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1.
Sci Rep ; 13(1): 6913, 2023 04 27.
Article in English | MEDLINE | ID: mdl-37106020

ABSTRACT

Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study we investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. Here we report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function.


Subject(s)
Extracellular Vesicles , Norepinephrine , Humans , Dopamine/metabolism , Neurons/metabolism , Epigenesis, Genetic , Extracellular Vesicles/metabolism
3.
Science ; 326(5960): 1707-11, 2009 Dec 18.
Article in English | MEDLINE | ID: mdl-19892943

ABSTRACT

The LKB1 tumor suppressor is a protein kinase that controls the activity of adenosine monophosphate-activated protein kinase (AMPK). LKB1 activity is regulated by the pseudokinase STRADalpha and the scaffolding protein MO25alpha through an unknown, phosphorylation-independent, mechanism. We describe the structure of the core heterotrimeric LKB1-STRADalpha-MO25alpha complex, revealing an unusual allosteric mechanism of LKB1 activation. STRADalpha adopts a closed conformation typical of active protein kinases and binds LKB1 as a pseudosubstrate. STRADalpha and MO25alpha promote the active conformation of LKB1, which is stabilized by MO25alpha interacting with the LKB1 activation loop. This previously undescribed mechanism of kinase activation may be relevant to understanding the evolution of other pseudokinases. The structure also reveals how mutations found in Peutz-Jeghers syndrome and in various sporadic cancers impair LKB1 function.


Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Calcium-Binding Proteins/chemistry , Protein Serine-Threonine Kinases/chemistry , AMP-Activated Protein Kinase Kinases , AMP-Activated Protein Kinases/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Allosteric Regulation , Amino Acid Sequence , Binding Sites , Calcium-Binding Proteins/metabolism , Crystallography, X-Ray , Enzyme Activation , Humans , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation , Phosphorylation , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary
4.
Cell Mol Life Sci ; 66(21): 3449-67, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19669618

ABSTRACT

The glycerophosphoinositols are cellular products of phospholipase A(2) and lysolipase activities on the membrane phosphoinositides. Their intracellular concentrations can vary upon oncogenic transformation, cell differentiation and hormonal stimulation. Specific glycerophosphodiester phosphodiesterases are involved in their catabolism, which, as with their formation, is under hormonal regulation. With their mechanisms of action including modulation of adenylyl cyclase, intracellular calcium levels, and Rho-GTPases, the glycerophosphoinositols have diverse effects in multiple cell types: induction of cell proliferation in thyroid cells; modulation of actin cytoskeleton organisation in fibroblasts; and reduction of the invasive potential of tumour cell lines. More recent investigations include their effects in inflammatory and immune responses. Indeed, the glycerophosphoinositols enhance cytokine-dependent chemotaxis in T-lymphocytes induced by SDF-1alpha-receptor activation, indicating roles for these compounds as modulators of T-cell signalling and T-cell responses.


Subject(s)
Cells/metabolism , Inositol Phosphates/metabolism , Inositol Phosphates/physiology , Animals , Cell Physiological Phenomena , Humans , Lipids/chemistry , Lipids/physiology , Models, Biological , Phosphatidylinositols/metabolism , Phosphatidylinositols/physiology , Second Messenger Systems/physiology
5.
PLoS Biol ; 7(6): e1000126, 2009 Jun 09.
Article in English | MEDLINE | ID: mdl-19513107

ABSTRACT

Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADalpha in complex with MO25alpha. The structure reveals an intricate web of interactions between STRADalpha and MO25alpha involving the alphaC-helix of STRADalpha, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADalpha binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADalpha for MO25alpha, and conversely, binding of MO25alpha promotes interaction of STRADalpha with ATP. Mutagenesis studies reveal that association of STRADalpha with either ATP or MO25alpha is essential for LKB1 activation. We conclude that ATP and MO25alpha cooperate to maintain STRADalpha in an "active" closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADalpha that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE) syndrome. We demonstrate this mutation destabilizes STRADalpha and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADalpha to activate LKB1 is dependent on a closed "active" conformation, aided by ATP and MO25alpha binding. Thus, the function of STRADalpha is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.


Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/metabolism , Adenosine Triphosphate/metabolism , Calcium-Binding Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , AMP-Activated Protein Kinase Kinases , Abnormalities, Multiple/enzymology , Adenosine Diphosphate/metabolism , Amino Acid Sequence , Binding Sites , Cell Line , Conserved Sequence , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Enzyme Activation , Enzyme Stability , Humans , Magnesium , Models, Biological , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Protein Binding , Protein Structure, Secondary , Surface Properties , Syndrome
6.
J Cell Biol ; 183(1): 7-9, 2008 Oct 06.
Article in English | MEDLINE | ID: mdl-18838550

ABSTRACT

A question preoccupying many researchers is how signal transduction pathways control metabolic processes and energy production. A study by Jang et al. (Jang, C., G. Lee, and J. Chung. 2008. J. Cell Biol. 183:11-17) provides evidence that in Drosophila melanogaster a signaling network controlled by the LKB1 tumor suppressor regulates trafficking of an Sln/dMCT1 monocarboxylate transporter to the plasma membrane. This enables cells to import additional energy sources such as lactate and butyrate, enhancing the repertoire of fuels they can use to power vital activities.


Subject(s)
Drosophila Proteins/metabolism , Monocarboxylic Acid Transporters/metabolism , Protein Kinases/metabolism , Signal Transduction/physiology , AMP-Activated Protein Kinase Kinases , Animals , Apoptosis/physiology , Butyrates/metabolism , Cell Polarity/physiology , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Humans , Lactic Acid/metabolism , Models, Biological , Protein Transport , Symporters/metabolism
7.
Biochim Biophys Acta ; 1783(12): 2311-22, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18722484

ABSTRACT

The glycerophosphoinositols are diffusible phosphoinositide metabolites reported to modulate actin dynamics and tumour cell spreading. In particular, the membrane permeant glycerophosphoinositol 4-phosphate (GroPIns4P) has been shown to act at the level of the small GTPase Rac1, to induce the rapid formation of membrane ruffles. Here, we have investigated the signalling cascade involved in this process, and show that it is initiated by the activation of Src kinase. In NIH3T3 cells, exogenous addition of GroPIns4P induces activation and translocation of Rac1 and its exchange factor TIAM1 to the plasma membrane; in addition, in in-vitro assays, GroPIns4P favours the formation of a protein complex that includes Rac1 and TIAM1. Neither of these processes involves direct actions of GroPIns4P on these proteins. Thus, through the use of specific inhibitors of tyrosine kinases and phospholipase C (and by direct evaluation of kinase activities and inositol 1,4,5-trisphosphate production), we show that GroPIns4P activates Src, and as a consequence, phospholipase Cgamma and Ca(2+)/calmodulin kinase II, the last of which directly phosphorylates TIAM1 and leads to TIAM1/Rac1-dependent ruffle formation.


Subject(s)
Actins/metabolism , Inositol Phosphates/pharmacology , Signal Transduction , rac1 GTP-Binding Protein/metabolism , src-Family Kinases/metabolism , Animals , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Membrane/metabolism , Cells, Cultured , Enzyme Activation/drug effects , Fluorescent Antibody Technique , Guanine Nucleotide Exchange Factors/metabolism , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Mice , NIH 3T3 Cells , Phospholipase C gamma/antagonists & inhibitors , Phospholipase C gamma/metabolism , Phosphorylation , Protein Transport , Proto-Oncogene Proteins c-vav/metabolism , T-Lymphoma Invasion and Metastasis-inducing Protein 1
8.
Cancer Res ; 67(24): 11769-78, 2007 Dec 15.
Article in English | MEDLINE | ID: mdl-18089807

