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Gary L Johnson

Publications and source records attributed to Gary L Johnson.

At least 19 recordsLinked to original sources
PubMed2006

MEKK1 mediates the ubiquitination and degradation of c-Jun in response to osmotic stress.

c-Jun, a major transcription factor in the activating protein 1 family of regulatory proteins, is activated by many physiologic and pathological stimuli. We show here that c-Jun was downregulated in response to osmotic stress via ubiquitination-dependent degradation by the PHD/RING finger domain of MEKK1, which exhibited E3 ubiquitin ligase activity toward c-Jun in vitro and in vivo. The reduced c-Jun protein level resulting from exogenous expression of wild-type MEKK1 and the opposite effect induced by expression of a MEKK1 PHD/RING finger domain mutant were consistent with a higher level of c-Jun protein in MEKK1(-/-) cells than in corresponding wild-type cells. The deficiency of MEKK1 blocked posttranslational downregulation of c-Jun in response to osmotic stress. Furthermore, apoptosis induced by osmotic stress was suppressed by overexpression of c-Jun, indicating that the downregulation of c-Jun promotes apoptosis.

Animals↗
PubMed2006

The use of rabbit polyclonal antibodies to assess neoantigenicity following viral reduction of an alpha-1-proteinase inhibitor preparation.

The objective of this study was to determine whether the viral reduction processes of nanofiltration and solvent/detergent treatment used in the manufacture of alpha-1 proteinase inhibitor (API) cause neoantigenic changes. Polyclonal antibodies were raised in rabbits against the treated API and quantitatively absorbed with an affinity column containing API that had not undergone viral reduction treatment. Antibodies before and after absorption were measured in a validated ELISA using the immunogen for antibody capture. Antibodies against novel API epitopes were not found after antiserum from rabbits inoculated with treated API was absorbed with untreated API. A positive control, consisting of serum obtained from rabbits inoculated with trinitrophenylated API, showed substantial amounts of measurable antibody following absorption with untreated API. The results suggest that the viral reduction process used does not result in the creation of API neoantigens.

Animals↗
PubMed2006

PB1 domain-dependent signaling complex is required for extracellular signal-regulated kinase 5 activation.

MEKK2, MEK5, and extracellular signal-regulated kinase 5 (ERK5) are members of a three-kinase cascade for the activation of ERK5. MEK5 is the only MAP2K to express a PB1 domain, and we have shown that it heterodimerizes with the PB1 domain of MEKK2. Here we demonstrate the MEK5 PB1 domain is a scaffold that also binds ERK5, functionally forming a MEKK2-MEK5-ERK5 complex. Reconstitution assays and CFP/YFP imaging (fluorescence resonance energy transfer [FRET]) measuring YFP-MEKK2/CFP-MEK5 and CFP-MEK5/YFP-ERK5 interactions define distinct MEK5 PB1 domain binding sites for MEKK2 and ERK5, with a C-terminal extension of the PB1 domain contributing to ERK5 binding. Stimulus-dependent CFP/YFP FRET in combination with mutational analysis was used to define MEK5 PB1 domain residues critical for the interaction of MEKK2/MEK5 and MEK5/ERK5 required for activation of the ERK5 pathway in living cells. Fusion of the MEK5 PB1 domain to the N terminus of MEK1 confers ERK5 regulation by a MAP2K normally regulating only ERK1/2. The MEK5 PB1 domain confers stringent MAP3K regulation of ERK5 relative to more promiscuous MAP3K control of ERK1/2, JNK, and p38.

Amino Acid Sequence↗
PubMed2005

MEKK4 is an effector of the embryonic TRAF4 for JNK activation.

TRAF4 has previously been shown to activate JNK through an unknown mechanism. Here, we show that endogenous TRAF4 and MEKK4 associate in both human K562 cells and mouse E10.5 embryos. TRAF4 interacts with the kinase domain of MEKK4. However, this association does not require MEKK4 kinase activity. The interaction of MEKK4 and TRAF4 are further demonstrated by the colocalization of TRAF4 and MEKK4 in cells. Importantly, although TRAF4 has little or no ability to activate JNK independently, coexpression of TRAF4 and MEKK4 results in synergistic activation of JNK that is inhibited by a kinase-inactive mutant of MEKK4, MEKK4K1361R. MEKK4 binds the TRAF domain of TRAF4 and MEKK4/TRAF4 activation of JNK is inhibited by expression of the TRAF domain. Furthermore, TRAF4 stimulates MEKK4 kinase activity by promoting MEKK4 oligomerization and JNK activation can be stimulated by chemical induction of MEKK4 dimerization. The findings identify MEKK4 as the MAPK kinase kinase for TRAF4 regulation of the JNK pathway.

