neutrophils at high purity without the need of sorting for isolation of mature neutrophils. Use of the OP9 stromal cell line significantly enhances the number, percentage, and duration of differentiated neutrophils produced from embryonic stem cells. Effective and sustained differentiation of ES cells into neutrophils provides a useful model system for studying neutrophil differentiation and function and the factors that regulate them. Morphologic and functional evaluation of these ES-derived neutrophils indicates that large numbers of mature neutrophils can be produced from pluripotent ES cells in vitro. Specifically, their morphology, ability to produce superoxides, flux calcium, undergo chemotaxis in response to macrophage inflammatory protein 2 (MIP-2), stain for the granulocyte-specific marker-specific chloroacetate esterase, and contain the neutrophil-specific markers Gr-1 and the mouse neutrophil-specific antigen indicates that they are comparable with purified mouse bone marrow neutrophils. They also express gelatinase and lactoferrin granule proteins. During the differentiation of these ES-derived neutrophils, regional areas of neutrophil production can be identified that have been designated as neutrophil generating regions (NGRs).

Animals↗

PubMed2003

PB1 domains of MEKK2 and MEKK3 interact with the MEK5 PB1 domain for activation of the ERK5 pathway.

MEKK2 and MEKK3 are MAPK kinase kinases that activate the ERK5 pathway by phosphorylating and activating the MAPK kinase, MEK5. Activated MEK5 then phosphorylates and activates ERK5. PB1 domains were first defined in the p67phox and Bem1p proteins and have been shown to mediate protein-protein heterodimerization. A PB1 domain is encoded within the N-terminal portion of MEKK2, MEKK3, and MEK5. Herein, we analyze the functional role of MEKK2, MEKK3, and MEK5 PB1 domains in the ERK5 activation pathway. The PB1 domains of MEKK2 and MEKK3 bind the PB1 domain of MEK5 but do not significantly homo- or heterodimerize with one another in vitro. Furthermore, co-immunoprecipitation of MEKK2 and MEK5 from cell lysates shows that they form a complex in vivo. Deletion or mutation of the MEKK2 PB1 domain abolishes MEKK2-MEK5 complexes, demonstrating that the PB1 domain interaction is required for MEKK2-MEK5 interactions. Expression in cells of the MEKK2 or MEKK3 PB1 domain inhibits ERK5 activation, whereas expression of a mutant MEKK2 unable to bind the MEK5 PB1 domain or expression of the p67phox PB1 domain has no effect on ERK5 activation. These findings demonstrate that the PB1 domain mediates the association of MEKK2 and MEKK3 with MEK5 and that the respective PB1 domains of these kinases are critical for regulation of the ERK5 pathway. The free PB1 domain of MEKK2 or MEKK3 functions effectively to inhibit the ERK5 pathway but not the p38 or JNK pathways, demonstrating the specific and unique requirement of the MEKK2 and MEKK3 PB1 domain in regulating ERK5 activation.

Amino Acid Sequence↗

PubMed2003

MEKK1 regulates calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of migrating fibroblasts.

Herein, we define how MEKK1, a MAPK kinase kinase, regulates cell migration. MEKK1 is associated with actin fibers and focal adhesions, localizing MEKK1 to sites critical in the control of cell adhesion and migration. EGF-induced ERK1/2 activation and chemotaxis are inhibited in MEKK1-/- fibroblasts. MEKK1 deficiency causes loss of vinculin in focal adhesions of migrating cells, increased cell adhesion and impeded rear-end detachment. MEKK1 is required for activation of the cysteine protease calpain and cleavage of spectrin and talin, proteins linking focal adhesions to the cytoskeleton. Inhibition of ERK1/2 or calpain, but not of JNK, mimics MEKK1 deficiency. Therefore, MEKK1 regulates calpain-mediated substratum release of migrating fibroblasts.

Animals↗

PubMed2003

MEF2C regulates c-Jun but not TNF-alpha gene expression in stimulated mast cells.

