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1.
Biochem Biophys Res Commun ; 469(1): 126-131, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26616053

ABSTRACT

Cytoplasmic dynein is a macromolecular motor complex with diverse functions in eukaryotic cells. Dynein plays essential roles in intracellular transport of organelles and mitosis, mediated in part by interactions between the dynein intermediate chain 2 (IC-2) subunits and adapter proteins that bind specific cargos. In experiments to identify phosphorylation-dependent binding partners for IC-2 we instead identified a phosphorylation-independent binding partner, the cytosolic chaperonin containing T complex protein 1 (CCT). CCT consists of eight subunits (CCT1-8) and facilitates folding of a subset of newly synthesized proteins. We confirmed interactions between IC-2 and CCT5 and CCT8 in co-immunoprecipitation experiments and determined that the C-terminal half of IC-2 is necessary and sufficient to bind CCT8. Interestingly, co-immunoiprecipitation of IC-2 and CCT is abolished by prior cycloheximide treatment of cells, suggesting that CCT participates in folding of nascent IC-2. In vitro translation experiments employing recombinant CCT complex demonstrated that CCT is able to bind newly synthesized IC-2 after release from the ribosome consistent with a role in folding of IC-2.


Subject(s)
Chaperonin Containing TCP-1/metabolism , Cytoplasmic Dyneins/metabolism , Molecular Motor Proteins/metabolism , Subcellular Fractions/metabolism , Animals , Binding Sites , Protein Binding , Protein Interaction Mapping , Rats
2.
Microbes Infect ; 16(4): 328-36, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24463270

ABSTRACT

Leishmania amazonensis infection promotes alteration of host cellular signaling and intracellular parasite survival, but specific mechanisms are poorly understood. We previously demonstrated that L. amazonensis infection of dendritic cells (DC) activated extracellular signal-regulated kinase (ERK), an MAP-kinase kinase kinase, leading to altered DC maturation and non-healing cutaneous leishmaniasis. Studies using growth factors and cell lines have shown that targeted, robust, intracellular phosphorylation of ERK1/2 from phagolysosomes required recruitment and association with scaffolding proteins, including p14/MP1 and MORG1, on the surface of late endosomes. Based on the intracellular localization of L. amazonensis within a parasitophorous vacuole with late endosome characteristics, we speculated that scaffolding proteins would be important for intracellular parasite-mediated ERK signaling. Our findings demonstrate that MP1, MORG1, and ERK all co-localized on the surface of parasite-containing LAMP2-positive phagolysosomes. Infection of MEK1 mutant fibroblasts unable to bind MP1 demonstrated dramatically reduced ERK1/2 phosphorylation following L. amazonensis infection but not following positive control EGF treatment. This novel mechanism for localization of intracellular L. amazonensis-mediated ERK1/2 phosphorylation required the endosomal scaffold protein MP1 and localized to L. amazonensis parasitophorous vacuoles. Understanding how L. amazonensis parasites hijack host cell scaffold proteins to modulate signaling cascades provides targets for antiprotozoal drug development.


Subject(s)
Dendritic Cells/immunology , Dendritic Cells/parasitology , Extracellular Signal-Regulated MAP Kinases/metabolism , Host-Pathogen Interactions , Leishmania mexicana/immunology , Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Line , Fibroblasts/immunology , Fibroblasts/parasitology , Lysosomes/chemistry , Lysosomes/parasitology , Mice, Inbred C3H , Phagosomes/chemistry , Phagosomes/parasitology
3.
Mol Med ; 19: 253-62, 2013 Aug 28.
Article in English | MEDLINE | ID: mdl-23821363