Animals↗
PubMed2005

CCM1 and CCM2 protein interactions in cell signaling: implications for cerebral cavernous malformations pathogenesis.

Cerebral cavernous malformations (CCMs) are sporadically acquired or inherited vascular lesions of the central nervous system consisting of clusters of dilated thin-walled blood vessels that predispose individuals to seizures and stroke. Familial CCM is caused by mutations in KRIT1 (CCM1) or in malcavernin (CCM2), the murine ortholog of which was concurrently characterized as osmosensing scaffold for MEKK3 (OSM). The roles of the CCM proteins in the pathogenesis of the disorder remain largely unknown. Here, we use co-immunoprecipitation, fluorescence resonance energy transfer and subcellular localization strategies to show that the CCM1 gene product, KRIT1, interacts with the CCM2 gene product, malcavernin/OSM. Analogous to the established interactions of CCM1 and beta1 integrin with ICAP1, the CCM1/CCM2 association is dependent upon the phosphotyrosine binding (PTB) domain of CCM2. A familial CCM2 missense mutation abrogates the CCM1/CCM2 interaction, suggesting that loss of this interaction may be critical in CCM pathogenesis. CCM2 and ICAP1 bound to CCM1 via their respective PTB domains differentially influence the subcellular localization of CCM1. Furthermore, we expand upon the established involvement of CCM2 in the p38 mitogen-activated protein kinase signaling module by demonstrating that CCM1 associates with CCM2 and MEKK3 in a ternary complex. These data indicate that the genetic heterogeneity observed in familial CCM may reflect mutation of different molecular members of a coordinated signaling complex.

Amino Acid Substitution↗
PubMed2005

Ablation of MEK kinase 1 suppresses intimal hyperplasia by impairing smooth muscle cell migration and urokinase plasminogen activator expression in a mouse blood-flow cessation model.

BACKGROUND: Migration, proliferation, and matrix-degrading protease expression of smooth muscle cells (SMCs) are major features of intimal hyperplasia after vascular injury. Although MEK kinase 1 (MEKK1) has been shown to regulate cell migration and urokinase plasminogen activator (uPA) expression, the precise role of MEKK1 in this process remains unknown. METHODS AND RESULTS: We triggered a vascular remodeling model by complete ligation of the right common carotid artery in wild-type (WT) and MEKK1-null (MEKK1-/-) mice. The intimal areas 28 days after ligation were significantly decreased in the ligated MEKK1-/- arteries compared with WT arteries (28+/-8 versus 65+/-17 microm2, P<0.05). There were no differences in the ratios of proliferating cell nuclear antigen (PCNA)-positive cells to total cells within the arterial wall between WT and MEKK1-/- arteries. Proliferation capacity also did not differ between WT and MEKK1-/- cultured aortic smooth muscle cells (AoSMCs). In contrast, the number of intimal PCNA-positive cells 7 days after ligation was significantly smaller in MEKK1-/- arteries. Three different migration assays revealed that migration and invasion of MEKK1-/- AoSMCs were markedly impaired. Addition of full-length MEKK1 restored the migration capacity of MEKK1-/- AoSMCs. The number of MEKK1-/- AoSMCs showing lamellipodia formation by epithelial growth factor was significantly smaller compared with those of WT SMCs. Furthermore, uPA expression after ligation was markedly decreased in MEKK1-/- arteries. CONCLUSIONS: MEKK1 is implicated in vascular remodeling after blood-flow cessation by regulating the migration and uPA expression of SMCs. MEKK1 is a potential target for drug development to prevent vascular remodeling.

Animals↗
PubMed2005

MEKK1 regulates the AP-1 dimer repertoire via control of JunB transcription and Fra-2 protein stability.