Mitogen-activated protein kinase (MAPK) cascades play essential roles in the transduction of extracellular signals to cytoplasmic and nuclear effectors. The MAPK kinase kinase MEKK2 is essential for activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 5 (ERK5). These pathways are important for expression of specific cytokine genes in mast cells following cross-linking of the high-affinity IgE receptor (FcepsilonRI). A consequence of ERK5 activation is activation of the transcriptional factor myocyte enhancing factor-2C (MEF2C), leading to increased c-Jun expression. We have investigated the role of MEF2C activation in mast cells and demonstrated that it requires sequential activation of the signaling cascade of MEKK2-MEK5-ERK5. Following phosphorylation of MEF2C, activated MEF2C regulates transcription of c-Jun but not TNF-alpha. Inhibition of ERK5, MEK5 activation or activation of MEKK2-deficient mast cells was associated with inhibition of MEF2C phosphorylation and a decrease in c-Jun expression. Thus, these data define an activation module, MEKK2-MEK5-ERK5-MEF2C in the transcriptional activation of c-Jun in mast cells following FcepsilonRI cross-linking. These results demonstrate the novel and important, MEKK2-dependent role of MEF2C in induction of c-Jun expression in mast cells activated through FcepsilonRI, a pathway distinct from that involving MEKK2-MEK5-ERK5 in the regulation of mast cell cytokine production.

Animals↗

PubMed2003

Reovirus-induced alteration in expression of apoptosis and DNA repair genes with potential roles in viral pathogenesis.

Reoviruses are a leading model for understanding cellular mechanisms of virus-induced apoptosis. Reoviruses induce apoptosis in multiple cell lines in vitro, and apoptosis plays a key role in virus-induced tissue injury of the heart and brain in vivo. The activation of transcription factors NF-kappaB and c-Jun are key events in reovirus-induced apoptosis, indicating that new gene expression is critical to this process. We used high-density oligonucleotide microarrays to analyze cellular transcriptional alterations in HEK293 cells after infection with reovirus strain T3A (i.e., apoptosis inducing) compared to infection with reovirus strain T1L (i.e., minimally apoptosis inducing) and uninfected cells. These strains also differ dramatically in their potential to induce apoptotic injury in hearts of infected mice in vivo-T3A is myocarditic, whereas T1L is not. Using high-throughput microarray analysis of over 12,000 genes, we identified differential expression of a defined subset of genes involved in apoptosis and DNA repair after reovirus infection. This provides the first comparative analysis of altered gene expression after infection with viruses of differing apoptotic phenotypes and provides insight into pathogenic mechanisms of virus-induced disease.

Animals↗

PubMed2003

MEK kinase 2 and the adaptor protein Lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src.

Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex.

Adaptor Proteins, Signal Transducing↗

PubMed2003

DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet.

Using a bisubstituted caspase-3 target sequence: aspartate-glutamate-valine-aspartate, (z-DEVD)2 peptide derivative of the fluorophore, cresyl violet, we have obtained a cell permeant, fluorogenic, caspase substrate capable of detecting the site-specific presence of functionally active, caspase-3 and caspase-7 up-regulation within intact apoptotic cells. Addition of this substrate to induced and noninduced cell culture populations allows for the rapid site-specific detection of caspase up-regulation without the requirement for a wash step. We demonstrate here the use of (z-DEVD)2-cresyl violet substrate for the detection of apoptosis induction in Jurkat, THP-1, and MCF-7 cells using fluorescence microscopy and 96-well fluorescence plate reader analysis. Intracellular up-regulated DEVDase activity, which was clearly visible by fluorescence microscopy and 96-well fluorescence plate reader measurements, showed greater than 6-fold increases in fluorescence output in induced versus noninduced Jurkat cell samples. A simple fluorogenic substrate conversion method is demonstrated here for detecting apoptosis induction within intact living cells.

Apoptosis↗

PubMed2002

MEKK1 is required for inducible urokinase-type plasminogen activator expression.

Urokinase-type plasminogen activator (uPA) regulates the remodeling of extracellular matrix and controls reparative processes such as wound healing and liver regeneration. Here we show inducible uPA expression is controlled by MEKK1, a MAPK kinase kinase that regulates the ERK1/2 and JNK pathways. MEKK1 is activated in response to growth factors and cytoskeletal changes. We have found MEKK1 to be necessary for uPA up-regulation in response to treatment with phorbol 12-myristate 13-acetate or basic fibroblast growth factor. We demonstrate that growth factor-treated MEKK1-deficient fibroblasts display greatly reduced uPA expression and activity compared with control fibroblasts. Further, we show that growth factor-induced uPA expression requires MEKK1-dependent MKK1 and JNK activity and that transfection of MEKK1 into knockout cells restores inducible uPA expression and activity. Importantly, disrupted expression of MEKK2, a related MAPK kinase kinase, had no effect on uPA activity. Therefore, we conclude that MEKK1 expression is required for PMA- or FGF-2-induced signals to control uPA expression and function.

