scholarly journals Sensing Host Arginine Is Essential for Leishmania Parasites’ Intracellular Development

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Adele Goldman-Pinkovich ◽  
Sriram Kannan ◽  
Roni Nitzan-Koren ◽  
Madhu Puri ◽  
Harsh Pawar ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Mitogen Activated Protein Kinase ◽  
Macrophage Infection ◽  
Hunger Games ◽  
Host Macrophage ◽  
Arginine Deprivation ◽  
Mitogen Activated Protein ◽  
Human Leishmaniasis ◽  
Host Innate Immune Response

ABSTRACT Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine “hunger games” by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis. IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the “hunger games” for arginine.

scholarly journals The hunger games: sensing host arginine is essential for Leishmania parasite virulence

2019 ◽  
Author(s):  
Adele Goldman-Pinkovich ◽  
Sriram Kannan ◽  
Roni Nitzan-Koren ◽  
Madhu Puri ◽  
Yael Bar-Avraham ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Wild Type ◽  
Intracellular Growth ◽  
Macrophage Infection ◽  
Hunger Games ◽  
Arginine Deprivation ◽  
The Hunger Games ◽  
Mtorc1 Activation ◽  
First Time ◽  
Functional Copy

AbstractArginine homeostasis in lysosomes is critical for growth and metabolism of mammalian cells. They employ a specific sensor (SLC38A9) that monitors intra-lysosome arginine sufficiency and subsequently up-regulates cellular mTORC1 activity. Lysosomes of macrophages (phagolysosomes) are the niche where the parasitic protozoan Leishmania resides and causes important human disease. Several years ago, we discovered that upon arginine starvation, cultured Leishmania parasites promptly activate a MAPK2-mediated Arginine Deprivation Response (ADR) pathway, resulting in up-regulation of the Leishmania arginine transporter (AAP3), as well as a small group of other transporters. Significantly, ADR is also activated during macrophage infection, implying that the intracellular parasite actively depletes arginine within the host phagolysosome, likely to prevent mTORC1 activation and enhance intracellular development. We hypothesize that ADR-mediated up-regulation of AAP3 activity is necessary to withstand the resultant arginine starvation. Both copies of the AAP3 genes are located (in tandem) on a tetrasomic chromosome (chr31), but only one (AAP3.2) is responsive to arginine deprivation. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport (mediated by AAP3.1), but lack a functional copy of AAP3.2 and are therefore not responsive to arginine starvation. While these mutants grow normally in culture as promastigotes, they were impaired in their ability to develop inside THP1 macrophages grown under physiological concentrations of arginine (0.1 mM). However, flooding the macrophage growth medium with arginine (1.5 mM) restored parasite infectivity and intracellular growth to that of wild type. The results indicate that inside the host macrophage, Leishmania must overcome the arginine “Hunger Games” by up-regulating transport of arginine via the ADR. Furthermore, the AAP3.2 mutants were ~70-80% less virulent in Balb/C mice, showing, for the first time, that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.

HIV-1 gp120 and chemokine activation of Pyk2 and mitogen-activated protein kinases in primary macrophages mediated by calcium-dependent, pertussis toxin–insensitive chemokine receptor signaling

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 2909-2916 ◽  
Author(s):  
Manuela Del Corno ◽  
Qing-Hua Liu ◽  
Dominique Schols ◽  
Erik de Clercq ◽  
Sandra Gessani ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Pertussis Toxin ◽  
Chemokine Receptor ◽  
Calcium Release ◽  
Mitogen Activated Protein Kinase ◽  
Macrophage Infection ◽  
Amino Terminal ◽  
Crac Channels ◽  
Mitogen Activated Protein ◽  
Hiv 1

Abstract Human immunodeficiency virus type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry. It was recently demonstrated that HIV-1 glycoprotein 120 (gp120) elevated calcium and activated several ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. This study shows that chemokine receptor engagement by both CCR5-dependent (R5) and CXCR4-dependent (X4) gp120 led to rapid phosphorylation of the focal adhesion-related tyrosine kinase Pyk2 in macrophages. Pyk2 phosphorylation was also induced by macrophage inflammatory protein-1β (MIP-1β) and stromal cell–derived factor-1α, chemokine ligands for CCR5 and CXCR4. Activation was blocked by EGTA and by a potent blocker of calcium release–activated Ca++(CRAC) channels, but was insensitive to pertussis toxin (PTX), implicating CRAC-mediated extracellular Ca++ influx but not Gαi protein-dependent mechanisms. Coreceptor engagement by gp120 and chemokines also activated 2 members of the mitogen-activated protein kinase (MAPK) superfamily, c-Jun amino-terminal kinase/stress-activated protein kinase and p38 MAPK. Furthermore, gp120-stimulated macrophages secreted the chemokines monocyte chemotactic protein-1 and MIP-1β in a manner that was dependent on MAPK activation. Thus, the gp120 signaling cascade in macrophages includes coreceptor binding, PTX-insensitive signal transduction, ionic signaling including Ca++ influx, and activation of Pyk2 and MAPK pathways, and leads to secretion of inflammatory mediators. HIV-1 Env signaling through these pathways may contribute to dysregulation of uninfected macrophage functions, new target cell recruitment, or modulation of macrophage infection.