Activator protein 1 (AP-1) transcription factor dimers are composed of Jun, Fos, and ATF member proteins, but the mechanisms that determine AP-1 composition are not clearly defined and the function of specific dimers is not well understood. MEKK1 is a mitogen-activated protein kinase (MAPK) kinase kinase and an ubiquitin ligase that regulates both the extracellular signal-regulated kinase 1/2 and the c-Jun amino-terminal kinase. Herein, we demonstrate that MEKK1 regulates the AP-1 protein repertoire. Both FGF-2 and phorbol ester-inducible urokinase-type plasminogen activator (uPA) expression requires AP-1 binding to an enhancer element in the uPA promoter, and we have previously shown that FGF-2 or PMA induction of uPA expression is strongly dependent on MEKK1. JunB mRNA is significantly increased in MEKK1-/- cells, demonstrating that MEKK1 suppresses JunB mRNA expression. Upregulation of JunB expression in MEKK1-/- cells forms an inhibitory AP-1 complex that binds to the uPA promoter and inhibits uPA transcription. MEKK1 also regulates Fra-2 protein stability by inducing Fra-2 ubiquitination and degradation. MEKK1 regulates AP-1-dependent gene expression by regulating the expression, activity and degradation of component members of the AP-1 complex. Controlling the repertoire of a transcription factor complex is a newly defined function for an MAPK kinase kinase.

Animals↗
PubMed2005

Structural and evolutionary division of phosphotyrosine binding (PTB) domains.

Proteins encoding phosphotyrosine binding (PTB) domains function as adaptors or scaffolds to organize the signaling complexes involved in wide-ranging physiological processes including neural development, immunity, tissue homeostasis and cell growth. There are more than 200 proteins in eukaryotes and nearly 60 human proteins having PTB domains. Six PTB domain encoded proteins have been found to have mutations that contribute to inherited human diseases including familial stroke, hypercholesteremia, coronary artery disease, Alzheimer's disease and diabetes, demonstrating the importance of PTB scaffold proteins in organizing critical signaling complexes. PTB domains bind both peptides and headgroups of phosphatidylinositides, utilizing two distinct binding motifs to mediate spatial organization and localization within cells. The structure of PTB domains confers specificity for binding peptides having a NPXY motif with differing requirements for phosphorylation of the tyrosine within this recognition sequence. In this review, we use structural, evolutionary and functional analysis to divide PTB domains into three groups represented by phosphotyrosine-dependent Shc-like, phosphotyrosine-dependent IRS-like and phosphotyrosine-independent Dab-like PTBs, with the Dab-like PTB domains representing nearly 75% of proteins encoding PTB domains. In addition, we further define the binding characteristics of the cognate ligands for each group of PTB domains. The signaling complexes organized by PTB domain encoded proteins are largely unknown and represents an important challenge in systems biology for the future.

Amino Acid Sequence↗
PubMed2005

MAPK kinase kinases (MKKKs) as a target class for small-molecule inhibition to modulate signaling networks and gene expression.

MAPKs are members of a three-kinase phosphorelay system composed of the MAPK, MAPK kinase (MKK) and MAPK kinase kinase (MKKK). MKKKs phosphorylate and activate MKKs, which in turn phosphorylate and activate MAPKs. Scaffolding proteins organize MKK-MAPK complexes for activation by specific MKKKs and do so in specific locations in the cell. It is the MKKK associated with the scaffolded complex that provides selectivity for activation by upstream stimuli including GTPases, additional kinases and receptors. The demonstration that specific MKKKs also exert control over gene expression by regulating the repertoire of required transcriptional machinery is a newly recognized function for MKKKs. Appreciation of MKKKs as signaling hubs for selectively integrating different upstream stimuli into the control of MAPK networks, and the ability of MKKKs to function as master planners of the transcription factor machinery suggest that MKKK inhibitors will provide selective therapeutic intervention of stimulus-specific MAPK responses.

Drug Design↗
PubMed2005

Analysis of orthologous gene expression between human pulmonary adenocarcinoma and a carcinogen-induced murine model.

Human adenocarcinoma (AC) is the most frequently diagnosed human lung cancer, and its absolute incidence is increasing dramatically. Compared to human lung AC, the A/J mouse-urethane model exhibits similar histological appearance and molecular changes. We examined the gene expression profiles of human and murine lung tissues (normal or AC) and compared the two species' datasets after aligning approximately 7500 orthologous genes. A list of 409 gene classifiers (P value <0.0001), common to both species (joint classifiers), showed significant, positive correlation in expression levels between the two species. A number of previously reported expression changes were recapitulated in both species, such as changes in glycolytic enzymes and cell-cycle proteins. Unexpectedly, joint classifiers in angiogenesis were uniformly down-regulated in tumor tissues. The eicosanoid pathway enzymes prostacyclin synthase (PGIS) and inducible prostaglandin E(2) synthase (PGES) were joint classifiers that showed opposite effects in lung AC (PGIS down-regulated; PGES up-regulated). Finally, tissue microarrays identified the same protein expression pattern for PGIS and PGES in 108 different non-small cell lung cancer biopsies, and the detection of PGIS had statistically significant prognostic value in patient survival. Thus, the A/J mouse-urethane model reflects significant molecular details of human lung AC, and comparison of changes in orthologous gene expression may provide novel insights into lung carcinogenesis.