ABSTRACT

Although a relationship between PDZK1 expression and estrogen receptor (ER)-α stimulation has been suggested, the nature of such a connection and the function of PDZK1 in breast cancer remain unknown. Human tissue microarrays (cancer tissue: 262 cores; normal tissue: 87 cores) and breast cancer cell lines were used to conduct the study. We show that PDZK1 protein expression is tightly correlated with human breast malignancy, is negatively correlated with age and had no significant correlation with ER-α expression levels. PDZK1 exhibited an exclusive epithelial expression with mostly cytosolic subcellular localization. Additionally, 17ß-estradiol induced PDZK1 expression above its basal level more than 24 h after treatment in MCF-7 cells. PDZK1 expression was indirectly regulated by ER-α stimulation, requiring insulinlike growth factor 1 receptor (IGF-1R) expression and function. The molecular link between PDZK1 and IGF-1R was supported by a significant correlation between protein and mRNA levels (r = 0.591, p < 0.001, and r = 0.537, p < 0.001, respectively) of the two factors in two different cohorts of human breast cancer tissues. Interestingly, PDZK1 knockdown in MCF-7 cells blocked ER-dependent growth and reduced c-Myc expression, whereas ectopic expression of PDZK1 enhanced cell proliferation in the presence or absence of 17ß-estradiol potentially through an increase in c-Myc expression, suggesting that PDZK1 has oncogenic activity. PDKZ1 also appeared to interact with the Src/ER-α/epidermal growth factor receptor (EGFR) complex, but not with IGF-1R and enhanced EGFR-stimulated MEK/ERK1/2 signaling. Collectively, our results clarify the relationship between ER-α and PDZK1, propose a direct relationship between PDZK1 and IGF-1R, and identify a novel oncogenic activity for PDZK1 in breast cancer.


Subject(s)
Breast Neoplasms/metabolism , Carrier Proteins/metabolism , Estrogen Receptor alpha/metabolism , Receptor, IGF Type 1/metabolism , Cell Line, Tumor , Estrogens/pharmacology , Female , Humans , Membrane Proteins , Tissue Array Analysis
4.
Int J Mol Sci ; 14(2): 3595-620, 2013 Feb 07.
Article in English | MEDLINE | ID: mdl-23434660

ABSTRACT

Extracellular-signal regulated kinase (ERK) signaling is required for a multitude of physiological and patho-physiological processes. However, the identities of the proteins that ERK phosphorylates to elicit these responses are incompletely known. Using an affinity purification methodology of general utility, here we identify cytoplasmic dynein intermediate chain 2 (DYNC1I-2, IC-2) as a novel substrate for ERK following epidermal growth factor receptor stimulation of fibroblasts. IC-2 is a subunit of cytoplasmic dynein, a minus-end directed motor protein necessary for transport of diverse cargos along microtubules. Emerging data support the hypothesis that post-translational modification regulates dynein but the signaling mechanisms used are currently unknown. We find that ERK phosphorylates IC-2 on a novel, highly conserved Serine residue proximal to the binding site for the p150Glued subunit of the cargo adapter dynactin. Surprisingly, neither constitutive phosphorylation nor a phosphomimetic substitution of this Serine influences binding of p150Glued to IC-2. These data suggest that ERK phosphorylation of IC-2 regulates dynein function through mechanisms other than its interaction with dynactin.

5.
J Biol Chem ; 288(3): 1458-68, 2013 Jan 18.
Article in English | MEDLINE | ID: mdl-23184953

ABSTRACT

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.


Subject(s)
Asthma/drug therapy , GATA3 Transcription Factor/genetics , Immunologic Factors/therapeutic use , Inflammation/drug therapy , Interleukin-4/genetics , Minocycline/therapeutic use , NF-kappa B/genetics , Receptors, Antigen, T-Cell/genetics , Animals , Asthma/complications , Asthma/genetics , Asthma/immunology , GATA3 Transcription Factor/agonists , GATA3 Transcription Factor/immunology , Gene Expression Regulation/drug effects , Immunoglobulin E/genetics , Immunoglobulin E/immunology , Immunologic Factors/pharmacology , Inflammation/complications , Inflammation/genetics , Inflammation/immunology , Interleukin-4/antagonists & inhibitors , Interleukin-4/immunology , Male , Mice , Mice, Inbred C57BL , Minocycline/pharmacology , NF-kappa B/agonists , NF-kappa B/immunology , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/immunology , Receptors, Antigen, T-Cell/antagonists & inhibitors , Receptors, Antigen, T-Cell/immunology , Signal Transduction/drug effects
6.
Cell Host Microbe ; 12(5): 657-68, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-23159055