Animals↗

PubMed2002

Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases.

Multicellular organisms have three well-characterized subfamilies of mitogen-activated protein kinases (MAPKs) that control a vast array of physiological processes. These enzymes are regulated by a characteristic phosphorelay system in which a series of three protein kinases phosphorylate and activate one another. The extracellular signal-regulated kinases (ERKs) function in the control of cell division, and inhibitors of these enzymes are being explored as anticancer agents. The c-Jun amino-terminal kinases (JNKs) are critical regulators of transcription, and JNK inhibitors may be effective in control of rheumatoid arthritis. The p38 MAPKs are activated by inflammatory cytokines and environmental stresses and may contribute to diseases like asthma and autoimmunity.

Animals↗

PubMed2002

Ubiquitylation of MEKK1 inhibits its phosphorylation of MKK1 and MKK4 and activation of the ERK1/2 and JNK pathways.

MEKK1 is a MAPK kinase kinase that is activated in response to stimuli that alter the cytoskeleton and cell shape. MEKK1 phosphorylates and activates MKK1 and MKK4, leading to ERK1/2 and JNK activation. MEKK1 has a plant homeobox domain (PHD) that has been shown to have E3 ligase activity. (Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H., and Hunter, T. (2002) Mol. Cell 9, 945-956). MEKK1 kinase activity is required for ubiquitylation of MEKK1. MEKK1 ubiquitylation is inhibited by mutation of cysteine 441 to alanine (C441A) within the PHD. The functional consequence of MEKK1 ubiquitylation is the inhibition of MEKK1 catalyzed phosphorylation of MKK1 and MKK4 resulting in inhibition of ERK1/2 and JNK activation. The C441A mutation within the PHD of MEKK1 prevents ubiquitylation and preserves the ability of MEKK1 to catalyze MKK1 and MKK4 phosphorylation. MEKK1 ubiquitylation represents a mechanism for inhibiting the ability of a protein kinase to phosphorylate substrates and regulate downstream signaling pathways.

Base Sequence↗

PubMed2002

MEKK1-induced apoptosis requires TRAIL death receptor activation and is inhibited by AKT/PKB through inhibition of MEKK1 cleavage.

MEK kinase 1 (MEKK1) induces apoptosis through the activation of caspases. The mechanism for MEKK1-induced apoptosis involves caspase-mediated cleavage of MEKK1, releasing a pro-apoptotic 91 kDa kinase fragment that serves to further amplify caspase activation in a feedback loop. Both cleavage of MEKK1 and increased expression of death receptor 4 (DR4, TRAILR1) and death receptor 5 (DR5, TRAILR2) occur following exposure of cells to genotoxins. Overexpression of kinase inactive MEKK1 inhibits MEKK1-mediated apoptosis and effectively blocks death receptor upregulation following etoposide treatment. Herein, we investigate the role of death receptor activation and the ability of AKT/PKB (AKT) to inhibit cell death in MEKK1-induced apoptosis. We show that by preventing DR4 and DR5 activation through expression of decoy receptor 1 (DcR1) and dominant negative FADD, we inhibit MEKK1-induced apoptosis. Furthermore, expression of 91 kDa MEKK1 increased DR4 and FAS mRNA and protein levels. MEKK1-induced apoptosis is amplified by blocking PI-3 kinase activation and overexpression of AKT blocked both MEKK1-induced apoptosis and caspase activation. AKT overexpression also prevented the cleavage of endogenous MEKK1 by genotoxins. AKT did not, however, block MEKK1-induced JNK activation, showing that regulation of the JNK pathway by MEKK1 is independent of its role in regulation of apoptosis. Thus, MEKK1-induced apoptosis requires TRAIL death receptor activation and is blocked by AKT through inhibition of MEKK1 cleavage.

Apoptosis↗

PubMed2002

Inhibition of Src family kinases blocks epidermal growth factor (EGF)-induced activation of Akt, phosphorylation of c-Cbl, and ubiquitination of the EGF receptor.