Adenocarcinoma↗
PubMed2005

Ablation of MEKK4 kinase activity causes neurulation and skeletal patterning defects in the mouse embryo.

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.

Animals↗
PubMed2004

CP-64131, an aminobenzazepine with cytokine-like properties, stimulates human neutrophil functions through the p38-MAPK pathway.

CP-64131 (CP), an aminobenzazepine with cytokine-like, physiologic effects similar to granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage (GM)-CSF, increases the number of neutrophils and stimulates marrow recovery after doxirubicin ablation. CP can also function as a neutrophil agonist, like formyl-Met-leu-Phe (fMLP). In these studies, we show that CP is unique in that it stimulates the p38-mitogen-activated protein kinase (MAPK) pathway but not extracellular signal-regulated kinase (ERK)1/2 or c-jun N-terminal kinase MAPKs in human neutrophils from peripheral blood. This is in contrast to other neutrophil agonists such as fMLP, interleukin (IL)-8, or GM-CSF, which stimulate multiple MAPK pathways. Like fMLP and IL-8, CP is capable of stimulating superoxide (O2-) production, CD11b expression, and cell polarization in human neutrophils. CP-stimulated O2- production is completely dependent on p38-MAPK activation, as determined by sensitivity to the p38-MAPK inhibitor SB203580. In contrast, SB203580 only partially inhibits expression of CD11b and has no effect on cell polarization stimulated by CP. Therefore, CP treatment of neutrophils activates p38-MAPK but has effects independent of p38-MAPK activation. In human embryonic kidney 293 cells, a human kidney epithelial cell line CP stimulates p38-MAPK and modestly activates ERK1/2. The findings define CP as a novel, small molecule, which has little cellular toxicity in vitro. CP has the ability to activate specific MAPK pathways in different cell types and should prove to be an effective agonist in combination with inhibitors to study biological responses regulated by MAPKs.

Adjuvants, Immunologic↗
PubMed2004

Rac2D57N, a dominant inhibitory Rac2 mutant that inhibits p38 kinase signaling and prevents surface ruffling in bone-marrow-derived macrophages.

Rac2 is a Rho GTPase that is expressed in cells of hematopoietic origin, including neutrophils and macrophages. We recently described an immunodeficient patient with severe, recurrent bacterial infections that had a point mutation in one allele of the Rac2 gene, resulting in the substitution of aspartate 57 with asparagine. To ascertain further the effects of Rac2D57N in leukocytes, Rac2D57N was expressed in primary murine bone-marrow-derived macrophages (cells that we show express approximately equal amounts of Rac1 and Rac2). Rac2D57N expression in macrophages inhibited membrane ruffling. Rac2D57N expression inhibited the formation of macropinosomes, demonstrating a functional effect of the loss of surface membrane dynamics. Surprisingly, Rac2D57N induced an elongated, spread morphology but did not affect microtubule networks. Rac2D57N also inhibited lipopolysaccharide-stimulated p38 kinase activation. Examination of guanine nucleotide binding to recombinant Rac2D57N revealed reduced dissociation of GDP and association of GTP. Coimmunoprecipitation studies of Rac2D57N with RhoGDI alpha and Tiam1 demonstrated increased binding of Rac2D57N to these upstream regulators of Rac signaling relative to the wild type. Enhanced binding of Rac2D57N to its upstream regulators would inhibit Rac-dependent effects on actin cytoskeletal dynamics and p38 kinase signaling.

Actins↗
PubMed2004

MEKK2 regulates the coordinate activation of ERK5 and JNK in response to FGF-2 in fibroblasts.