ABSTRACT

After host cell entry, Salmonella replicate in membrane-bound compartments, which accumulate a dense meshwork of F-actin through the kinase activity of the Salmonella SPI-2 type III secretion effector SteC. We find that SteC promotes actin cytoskeleton reorganization by activating a signaling pathway involving the MAP kinases MEK and ERK, myosin light chain kinase (MLCK) and Myosin IIB. Specifically, SteC phosphorylates MEK directly on serine 200 (S200), a previously unstudied phosphorylation site. S200 phosphorylation is predicted to displace a negative regulatory helix causing autophosphorylation on the known MEK activatory residues, S218 and S222. In support of this, substitution of S200 with alanine prevented phosphorylation on S218 and S222, and phosphomimetic mutations of S200 stimulated phosphorylation of these residues. Both steC-null and kinase-deficient mutant strains displayed enhanced replication in infected cells, suggesting that SteC manipulates the actin cytoskeleton to restrain bacterial growth, thereby regulating virulence.


Subject(s)
Actin Cytoskeleton/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Protein Kinases/metabolism , Salmonella typhimurium/metabolism , Salmonella typhimurium/pathogenicity , 3T3 Cells , Amino Acid Substitution , Animals , Cell Line, Tumor , Female , HeLa Cells , Humans , MAP Kinase Signaling System , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Molecular Dynamics Simulation , Myosin-Light-Chain Kinase/metabolism , Nonmuscle Myosin Type IIB/metabolism , Phosphorylation , Salmonella Infections, Animal/microbiology , Salmonella typhimurium/genetics
7.
J Neurosci ; 32(44): 15495-510, 2012 Oct 31.
Article in English | MEDLINE | ID: mdl-23115187

ABSTRACT

The retrograde transport of Trk-containing endosomes from the axon to the cell body by cytoplasmic dynein is necessary for axonal and neuronal survival. We investigated the recruitment of dynein to signaling endosomes in rat embryonic neurons and PC12 cells. We identified a novel phosphoserine on the dynein intermediate chains (ICs), and we observed a time-dependent neurotrophin-stimulated increase in intermediate chain phosphorylation on this site in both cell types. Pharmacological studies, overexpression of constitutively active MAP kinase kinase, and an in vitro assay with recombinant proteins demonstrated that the intermediate chains are phosphorylated by the MAP kinase ERK1/2, extracellular signal-regulated kinase, a major downstream effector of Trk. Live cell imaging with fluorescently tagged IC mutants demonstrated that the dephosphomimic mutants had significantly reduced colocalization with Trk and Rab7, but not a mitochondrial marker. The phosphorylated intermediate chains were enriched on immunoaffinity-purified Trk-containing organelles. Inhibition of ERK reduced the amount of phospho-IC and the total amount of dynein that copurified with the signaling endosomes. In addition, inhibition of ERK1/2 reduced the motility of Rab7- and TrkB-containing endosomes and the extent of their colocalization with dynein in axons. NGF-dependent survival of sympathetic neurons was significantly reduced by the overexpression of the dephosphomimic mutant IC-1B-S80A, but not WT IC-1B, further demonstrating the functional significance of phosphorylation on this site. These results demonstrate that neurotrophin binding to Trk initiates the recruitment of cytoplasmic dynein to signaling endosomes through ERK1/2 phosphorylation of intermediate chains for their subsequent retrograde transport in axons.


Subject(s)
Axonal Transport/physiology , Cytoplasm/physiology , Dyneins/physiology , Endosomes/physiology , MAP Kinase Signaling System/physiology , Receptor, trkA/physiology , Animals , Blotting, Western , Cell Membrane/metabolism , Cell Membrane/physiology , Cell Survival/physiology , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , MAP Kinase Signaling System/genetics , Nerve Growth Factor/physiology , Nerve Growth Factors/pharmacology , Neurons/physiology , Organelles/physiology , PC12 Cells , Phosphorylation , Plasmids/genetics , RNA, Small Interfering/genetics , Rats , Signal Transduction/physiology , Transfection
8.
PLoS One ; 6(6): e20710, 2011.
Article in English | MEDLINE | ID: mdl-21677780

