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1.
Cell Growth Differ ; 11(1): 19-30, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10672900

ABSTRACT

Thrombin causes rapid pseudopod detachment and shortening in Dunning rat prostatic carcinoma (MAT-Lu) cells. As seen by interference reflection microscopy and by immunofluorescence analysis with antibodies to paxillin and talin, the primary event is disassembly of adhesion sites. Biochemically, thrombin is a potent activator of cytosolic phospholipase A2 and increases eicosanoid production in these cells. The pseudopod effects are blocked by lipoxygenase (but not cyclooxygenase) inhibitors. Arachidonic acid and 12(S)-hydroxyeicosatetraenoic acid or 15(S)-hydroxyeicosatetraenoic acid mimic the thrombin effect. We conclude that in certain cancer cells, thrombin is a pseudopod repellent that exerts its effect via a cascade involving cytosolic phospholipase A2, 12/15-lipoxygenase, and 12(S)- and/or 15(S)-hydroxyeicosatetraenoic acid.


Subject(s)
Arachidonate 12-Lipoxygenase/metabolism , Arachidonate 15-Lipoxygenase/metabolism , Phospholipases A/metabolism , Pseudopodia/drug effects , Thrombin/pharmacology , Animals , Arachidonic Acids/pharmacology , Cell Adhesion/drug effects , Cell Movement , Cytoskeletal Proteins/immunology , Eicosanoids/pharmacology , Fatty Acids/pharmacology , Fluorescent Antibody Technique , Male , Microscopy, Phase-Contrast , Paxillin , Peptide Fragments/pharmacology , Phospholipases A2 , Phosphoproteins/immunology , Prostatic Neoplasms , Rats , Rats, Inbred Strains , Talin/immunology , Tumor Cells, Cultured
2.
J Neurosci ; 19(24): 10843-55, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10594066

ABSTRACT

The studies presented here explore intracellular signals resulting from the action of repellents on growth cones. Growth cone challenge with thrombin or thrombin receptor-activating peptide (TRAP) triggers collapse via a receptor-mediated process. The results indicate that this involves activation of cytosolic phospholipase A(2) (PLA(2)) and eicosanoid synthesis. The collapse response to repellents targets at least two functional units of the growth cone, the actin cytoskeleton and substratum adhesion sites. We show in a cell-free assay that thrombin and TRAP cause the detachment of isolated growth cones from laminin. Biochemical analyses of isolated growth cones reveal that thrombin and TRAP stimulate cytosolic PLA(2) but not phospholipase C. In addition, thrombin stimulates synthesis of 12- and 15-hydroxyeicosatetraenoic acid (HETE) from the released arachidonic acid via a lipoxygenase (LO) pathway. A selective LO inhibitor blocks 12/15-HETE synthesis in growth cones and inhibits thrombin-induced growth cone collapse. Exogenously applied 12(S)-HETE mimics the thrombin effect and induces growth cone collapse in culture. These observations indicate that thrombin-induced growth cone collapse occurs by a mechanism that involves the activation of cytosolic PLA(2) and the generation of 12/15-HETE.


Subject(s)
Eicosanoids/biosynthesis , Growth Cones/physiology , Phospholipases A/biosynthesis , Thrombin/physiology , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/biosynthesis , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/pharmacology , Animals , Arachidonate 12-Lipoxygenase/metabolism , Arachidonate 15-Lipoxygenase/metabolism , Cells, Cultured , Cerebral Cortex/embryology , Embryo, Mammalian/cytology , Enzyme Activation/physiology , Growth Cones/drug effects , Growth Cones/ultrastructure , Hydroxyeicosatetraenoic Acids/biosynthesis , Peptide Fragments/pharmacology , Phospholipases A/metabolism , Rats , Rats, Sprague-Dawley , Thrombin/pharmacology
3.
J Neurochem ; 67(6): 2599-608, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8931495