Stimulation of T47D cells with epidermal growth factor (EGF) results in the activation of the intrinsic tyrosine kinases of the receptor and the phosphorylation of multiple cellular proteins including the receptor, scaffold molecules such as c-Cbl, adapter molecules such as Shc, and the serine/threonine protein kinase Akt. We demonstrate that EGF stimulation of T47D cells results in the activation of the Src protein-tyrosine kinase and that the Src kinase inhibitor PP1 blocks the EGF-induced phosphorylation of c-Cbl but not the activation/phosphorylation of the EGF receptor itself. PP1 also blocks EGF-induced ubiquitination of the EGF receptor, which is presumably mediated by phosphorylated c-Cbl. Src is associated with c-Cbl, and we have previously demonstrated that the Src-like kinase Fyn can phosphorylate c-Cbl at a preferred binding site for the p85 subunit of phosphatidylinositol 3'-kinase. PP1 treatment blocks EGF-induced activation of the anti-apoptotic protein kinase Akt suggesting that Src may regulate activation of Akt, perhaps by a Src --> c-Cbl --> phosphatidylinositol 3'-kinase --> Akt pathway.

Epidermal Growth Factor↗

PubMed2002

MEKK1 is essential for cardiac hypertrophy and dysfunction induced by Gq.

Signaling via mitogen-activated protein kinases is implicated in heart failure induced by agonists for G protein-coupled receptors that act via the G protein Galphaq. However, this assertion relies heavily on pharmacological inhibitors and dominant-interfering proteins and not on gene deletion. Here, we show that endogenous cardiac MAPK/ERK kinase kinase-1 (MEKK1)/(MAP3K1), a mitogen-activated protein kinase kinase kinase, is activated by heart-restricted overexpression of Galphaq in mice. In cardiac myocytes derived from embryonic stem cells in culture, homozygous disruption of MEKK1 selectively impaired c-Jun N-terminal kinase activity in the absence or presence of phenlyephrine, a Galphaq-dependent agonist. Other terminal mitogen-activated protein kinases were unaffected. In mice, the absence of MEKK1 abolished the increase in cardiac mass, myocyte size, hypertrophy-associated atrial natriuretic factor induction, and c-Jun N-terminal kinase activation by Galphaq, and improved ventricular mechanical function. Thus, MEKK1 mediates cardiac hypertrophy induced by Galphaq in vivo and is a logical target for drug development in heart disease involving this pathway.

Animals↗

PubMed2002

Kinetics of HL-60 cell entry to apoptosis during treatment with TNF-alpha or camptothecin assayed by the stathmo-apoptosis method.

BACKGROUND: Duration of apoptosis, from onset to final disintegration of the cell, is often short and variable. The apoptotic index (AI), as a snapshot of a transient event of variable length, does not truly represent incidence of apoptosis in the studied cell population. We recently proposed to estimate the cumulative apoptotic index (CAI) by inducing stathmo-apoptosis. A fluorescent inhibitor of caspases (FLICA) FAM-VAD-FMK is used to arrest the process of apoptosis and thereby prevent cell disintegration. Simultaneously, the arrested/apoptotic cells become FLICA-labeled. In the present study, this approach was applied to measure kinetics of HL-60 cell entrance into apoptosis induced via cell surface death receptor or a mitochondria-initiated pathway. Materials and Methods Cultures of HL-60 cells were treated with either TNF-alpha or camptothecin (CPT) in the absence or constant presence of 10-50 microM FLICA. The CAI was measured at different time points for up to 48 h by flow cytometry. Bivariate analysis of DNA content and cell labeling with FLICA was used to correlate apoptosis with the cell-cycle position. RESULTS: Selective loss of apoptotic cells seen in HL-60 cell cultures exposed to either TNF-alpha or CPT alone was prevented in cultures containing FLICA. Addition of FLICA alone had no effect on cell viability. The percentage of FLICA-labeled cells was plotted as a function of time after addition of TNF-alpha or CPT. The rate of cell entry to apoptosis was subsequently estimated from the slopes of the stathmo-apoptotic plot. The slopes revealed that the TNF-alpha or CPT-treated cells asynchronously underwent apoptosis with a stochastic-like kinetics and at two different rates. About 50% of cells in the TNF-alpha-treated cultures underwent apoptosis during the initial 6 h at a rate of approximately 8% of cells per hour; the remaining cells were undergoing apoptosis at a rate of approximately 2.5% of cells per hour for up to 24 h. Also, about 50% of the CPT-treated cells, predominantly those in S phase of the cell cycle, underwent apoptosis within the initial 8 h of CPT exposure, at a rate of approximately 7% of cells per hour. Remaining cells were undergoing apoptosis at a rate of approximately 1% of cells per hour during up to 48 h exposure to CPT. Spontaneous apoptosis in the untreated cultures occurred at a rate of 0.2% of cells per hour. CONCLUSIONS: This approach provides a means for measuring the kinetics of cell entrance to apoptosis (caspase activation) in large populations of cells in relation to the cell-cycle position.