ABSTRACT

RACK1 proteins belong to the eukaryote WD40-repeat protein family and function as spatial regulators of multiple cellular events, including signaling pathways, the cell cycle and translation. For this latter role, structural and genetic studies indicate that RACK1 associates with the ribosome through two conserved positively charged amino acids in its first WD40 domain. Unlike RACK1s, including Trypanosoma brucei RACK1 (TbRACK1), only one of these two positively-charged residues is conserved in the first WD40 domain of the Leishmania major RACK1 ortholog, LACK. We compared virulence-attenuated LACK single copy (LACK/-) L. major, with L. major expressing either two LACK copies (LACK/LACK), or one copy each of LACK and TbRACK1 (LACK/TbRACK1), to evaluate the function of these structurally distinct RACK1 orthologs with respect to translation, viability at host temperatures and pathogenesis. Our results indicate that although the ribosome-binding residues are not fully conserved in LACK, both LACK and TbRACK1 co-sedimented with monosomes and polysomes in LACK/LACK and LACK/TbRACK1 L. major, respectively. LACK/LACK and LACK/TbRACK1 strains differed in their sensitivity to translation inhibitors implying that minor sequence differences between the RACK1 proteins can alter their functional properties. While biochemically distinguishable, both LACK/LACK and LACK/TbRACK1 lines were more tolerant of elevated temperatures, resistant to translation inhibitors, and displayed robust pathogenesis in vivo, contrasting to LACK/- parasites.


Subject(s)
Antigens, Protozoan/metabolism , Leishmania major/physiology , Leishmania major/pathogenicity , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Antigens, Protozoan/genetics , Cell Cycle/physiology , Female , Leishmania major/cytology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Polyribosomes/metabolism , Protein Biosynthesis , Protozoan Proteins/genetics , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Sequence Alignment , Temperature , Transcription, Genetic , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolism
9.
J Immunol ; 185(3): 1894-902, 2010 Aug 01.
Article in English | MEDLINE | ID: mdl-20610652

ABSTRACT

The role of NF-kappaB in the expression of inflammatory genes and its participation in the overall inflammatory process of chronic diseases and acute tissue injury are well established. We and others have demonstrated a critical involvement of poly(ADP-ribose) polymerase (PARP)-1 during inflammation, in part, through its relationship with NF-kappaB. However, the mechanism by which PARP-1 affects NF-kappaB activation has been elusive. In this study, we show that PARP-1 inhibition by gene knockout, knockdown, or pharmacologic blockade prevented p65 NF-kappaB nuclear translocation in smooth muscle cells upon TLR4 stimulation, NF-kappaB DNA-binding activity, and subsequent inducible NO synthase and ICAM-1 expression. Such defects were reversed by reconstitution of PARP-1 expression. PARP-1 was dispensable for LPS-induced IkappaBalpha phosphorylation and subsequent degradation but was required for p65 NF-kappaB phosphorylation. A perinuclear p65 NF-kappaB localization in LPS-treated PARP-1(-/-) cells was associated with an export rather an import defect. Indeed, whereas PARP-1 deficiency did not alter expression of importin alpha3 and importin alpha4 and their cytosolic localization, the cytosolic levels of exportin (Crm)-1 were increased. Crm1 inhibition promoted p65 NF-kappaB nuclear accumulation as well as reversed LPS-induced p65 NF-kappaB phosphorylation and inducible NO synthase and ICAM-1 expression. Interestingly, p65 NF-kappaB poly(ADP-ribosyl)ation decreased its interaction with Crm1 in vitro. Pharmacologic inhibition of PARP-1 increased p65 NF-kappaB-Crm1 interaction in LPS-treated smooth muscle cells. These results suggest that p65 NF-kappaB poly(ADP-ribosyl)ation may be a critical determinant for the interaction with Crm1 and its nuclear retention upon TLR4 stimulation. These results provide novel insights into the mechanism by which PARP-1 promotes NF-kappaB nuclear retention, which ultimately can influence NF-kappaB-dependent gene regulation.