ABSTRACT

Modulation of cytosolic phospholipase A(2) (PLA(2)) expression levels and production of its metabolites have been reported in several tumor types, indicating involvement of arachidonic acid and its derivatives in tumorigenesis. Following our demonstration that the PLA(2) group IV isoform alpha (PLA(2)IV alpha) controls TSH-independent growth of normal thyroid (PCCl(3)) cells, we have investigated the mitogenic role of PLA(2)IV alpha in rat thyroid cells transformed by the RET/PTC oncogenes (PC-PTC cells). We now report that PLA(2)IV alpha acts downstream of the RET/PTC oncogenes in a novel pathway controlling RET-dependent cell proliferation. In addition, we show that PLA(2)IV alpha is in its phosphorylated/active form not only in RET/PTC-transformed cells and in cells derived from human papillary carcinomas but also in lysates from tumor tissues, thus relating constitutive activation of PLA(2)IV alpha to RET/PTC-dependent tumorigenesis. Moreover, p38 stress-activated protein kinase is the downstream effector of RET/PTC that is responsible for PLA(2)IV alpha phosphorylation and activity. In summary, our data elucidate a novel mechanism in the control of thyroid tumor cell growth that is induced by the RET/PTC oncogenes and which is distinguishable from that of other oncogenes, such as BRAF. This mechanism is mediated by PLA(2)IV alpha and should be amenable to targeted pharmacologic intervention.


Subject(s)
Cell Transformation, Neoplastic/pathology , Group IV Phospholipases A2/metabolism , Thyroid Gland/cytology , Thyroid Gland/pathology , Animals , Cell Division/physiology , Cell Line , Cell Line, Transformed , Cytosol/enzymology , Rats , Thymidine/metabolism
9.
FASEB J ; 20(14): 2567-9, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17060404

ABSTRACT

The phosphoinositides have well-defined roles in the control of cellular functions, including cytoskeleton dynamics, membrane trafficking, and cell signaling. However, the interplay among the phosphoinositides and their diffusible derivatives that originate through phospholipase A2 action (the lysophosphoinositides and glycerophosphoinositols) remains to be fully elucidated. Here we demonstrate that in PCCl3 rat thyroid cells, the intracellular levels of glycerophosphoinositol are finely modulated by ATP and norepinephrine through the P2Y metabotropic and alpha-adrenergic receptors, respectively. The enzyme involved here is phospholipase A2 IValpha (PLA2 IValpha), which in these cells specifically hydrolyzes phosphatidylinositol, forming lysophosphatidylinositol, glycerophosphoinositol, and arachidonic acid. This receptor-mediated activation of PLA2 IValpha leads to stimulation of PCCl3 cell growth. The involvement of a PLA2 IValpha-mediated pathway is demonstrated by inhibition of the increase in intracellular glycerophosphoinositol levels and cell proliferation by specific inhibitors, RNA interference, and overexpression of the dominant-negative PLA2 IValpha(1-522). Modulation of PCCl3 cell growth is not seen with inhibitors of arachidonic acid metabolism. In conclusion, these data characterize glycerophosphoinositol as a mediator of the purinergic and adrenergic regulation of PCCl3 cell proliferation, defining a novel regulatory cascade specifically involving this soluble phosphoinositide derivative and widening the involvement of the phosphoinositides in the regulation of cell function.


Subject(s)
Epithelial Cells/metabolism , Inositol Phosphates/metabolism , Phosphatidylinositols/metabolism , Phospholipases A/metabolism , Animals , CHO Cells , Cell Differentiation , Cell Line , Cricetinae , Cricetulus , Epithelial Cells/cytology , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation , Genetic Markers/physiology , Group IV Phospholipases A2 , Phospholipases A2 , RNA, Messenger/metabolism , Rats , Receptors, Purinergic/metabolism , Thyroid Gland/cytology
10.
Mol Biol Cell ; 14(2): 503-15, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12589050

ABSTRACT

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P.


Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Inositol Phosphates/metabolism , Phosphatidylinositols/chemistry , 3T3 Cells , Animals , Cell Line, Transformed , Cell Movement , Dose-Response Relationship, Drug , GTP Phosphohydrolases/chemistry , Green Fluorescent Proteins , Growth Substances/metabolism , Luminescent Proteins/metabolism , Mice , Microscopy, Fluorescence , Neoplasm Invasiveness , Neoplasm Metastasis , Recombinant Proteins/metabolism , Time Factors , Transfection , cdc42 GTP-Binding Protein/metabolism
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