ABSTRACT

Nerve growth cones isolated from fetal rat brain exhibit in their cytosol a robust level of phospholipase A2 activity hydrolyzing phosphatidylinositol (PI) and phosphatidylethanolamine (PE) but not phosphatidylcholine (PC). Western blot analysis with an antibody to the well-characterized cytosolic phospholipase A2 (mol wt 85,000) reveals only trace amounts of this PC- and PE-selective enzyme in growth cones. By gel filtration on Superose 12, growth cone phospholipase A2 activity elutes essentially as two peaks of high molecular mass, at approximately 65 kDa and at well over 100 kDa. Anion exchange chromatography completely separates a PI-selective from a PE-selective activity, indicating the presence of two different, apparently monoselective phospholipase A2 species. The PI-selective enzyme, the predominant phospholipase A2 activity in whole growth cones, is enriched greatly in these structures relative to their parent fractions from fetal brain. This phospholipase A2 is resistant to reducing agents and is found in the cytosol as well as membrane-associated in the presence of Ca2+. However, its catalytic activity is Ca(2+)-independent regardless of whether the enzyme is associated with pure substrate or mixed-lipid growth cone vesicles. The PE-selective phospholipase A2 in growth cones was studied in less detail but shares with the PI-selective enzyme several properties, including intracellular localization, the existence of cytosolic and membrane-associated forms, and Ca2+ independence. Our data indicate growth cones contain two high-molecular-weight forms of phospholipase A2 that share many properties with known, Ca(2+)-independent cytosolic phospholipase A2 species but that appear to be monoselective for PI and PE, respectively. In particular, the PI-selective enzyme may represent a new member of the growing family of cytoplasmic phospholipases A2. The enrichment of the PI-selective phospholipase A2 in growth cones suggests it plays a major role in the regulation of growth cone function.


Subject(s)
Brain/cytology , Neurites/enzymology , Phospholipases A/chemistry , Phospholipases A/metabolism , Animals , Arachidonic Acid/metabolism , Calcium/pharmacology , Chromatography, Gel , Chromatography, Ion Exchange , Cytosol/enzymology , Fetus/cytology , Fetus/enzymology , Molecular Weight , Neurons/enzymology , Neurons/ultrastructure , Phosphatidylinositols/metabolism , Phospholipases A/drug effects , Phospholipases A2 , Rats , Subcellular Fractions/enzymology , Substrate Specificity
4.
Brain Res Dev Brain Res ; 96(1-2): 83-96, 1996 Oct 23.
Article in English | MEDLINE | ID: mdl-8922671

ABSTRACT

The investigation of the molecular properties of nerve growth cones depends to a significant degree on their isolation from fetal brain in the form of 'growth cone particles' (GCPs). The availability of markers for developing axons and dendrites, as well as glial cells, has made it possible to characterize the GCP fraction in much greater detail than before and to optimize its yield. Marker analyses show that a member of the N-CAM family (5B4-CAM), synaptophysin, and especially GAP-43 and non-phosphorylated tau, are enriched in the GCP fraction. In contrast, MAP2 and, particularly, glial fibrillary acidic protein and vimentin are fractionated away from GCPs. Furthermore, GCP yield can be doubled relative to the original procedure, without compromising purity, by raising the sucrose concentration of the fractionation gradient's uppermost layer. The results indicate that GCPs are highly purified growth cone fragments with very little glial contamination, and that they are primarily of axonal origin.


Subject(s)
Axons/ultrastructure , Brain/embryology , Nerve Tissue Proteins/analysis , Animals , Astrocytes/cytology , Biomarkers , Brain/ultrastructure , Cell Differentiation/physiology , Cell Fractionation , Embryonic and Fetal Development/physiology , GAP-43 Protein , Glial Fibrillary Acidic Protein/analysis , Membrane Glycoproteins/analysis , Microtubule-Associated Proteins/analysis , Neuroglia/ultrastructure , Oligodendroglia/cytology , Rats , Synaptophysin/analysis
5.
Mol Neurobiol ; 5(2-4): 143-51, 1991.
Article in English | MEDLINE | ID: mdl-1823138