Subject(s)
Cell Nucleus/metabolism , Karyopherins/physiology , Poly(ADP-ribose) Polymerases/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Toll-Like Receptor 4/physiology , Transcription Factor RelA/metabolism , Active Transport, Cell Nucleus/genetics , Active Transport, Cell Nucleus/immunology , Animals , Cell Line , Cell Nucleus/enzymology , Cell Nucleus/immunology , Cells, Cultured , DNA-Binding Proteins/biosynthesis , Gene Expression Regulation/immunology , Humans , Intercellular Adhesion Molecule-1/biosynthesis , Karyopherins/antagonists & inhibitors , Lipopolysaccharides/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocytes, Smooth Muscle/enzymology , Myocytes, Smooth Muscle/immunology , Myocytes, Smooth Muscle/metabolism , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/deficiency , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Transcription Factor RelA/antagonists & inhibitors , Transcription Factor RelA/physiology , Exportin 1 Protein
10.
Mol Cell Biol ; 30(13): 3233-48, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20439493

ABSTRACT

Cell migration is critical for normal development and for pathological processes including cancer cell metastasis. Dynamic remodeling of focal adhesions and the actin cytoskeleton are crucial determinants of cell motility. The Rho family and the mitogen-activated protein kinase (MAPK) module consisting of MEK-extracellular signal-regulated kinase (ERK) are important regulators of these processes, but mechanisms for the integration of these signals during spreading and motility are incompletely understood. Here we show that ERK activity is required for fibronectin-stimulated Rho-GTP loading, Rho-kinase function, and the maturation of focal adhesions in spreading cells. We identify p190A RhoGAP as a major target for ERK signaling in adhesion assembly and identify roles for ERK phosphorylation of the C terminus in p190A localization and activity. These observations reveal a novel role for ERK signaling in adhesion assembly in addition to its established role in adhesion disassembly.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesions/metabolism , Repressor Proteins/metabolism , rhoA GTP-Binding Protein/metabolism , Actins/metabolism , Amino Acid Sequence , Animals , Butadienes/metabolism , Cell Adhesion/physiology , Cell Line , Enzyme Inhibitors/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Fibronectins/metabolism , Humans , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Molecular Sequence Data , Nitriles/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Sequence Alignment , Vinculin/metabolism , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/genetics
11.
Behav Pharmacol ; 21(2): 121-34, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20177375

ABSTRACT

In contrast to estrogen in female rats, testosterone in male rats may decrease cholinergic activity in the brain, thereby attenuating behaviors mediated by the cholinergic system. To investigate this possibility, the interactive effects of the gonadal hormones and donepezil, an acetylcholinesterase (AChE) inhibitor, on the responding of male rats were examined under a multiple schedule of repeated acquisition and performance of response sequences and on AChE activity in specific brain regions. Donepezil dose-effect curves (0.56-10 mg/kg) were determined in males that were gonadally intact, gonadectomized (GX), GX with testosterone replacement (GX+T) or GX with estradiol replacement (GX+E). In all four groups, donepezil produced dose-dependent rate-decreasing and error-increasing effects in the acquisition and performance components. However, disruptions of response rate and accuracy in both components occurred at lower doses in GX and GX+E males than in intact males. The GX+E males also had the highest percentage of errors under control (saline) conditions in the acquisition components. In terms of AChE activity, GX males had higher levels in the prefrontal cortex, striatum and hippocampus, but lower levels in the midbrain, compared with intact males; hypothalamic and cortical levels were comparable for the GX and intact groups. Together, these results in male rats indicate that the potency of donepezil's disruptive effects on the responding under a complex operant procedure requiring learning and performance of response sequences depends upon the gonadal hormone status, and that the effects of testosterone on cholinergic activity vary among brain regions.


Subject(s)
Brain/enzymology , Cholinesterase Inhibitors/pharmacology , Conditioning, Operant/drug effects , Gonadal Hormones/pharmacology , Indans/pharmacology , Piperidines/pharmacology , Animals , Brain/drug effects , Donepezil , Dose-Response Relationship, Drug , Drug Interactions , Estradiol/pharmacology , Gonadal Hormones/deficiency , Hormone Replacement Therapy/psychology , Male , Rats , Rats, Long-Evans , Reinforcement Schedule , Testosterone/pharmacology
12.
Cell Commun Signal ; 7: 26, 2009 Nov 23.
Article in English | MEDLINE | ID: mdl-19930650

ABSTRACT

ERK signaling regulates focal adhesion disassembly during cell movement, and increased ERK signaling frequently contributes to enhanced motility of human tumor cells. We previously found that the ERK scaffold MEK Partner 1 (MP1) is required for focal adhesion disassembly in fibroblasts. Here we test the hypothesis that MP1-dependent ERK signaling regulates motility of DU145 prostate cancer cells. We find that MP1 is required for motility on fibronectin, but not for motility stimulated by serum or EGF. Surprisingly, MP1 appears not to function through its known binding partners MEK1 or PAK1, suggesting the existence of a novel pathway by which MP1 can regulate motility on fibronectin. MP1 may function by regulating the stability or expression of paxillin, a key regulator of motility.