Mitogen-activated protein kinases (MAPKs) are regulated by MAPK kinases (MKKs), which are in turn regulated by MKK kinases (MKKKs). While a single MKKK can regulate several different MAPK family members, and several MKKKs can often activate the same MAPK, emerging evidence indicates a unique role for individual MKKKs in acting as signaling nodes to coordinately activate different subsets of MAPKs in response to specific cellular stimuli. Thus, while there is much apparent overlap in MAPK regulation by different MKKKs, each MKKK serves a specific purpose in regulation of unique cellular functions. The purpose of this study was to define the specific role of MEKK2, an MKKK, in MAPK regulation and cell function. MEKK2 coordinately activates the ERK5 and JNK pathways. Targeted disruption of MEKK2 expression causes loss of ERK5 and JNK activation in response to FGF-2 in mouse embryonic fibroblasts (MEFs). FGF-2 receptor signaling requires MEKK2 for induction of mRNA for c-Jun, Fra-1, and Fra-2, components of the AP-1 transcription complex. In FGF-2-stimulated MEKK2-/- fibroblasts, c-Jun phosphorylation is inhibited, consistent with a loss of JNK activation. Thus, MEKK2 regulates AP-1 activity at two levels, by regulating both expression of AP-1 components and c-Jun N-terminal phosphorylation. One function of the AP-1 transcription complex is to regulate cytokine gene expression. Expression of IL-1alpha, IL-1beta, IL-6, and TNFalpha is inhibited in MEKK2-/- fibroblasts. Bacterial lipopolysaccharide (LPS) and TNFalpha neither activate ERK5 nor require MEKK2 for JNK activation, demonstrating specificity of MEKK2 in FGF-2 receptor signaling and control of cytokine gene expression.

Animals↗
PubMed2004

JNK regulates the release of proapoptotic mitochondrial factors in reovirus-infected cells.

Reovirus-induced apoptosis is associated with activation of the proapoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) and the JNK-associated transcription factor c-Jun. Here we show that reovirus-induced apoptosis and activation of caspase 3 are inhibited in cells deficient in MEK kinase 1, an upstream activator of JNK in reovirus-infected cells. Inhibition of JNK activity following reovirus infection delays the release of proapoptotic mitochondrial factors and the subsequent onset of apoptosis. In contrast, reovirus-induced apoptosis is not blocked by infection with adenovirus expressing dominant-negative c-Jun, and c-Jun activation does not correlate with apoptosis in reovirus-infected cells. This is the first report demonstrating that JNK is associated with regulation of mitochondrial pathways of apoptosis following viral infection.

Apoptosis↗
PubMed2004

Wiring diagrams of MAPK regulation by MEKK1, 2, and 3.

Mitogen-activated protein kinase (MAPK) pathways are activated by a plethora of stimuli. The literature is filled with papers describing the activation of different MAPKs by almost any stimulus or insult imaginable to cells. In this review, we use signal transduction wiring diagrams to illustrate putative upstream regulators for the MAPK kinase kinases, MEKK1, 2, and 3. Targeted gene disruption of MEKK1, 2, or 3 defined phenotypes for each MEKK associated with loss of specific MAPK regulation. Genetic analysis of MEKK function clearly defines specific components of the wiring diagram that require MEKK1, 2, or 3 for physiological responses. We propose that signal transduction network wiring diagrams are valuable tools for hypothesis building and filtering physiologically relevant phenotypic responses from less connected protein relations in the regulation of MAPK pathways.

Animals↗
PubMed2003

Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock.

Sensing the osmolarity of the environment is a critical response for all organisms. Whereas bacteria will migrate away from high osmotic conditions, most eukaryotic cells are not motile and use adaptive metabolic responses for survival. The p38 MAPK pathway is a crucial mediator of survival during cellular stress. We have discovered a novel scaffold protein that binds to actin, the GTPase Rac, and the upstream kinases MEKK3 and MKK3 in the p38 MAPK phospho-relay module. RNA interference (RNAi) demonstrates that MEKK3 and the scaffold protein are required for p38 activation in response to sorbitol-induced hyperosmolarity. FRET identifies a cytoplasmic complex of the MEKK3 scaffold protein that is recruited to dynamic actin structures in response to sorbitol treatment. Through its ability to bind actin, relocalize to Rac-containing membrane ruffles and its obligate requirement for p38 activation in response to sorbitol, we have termed this protein osmosensing scaffold for MEKK3 (OSM). The Rac-OSM-MEKK3-MKK3 complex is the mammalian counterpart of the CDC42-STE50-STE11-Pbs2 complex in Saccharomyces cerevisiae that is required for the regulation of p38 activity.

Actin Cytoskeleton↗