scholarly journals Nuclear Translocation of Soybean MPK6, GmMPK6, Is Mediated by Hydrogen Peroxide in Salt Stress

Plants ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2611
Author(s):  
Jong Hee Im ◽  
Seungmin Son ◽  
Jae-Heung Ko ◽  
Kyung-Hwan Kim ◽  
Chung Sun An ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Hydrogen Peroxide ◽  
Salt Stress ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Signaling Molecules ◽  
Mitogen Activated Protein Kinase ◽  
Signal Transduction System ◽  
Diphenylene Iodonium ◽  
Mitogen Activated Protein

The plant mitogen-activated protein kinase (MPK) cascade, a highly conserved signal transduction system in eukaryotes, plays a crucial role in the plant’s response to environmental stimuli and phytohormones. It is well-known that nuclear translocation of MPKs is necessary for their activities in mammalian cells. However, the mechanism underlying nuclear translocation of plant MPKs is not well elucidated. In the previous study, it has been shown that soybean MPK6 (GmMPK6) is activated by phosphatidic acid (PA) and hydrogen peroxide (H2O2), which are two signaling molecules generated during salt stress. Using the two signaling molecules, we investigated how salt stress triggers its translocation to the nucleus. Our results show that the translocation of GmMPK6 to the nucleus is mediated by H2O2, but not by PA. Furthermore, the translocation was interrupted by diphenylene iodonium (DPI) (an inhibitor of RBOH), confirming that H2O2 is the signaling molecule for the nuclear translocation of GmMPK6 during salt stress.

Mesodermal patterning defect in mice lacking the Ste20 NCK interacting kinase (NIK)

Development ◽  
2001 ◽  
Vol 128 (9) ◽  
pp. 1559-1572 ◽  
Author(s):  
Y. Xue ◽  
X. Wang ◽  
Z. Li ◽  
N. Gotoh ◽  
D. Chapman ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Mammalian Cells ◽  
Essential Gene ◽  
Primitive Streak ◽  
Mitogen Activated Protein Kinase ◽  
Drosophila Embryo ◽  
Mammalian Development ◽  
Mitogen Activated Protein ◽  
Patterning Defect ◽  
Correct Location

We have previously shown that the Drosophila Ste20 kinase encoded by misshapen (msn) is an essential gene in Drosophila development. msn function is required to activate the Drosophila c-Jun N-terminal kinase (JNK), basket (Bsk), to promote dorsal closure of the Drosophila embryo. Later in development, msn expression is required in photoreceptors in order for their axons to project normally. A mammalian homolog of msn, the NCK-interacting kinase (NIK) (recently renamed to mitogen-activated protein kinase kinase kinase kinase 4; Map4k4), has been shown to activate JNK and to bind the SH3 domains of the SH2/SH3 adapter NCK. To determine whether NIK also plays an essential role in mammalian development, we created mice deficient in NIK by homologous recombination at the Nik gene. Nik(−/−) mice die postgastrulation between embryonic day (E) 9.5 and E10.5. The most striking phenotype in Nik(−/−) embryos is the failure of mesodermal and endodermal cells that arise from the anterior end of the primitive streak (PS) to migrate to their correct location. As a result Nik(−/−)embryos fail to develop somites or a hindgut and are truncated posteriorly. Interestingly, chimeric analysis demonstrated that NIK has a cell nonautonomous function in stimulating migration of presomitic mesodermal cells away from the PS and a second cell autonomous function in stimulating the differentiation of presomitic mesoderm into dermomyotome. These findings indicate that despite the large number of Ste20 kinases in mammalian cells, members of this family play essential nonredundant function in regulating specific signaling pathways. In addition, these studies provide evidence that the signaling pathways regulated by these kinases are diverse and not limited to the activation of JNK because mesodermal and somite development are not perturbed in JNK1-, and JNK2-deficient mice.

scholarly journals Two human cDNAs, including a homolog of Arabidopsis FUS6 (COP11), suppress G-protein- and mitogen-activated protein kinase-mediated signal transduction in yeast and mammalian cells.