13.
Cardiovasc Res ; 78(3): 429-39, 2008 Jun 01.
Article in English | MEDLINE | ID: mdl-18245064

ABSTRACT

AIMS: The aim of this study was to take a combination of animal and cell culture approaches to examine the individual responses of vascular cells to varying inflammatory factors in order to gain insights on the mechanism(s) by which poly(ADP-ribose) polymerase (PARP) inhibition promotes factors of plaque stability. METHODS AND RESULTS: Apolipoprotein (ApoE(-/-)) mice fed a high-fat diet were used as a model of atherosclerosis. Primary endothelial cells, smooth muscle cells (SMCs), and ex-vivo generated foam cells (FCs) were used in our in vitro studies. PARP inhibition significantly decreased the markers of oxidative stress and caspase-3 activation and increased smooth muscle actin within plaques from ApoE(-/-) mice fed a high-fat diet. PARP inhibition protected against apoptosis and/or necrosis in SMCs and endothelial cells in response to H(2)O(2) or tumour necrosis factor (TNF). Remarkably, PARP inhibition in FCs resulted in significant sensitization to 7-ketocholesterol (7-KC) by increasing cellular-toxic-free cholesterol, potentially through a down-regulation of acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) expression. 7-KC induced necrosis exclusively in endothelial cells, which was, surprisingly, unaffected by PARP inhibition indicating that PARP inhibition does not prevent all forms of necrotic cell death. In SMCs, PARP-1 inhibition by gene deletion conferred protection against 7-KC or TNF, potentially by reducing caspase-3-like activation, preventing induction of c-Jun N-terminal protein kinase phosphorylation, and inducing extracellular signal-regulated kinase phosphorylation independently of PARP classical enzymatic activity. CONCLUSIONS: These data present PARP-1 as an important player in the death of cells constituting atherosclerotic plaques contributing to plaque dynamics. PARP inhibition may be a protective, a neutral, or a sensitizing factor. Additionally, PARP-1 may be a novel factor that can alter lipid metabolism. These novel functions of PARP not only challenge the current understanding of the role of the enzyme in cell death but also provide insights on the intricate contribution of PARP in cellular responses to predominant inflammatory factors within atherosclerotic plaques, presenting additional evidence for the viability of PARP inhibition as a therapeutic strategy for atherosclerosis.


Subject(s)
Acetyl-CoA C-Acetyltransferase/metabolism , Atherosclerosis/prevention & control , Endothelial Cells/drug effects , Enzyme Inhibitors/pharmacology , Foam Cells/drug effects , Isoquinolines/pharmacology , Muscle, Smooth, Vascular/drug effects , Poly(ADP-ribose) Polymerase Inhibitors , Thiophenes/pharmacology , Actins/metabolism , Animals , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Apoptosis/drug effects , Atherosclerosis/enzymology , Atherosclerosis/pathology , Caspase 3/metabolism , Cells, Cultured , DNA Damage/drug effects , Disease Models, Animal , Endothelial Cells/enzymology , Endothelial Cells/pathology , Foam Cells/enzymology , Foam Cells/pathology , Hydrogen Peroxide/metabolism , Ketocholesterols/metabolism , MAP Kinase Kinase 4/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/enzymology , Necrosis , Oxidative Stress/drug effects , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Time Factors , Tumor Necrosis Factor-alpha/metabolism
14.
Cell Signal ; 19(8): 1621-32, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17553668

ABSTRACT

Cell migration is critical for many physiological processes and is often misregulated in developmental disorders and pathological conditions including cancer and neurodegeneration. MAPK signaling and the Rho family of proteins are known regulators of cell migration that exert their influence on cellular cytoskeleton during cell adhesion and migration. Here we review data supporting the view that localized ERK signaling mediated through recently identified scaffold proteins may regulate cell migration.