Apoptosis↗

PubMed2002

TRAIL and inhibitors of apoptosis are opposing determinants for NF-kappaB-dependent, genotoxin-induced apoptosis of cancer cells.

Opposing pro- and anti-apoptotic actions of TRAIL and the inhibitors of apoptosis (IAPs) contribute to the cell's decision to survive or die. We demonstrate that in H157 human lung carcinoma cells, etoposide and doxorubicin induce the NF-kappaB-dependent expression of both pro- and anti-apoptotic proteins including TRAIL and its death receptor, DR5, and IAPs. Inhibition of NF-kappaB activation in H157 cells in response to genotoxin resulted in loss of cell surface expression of TRAIL and DR5, aggressive growth and chemotherapy resistance of tumors in nude mice. Similar to the paracrine TRAIL response in H157 cells, the sensitivity of normal lung and breast epithelium and carcinomas to undergo genotoxin-induced apoptosis correlates strongly with cell surface expression of TRAIL. Suppression of TRAIL signaling by expression of the TRAIL decoy receptor, DcR1, confers chemoresistance to cancer cells. These findings demonstrate that TRAIL signaling via its death receptors is a significant contributor to genotoxin-induced apoptosis in human epithelial carcinomas.

Apoptosis↗

PubMed2002

Apoptosis stimulated by the 91-kDa caspase cleavage MEKK1 fragment requires translocation to soluble cellular compartments.

MEKK1, a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase, generates anti-apoptotic signaling as a full-length protein but induces apoptosis when cleaved by caspases. Here, we show that caspase-dependent cleavage of MEKK1 relocalizes the protease-generated 91-kDa kinase fragment from a particulate fraction to a soluble cytoplasmic fraction. Relocalization of MEKK1 catalytic activity is necessary for the pro-apoptotic function of MEKK1. The addition of a membrane-targeting signal to the 91-kDa fragment inhibits caspase activation and the induction of apoptosis but does not change the activation of JNK, ERK, NFkappaB, or p300. These results identify the caspase cleavage of MEKK1 as a dynamic regulatory mechanism that alters the subcellular distribution of MEKK1, changing its function to pro-apoptotic signaling, which does not depend on the currently described MEKK1 effectors.

Animals↗

PubMed2002

Role of the amino-terminal domains of MEKKs in the activation of NF kappa B and MAPK pathways and in the regulation of cell proliferation and apoptosis.

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) kinases (MEKKs) are serine/threonine kinases that are upstream regulators of MAPKs. Here, the role of the amino-terminal (N-terminal) domain of MEKK1-4 on the regulation of different intracellular signaling pathways, apoptosis, and cell proliferation has been assessed by comparing the responses induced by the full-length (FL) MEKKs to those induced by the kinase domains only. For each MEKK, the pattern of activation of NF kappa B, the ERK MAPK pathway, and the c-Jun N-terminal kinase (JNK) MAPK pathway markedly differed between the kinase domain and the FL form. Similarly, cell proliferation and apoptosis were differently regulated by the FL MEKK and the corresponding kinase domain. Our data show that the N-terminal domain of the MEKKs determines the specificity and the strength of activation of various intracellular signaling pathways and cellular responses.

Apoptosis↗

PubMed2002

Mixed-lineage kinase control of JNK and p38 MAPK pathways.

Mixed-lineage kinases (MLKs) are serine/threonine protein kinases that regulate signalling by the c-Jun amino-terminal kinase (JNK) and p38 mitogen-activated-protein kinase (MAPK) pathways. MLKs are represented in the genomes of both Caenorhabditis elegans and Drosophila melanogaster. The Drosophila MLK Slipper regulates JNK to control dorsal closure during embryonic morphogenesis. In mammalian cells, MLKs are implicated in the control of apoptosis and are potential drug targets for many neurodegenerative diseases.

Animals↗