ABSTRACT

Growth-regulated proteins (GRPs) of the neuron are synthesized during outgrowth and regeneration at an increased rate and enriched in nerve growth cones. Therefore, they can be used to some degree as markers of neurite growth. However, these proteins are not unique to the growing neuron, and their properties are not known sufficiently to assign them a functional and/or causal role in the mechanisms of outgrowth. During synaptogenesis, GRPs decrease in abundance, and growth cone functions of motility and organelle assembly are being replaced by junctional contact and transmitter release. However, there is a stage during which growth cone and synaptic properties overlap to some degree. We propose that it is this overlap and its continuation that allow for synaptic plasticity in developing and adult nervous systems. We also propose a hypothesis involving (a) trophic factor(s) that might explain the regulation of synaptic sizes and collateral sprouting. Some GRPs, especially GAP43/B50/pp46/F1, are more prominent in adult brain regions of high plasticity, and they undergo change, such as phosphorylation, during long-term potentiation (LTP). Without precise functional knowledge of GRPs, it is impossible to use changes in such proteins to explain the plasticity mechanism. However, changes in these "growth markers" are likely to be an indication of sprouting activity, which would explain well the various phenomena associated with plasticity and learning in the adult. Thus, plasticity and memory may be viewed as a continuation of the developmental process into adulthood.


Subject(s)
Nerve Tissue Proteins/biosynthesis , Neuronal Plasticity , Neurons/metabolism , Animals , Axons/metabolism , Biomarkers , Cell Differentiation , Gene Expression Regulation , Humans , Memory , Models, Neurological , Nerve Regeneration , Nerve Tissue Proteins/genetics , Neurons/cytology , Synapses/metabolism
6.
Biochem Biophys Res Commun ; 116(2): 675-81, 1983 Oct 31.
Article in English | MEDLINE | ID: mdl-6606426

ABSTRACT

The calcium/phospholipid-dependent protein kinase (PKC) and the H4 protease-activated protein kinase (H4PK) from lymphosarcoma cells were separated by CM Sephadex chromatography. PKC activity was increased 10-fold in the presence of calcium and phosphatidylserine, but no activation by Mg+2-ATP preincubation or inhibition by NaF was observed. In contrast, H4PK activity was increased 8-fold by preincubation with Mg+2ATP and NaF completely inhibited this enzyme. Activators and inhibitors of PKC did not affect H4PK activity. The substrate specificity of the H4PK and PKC also differed substantially. On the basis of these data it is concluded that PKC and H4PK are not related enzymes.


Subject(s)
Calcium/metabolism , Lymphoma, Non-Hodgkin/enzymology , Phospholipids/metabolism , Protein Kinases/metabolism , Adenosine Triphosphate/pharmacology , Animals , Enzyme Activation , Mice , Mice, Inbred BALB C , Phosphatidylserines/pharmacology , Sodium Fluoride/pharmacology , Substrate Specificity
7.
Cancer Res ; 43(8): 3514-20, 1983 Aug.
Article in English | MEDLINE | ID: mdl-6305487

ABSTRACT

The kinetics for activation of the cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase (PKA) and thymidine incorporation into DNA was investigated in epinephrine- and prostaglandin E1 (PGE1)-treated murine P1798 lymphosarcoma cells. A positive correlation between the duration and extent of PKA activation and accumulation of cyclic AMP and inhibition of thymidine incorporation into DNA was observed with both hormones. Epinephrine and PGE1 elevated intracellular cyclic AMP 34- and 14-fold, respectively. All hormone concentrations which increased cyclic AMP accumulation also promoted inhibition of thymidine incorporation into DNA. In addition, dibutyryl cyclic AMP (50 microM) inhibited thymidine incorporation. No difference in the kinetics for activation of PKA was observed when cells were treated with microM epinephrine or PGE1. With both agents, 50% PKA activation was observed when intracellular cyclic AMP concentrations were elevated 6.5-fold, or to 9 pmol/10(6) cells. In the presence of microM epinephrine, the cyclic AMP concentration was approximately 3-fold greater than that required for maximal PKA activation. In this case, the duration of the activation time for PKA was also 3- to 4-fold longer than that observed with 0.1 microM epinephrine. The data are consistent with a mechanism wherein both epinephrine and PGE1 suppress DNA synthesis by a cyclic AMP-mediated cascade of protein phosphorylation. No evidence for independent cyclic AMP or PKA pools which respond independently to either epinephrine or PGE1 could be detected.