Subject(s)
Cytoskeleton/physiology , Mitogen-Activated Protein Kinases/physiology , Signal Transduction , Actins/metabolism , Animals , Cell Adhesion/physiology , Cell Movement/physiology , Humans , Models, Biological , rho GTP-Binding Proteins/physiology
15.
Cell Signal ; 19(7): 1488-96, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17314031

ABSTRACT

Extracellular signal-Regulated Kinase (ERK) controls a variety of cellular processes, including cell proliferation and cell motility. While oncogenic mutations in Ras and B-Raf result in deregulated ERK activity and proliferation and migration in some tumor cells, other tumors exhibit elevated ERK signaling in the absence of these mutations. Here we provide evidence that PAK can directly activate MEK1 by a mechanism distinct from conventional Ras/Raf mediated activation. We find that PAK phosphorylation of MEK1 serine 298 stimulates MEK1 autophosphorylation on the activation loop, and activation of MEK1 activity towards ERK in in vitro reconstitution experiments. Serines 218 and/or 222 in the MEK1 activation loop are required for PAK-stimulated MEK1 activity towards ERK. MEK2, which is a poor target for PAK phosphorylation in cells, is not activated in this manner. Tissue culture experiments verify that this mechanism is used in suspended fibroblasts expressing mutationally activated PAK1. We speculate that aberrant signaling through PAK may directly induce anchorage-independent MEK1 activation in tumor cells lacking oncogenic Ras or Raf mutations, and that this mechanism may contribute to localized MEK signaling in focal contacts and adhesions during cell adhesion or migration.


Subject(s)
MAP Kinase Kinase 1/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , COS Cells , Cell Adhesion , Chlorocebus aethiops , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts/cytology , Fibroblasts/enzymology , Fibronectins/metabolism , Humans , Phosphorylation , Proto-Oncogene Proteins B-raf/metabolism , Rats , Serine/metabolism , p21-Activated Kinases , ras Proteins/metabolism
16.
Mol Cell Biol ; 25(12): 5119-33, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15923628

ABSTRACT

How the extracellular signal-regulated kinase (ERK) cascade regulates diverse cellular functions, including cell proliferation, survival, and motility, in a context-dependent manner remains poorly understood. Compelling evidence indicates that scaffolding molecules function in yeast to channel specific signals through common components to appropriate targets. Although a number of putative ERK scaffolding proteins have been identified in mammalian systems, none has been linked to a specific biological response. Here we show that the putative scaffold protein MEK partner 1 (MP1) and its partner p14 regulate PAK1-dependent ERK activation during adhesion and cell spreading but are not required for ERK activation by platelet-derived growth factor. MP1 associates with active but not inactive PAK1 and controls PAK1 phosphorylation of MEK1. Our data further show that MP1, p14, and MEK1 serve to inhibit Rho/Rho kinase functions necessary for the turnover of adhesion structures and cell spreading and reveal a signal-channeling function for a MEK1/ERK scaffold in orchestrating cytoskeletal rearrangements important for cell motility.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , MAP Kinase Kinase 1/metabolism , Protein Serine-Threonine Kinases/metabolism , rho GTP-Binding Proteins/metabolism , Actin Depolymerizing Factors , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Animals , Cell Adhesion/physiology , Cell Line , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesions/metabolism , Humans , Intracellular Signaling Peptides and Proteins , MAP Kinase Kinase 1/genetics , Microfilament Proteins/metabolism , Molecular Sequence Data , Protein Binding , Protein Serine-Threonine Kinases/genetics , Proteins/genetics , Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Sequence Alignment , Signal Transduction/physiology , Two-Hybrid System Techniques , p21-Activated Kinases , rho GTP-Binding Proteins/genetics , rho-Associated Kinases
17.
J Cell Biochem ; 94(4): 708-19, 2005 Mar 01.
Article in English | MEDLINE | ID: mdl-15547943

ABSTRACT

Specificity in signal transduction can be achieved through scaffolds, anchors, and adapters that assemble generic signal transduction components in specific combinations and locations. MEK Partner-1 (MP1) was identified as a potential "scaffold" protein for the mammalian extracellular signal-regulated kinase (ERK) pathway. To gain insight into the interactions of MP1 with the ERK pathway, we analyzed the ability of MP1 to bind to MEK1, ERK1, and to itself, and the regulation of these interactions. Gel filtration of cell lysates revealed two major MP1 peaks: a broad high molecular weight peak and a 28 kDa complex. An MP1 mutant that lost MEK1 binding no longer enhanced RasV12-stimulated ERK1 activity, and functioned as a dominant negative, consistent with the concept that MP1 function depends on facilitating these oligomerizations. Activation of the ERK pathway by serum or by RasV12 did not detectably affect MP1-MP1 dimerization or MP1-MEK1 interactions, but caused the dissociation of the MP1-ERK1 complex. Surprisingly, pharmacological inhibition of ERK activation did not restore the complex, suggesting that regulation of complex formation occurs independently of ERK phosphorylation. These results support the concept that MP1 functions as a regulator of MAP kinase signaling by binding to MEK1 and regulating its association with a larger signaling complex that may sequentially service multiple molecules of ERK.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , MAP Kinase Signaling System , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line , Cricetinae , Enzyme Activation , Gene Deletion , Humans , Mitogen-Activated Protein Kinase 3/metabolism , Mutation/genetics , Phosphorylation , Protein Binding , Serum , ras Proteins/genetics , ras Proteins/metabolism
18.
Mol Cell Biol ; 24(6): 2308-17, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14993270

ABSTRACT

Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1. It was also shown that activation of MEK1 and ERK by integrins depends on PAK phosphorylation of S298 in the PRS. Here we report a novel MEK1-specific regulatory feedback mechanism that provides a means by which activated ERK can terminate continued PAK phosphorylation of MEK1. Activated ERK can phosphorylate T292 in the PRS, and this blocks the ability of PAK to phosphorylate S298 and of Rac-PAK signaling to enhance MEK1-ERK complex formation. Preventing ERK feedback phosphorylation on T292 during cellular adhesion prolonged phosphorylation of S298 by PAK and phosphorylation of S218 and S222, the MEK1 activating sites. We propose that activation of ERK during adhesion creates a feedback system in which ERK phosphorylates MEK1 on T292, and this in turn blocks additional S298 phosphorylation in response to integrin signaling.


Subject(s)
Cell Adhesion/physiology , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Animals , Binding Sites/genetics , COS Cells , Enzyme Activation , Feedback , MAP Kinase Kinase 1 , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase Kinases/chemistry , Mitogen-Activated Protein Kinase Kinases/genetics , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Mutagenesis, Site-Directed , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , p21-Activated Kinases
19.
J Cell Biol ; 162(2): 281-91, 2003 Jul 21.
Article in English | MEDLINE | ID: mdl-12876277

ABSTRACT

Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane-proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.


Subject(s)
Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , COS Cells , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cell Line , Chlorocebus aethiops , Enzyme Activation , Epidermal Growth Factor/pharmacology , Fibroblasts/cytology , Fibroblasts/enzymology , Fibronectins/metabolism , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Focal Adhesions/metabolism , Gene Expression Regulation , Insulin-Like Growth Factor I/pharmacology , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase Kinases/genetics , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-raf/drug effects , Proto-Oncogene Proteins c-raf/metabolism , Pyrimidines/pharmacology , Rats , Recombinant Proteins/metabolism , p21-Activated Kinases , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/drug effects , src-Family Kinases/metabolism
20.
Mol Cell Biol ; 22(17): 6023-33, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12167697

ABSTRACT

Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-delta19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-delta19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.


Subject(s)
MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase 1/physiology , Mitogen-Activated Protein Kinase Kinases/physiology , Protein Serine-Threonine Kinases/physiology , rac GTP-Binding Proteins/physiology , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Cell Adhesion , Chlorocebus aethiops , Culture Media, Serum-Free/pharmacology , Enzyme Activation , Epidermal Growth Factor/pharmacology , MAP Kinase Kinase 1 , Macromolecular Substances , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase Kinases/genetics , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Proto-Oncogene Proteins p21(ras)/physiology , Recombinant Fusion Proteins/physiology , Sequence Deletion , Transfection , p21-Activated Kinases
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