Subject(s)
Cyclic AMP/metabolism , DNA/metabolism , Lymphoma, Non-Hodgkin/metabolism , Protein Kinases/metabolism , Thymidine/metabolism , Alprostadil , Animals , DNA Replication/drug effects , Enzyme Activation , Epinephrine/pharmacology , Kinetics , Mice , Mice, Inbred BALB C , Prostaglandins E/pharmacology
8.
J Biol Chem ; 258(7): 4272-8, 1983 Apr 10.
Article in English | MEDLINE | ID: mdl-6300081

ABSTRACT

The activation of a cyclic AMP-independent protein kinase by an endogenous protease is described. The H4 phosphotransferase (Masaracchia, R. A., Kemp, B., and Walsh, D. A. (1977) J. Biol. Chem. 252, 7109-7117) from lymphosarcoma cells was isolated in a nonactive form. Activation required ATP and Mg2+ and was shown to be time-dependent. Although Mn2+ was capable of substituting for Mg2+ in the protein kinase reaction, no activation was observed when Mn2+ replaced Mg2+. The protein substrate histone H4 inhibited phosphotransferase activation at concentrations greater than 60 microM. The inhibition was complete in the presence of 100 microM H4. Comparable concentrations of bovine serum albumin did not inhibit the activation. The selective dependence on Mg2+ suggested separate activating and phosphotransferase activities. This was confirmed by heat denaturation in which the activation reaction was shown to be more sensitive to heat inactivation than was the phosphotransferase reaction. The activating enzyme was separated from the protein kinase by column chromatofocusing in the pH range 7-4. The pI of the activating enzyme was greater than 7.0. The pI values of the activated and nonactivated phosphotransferase were 4.8 and 5.3, respectively. The apparent molecular weight of the nonactivated phosphotransferase was 68,000; the activated enzyme was eluted from an S-200 Sephadex column with an apparent Mr = 52,000. Despite many similarities to a protease-activated Ca2+/phospholipid-dependent enzyme isolated from lymphocytes (Ogawa, Y., Takai, Y., Kawahara, Y., Kimura, S., and Nishizuka, Y. (1981) J. Immunol. 127, 1369-1374), the H4 phosphotransferase was not activated by Ca2+, phospholipids, or any combination thereof.


Subject(s)
Endopeptidases/metabolism , Heat-Shock Proteins , Lymphoma, Non-Hodgkin/enzymology , Protein Kinases/metabolism , Serine Endopeptidases , ATP-Dependent Proteases , Animals , Cyclic AMP/pharmacology , Enzyme Activation , Histones/metabolism , Kinetics , Mice , Mice, Inbred BALB C , Sarcoma, Experimental/enzymology
9.
Anal Biochem ; 128(1): 54-9, 1983 Jan.
Article in English | MEDLINE | ID: mdl-6303156

ABSTRACT

Optimal assay conditions for analyses of the catalytic subunit activity of the cyclic AMP-dependent protein kinase using a well-defined, commercially available synthetic peptide as the phosphate acceptor are defined. Activity of purified catalytic subunit toward the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (PK-1; Kemptide) was 1.5- to 45-fold greater than activity toward other commonly used substrates such as histone fractions, casein, and protamine. The effects of buffer, pH, Mg2+, and protein kinase concentration on activity toward PK-1 were investigated. The optimal assay conditions determined were as follows: 20 mM Hepes or phosphate buffer, pH 7.5, 100 microM PK-1, 100 microM [gamma-32P]ATP, 3 mM MgCl2, 12 mM KCl, and 20-200 ng of catalytic subunit assayed at 30 degrees C. Since PK-1 is the only commercially available, well-defined substrate for this enzyme, adaption of the proposed standard assay conditions for the analyses of purified catalytic subunit activity will permit direct comparison of kinetic parameters and purity of enzyme preparations from multiple preparations.


Subject(s)
Carrier Proteins/metabolism , Cyclic AMP/pharmacology , Intracellular Signaling Peptides and Proteins , Muscles/enzymology , Protein Kinases/metabolism , Animals , Buffers , Hydrogen-Ion Concentration , Magnesium , Magnesium Chloride , Methods , Peptides/metabolism , Phosphorylation , Rabbits , Substrate Specificity
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