1996 ◽  
Vol 16 (12) ◽  
pp. 6698-6706 ◽  
Author(s):  
B H Spain ◽  
K S Bowdish ◽  
A R Pacal ◽  
S F Staub ◽  
D Koo ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
G Protein ◽  
Mammalian Cells ◽  
Enhanced Recovery ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Striking Similarity ◽  
Protein Subunit ◽  
Mitogen Activated Protein

We have isolated two novel human cDNAs, gps1-1 and gps2, that suppress lethal G-protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Suppression of other pathway-activating events was examined. In wild-type cells, expression of either gps1-1 or gps2 led to enhanced recovery from cell cycle arrest induced by pheromone. Sequence analysis indicated that gps1-1 contains only the carboxy-terminal half of the gps1 coding sequence. The predicted gene product of gps1 has striking similarity to the protein encoded by the Arabidopsis FUS6 (COP11) gene, a negative regulator of light-mediated signal transduction that is known to be essential for normal development. A chimeric construct containing gps1 and FUS6 sequences also suppressed the yeast pheromone pathway, indicating functional conservation between these human and plant genes. In addition, when overexpressed in mammalian cells, gps1 or gps2 potently suppressed a RAS- and mitogen-activated protein kinase-mediated signal and interfered with JNK activity, suggesting that signal repression is part of their normal function. For gps1, these results are consistent with the proposed function of FUS6 (COP11) as a signal transduction repressor in plants.

Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A

2001 ◽  
Vol 359 (3) ◽  
pp. 497-505 ◽  
Author(s):  
Sunke HIMPEL ◽  
Pascal PANZER ◽  
Klaus EIRMBTER ◽  
Hanna CZAJKOWSKA ◽  
Muhammed SAYED ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Molecular Model ◽  
Enzymic Activity ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Family ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Protein kinases of the DYRK (‘dual-specificity tyrosine-regulated kinase’) family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319–Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

scholarly journals Extracellular Signal-Regulated Kinase 2 Interacts with and Is Negatively Regulated by the LIM-Only Protein FHL2 in Cardiomyocytes

2004 ◽  
Vol 24 (3) ◽  
pp. 1081-1095 ◽  
Author(s):  
Nicole H. Purcell ◽  
Dina Darwis ◽  
Orlando F. Bueno ◽  
Judith M. Müller ◽  
Roland Schüle ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Erk Signaling ◽  
Interacting Protein ◽  
Hypertrophic Response ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT The mitogen-activated protein kinase (MAPK) signaling pathway regulates diverse biologic functions including cell growth, differentiation, proliferation, and apoptosis. The extracellular signal-regulated kinases (ERKs) constitute one branch of the MAPK pathway that has been implicated in the regulation of cardiac differentiated growth, although the downstream mechanisms whereby ERK signaling affects this process are not well characterized. Here we performed a yeast two-hybrid screen with ERK2 bait and a cardiac cDNA library to identify novel proteins involved in regulating ERK signaling in cardiomyocytes. This screen identified the LIM-only factor FHL2 as an ERK interacting protein in both yeast and mammalian cells. In vivo, FHL2 and ERK2 colocalized in the cytoplasm at the level of the Z-line, and interestingly, FHL2 interacted more efficiently with the activated form of ERK2 than with the dephosphorylated form. ERK2 also interacted with FHL1 and FHL3 but not with the muscle LIM protein. Moreover, at least two LIM domains in FHL2 were required to mediate efficient interaction with ERK2. The interaction between ERK2 and FHL2 did not influence ERK1/2 activation, nor was FHL2 directly phosphorylated by ERK2. However, FHL2 inhibited the ability of activated ERK2 to reside within the nucleus, thus blocking ERK-dependent transcriptional responsiveness of ELK-1, GATA4, and the atrial natriuretic factor promoter. Finally, FHL2 partially antagonized the cardiac hypertrophic response induced by activated MEK-1, GATA4, and phenylephrine agonist stimulation. Collectively, these results suggest that FHL2 serves a repressor function in cardiomyocytes through its ability to inhibit ERK1/2 transcriptional coupling.

ATR (ataxia telangiectasia mutated- and Rad3-related kinase) is activated by mild hypothermia in mammalian cells and subsequently activates p53

2011 ◽  
Vol 435 (2) ◽  
pp. 499-508 ◽  
Author(s):  
Anne Roobol ◽  
Jo Roobol ◽  
Martin J. Carden ◽  
Amandine Bastide ◽  
Anne E. Willis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ataxia Telangiectasia ◽  
Mammalian Cells ◽  
Mild Hypothermia ◽  
Chinese Hamster ◽  
Membrane Lipid ◽  
Mitogen Activated Protein Kinase ◽  
Ataxia Telangiectasia Mutated ◽  
Mitogen Activated Protein

In vitro cultured mammalian cells respond to mild hypothermia (27–33 °C) by attenuating cellular processes and slowing and arresting the cell cycle. The slowing of the cell cycle at the upper range (31–33 °C) and its complete arrest at the lower range (27–28 °C) of mild hypothermia is effected by the activation of p53 and subsequent expression of p21. However, the mechanism by which cold is perceived in mammalian cells with the subsequent activation of p53 has remained undetermined. In the present paper, we report that the exposure of Chinese-hamster ovary-K1 cells to mildly hypothermic conditions activates the ATR (ataxia telangiectasia mutated- and Rad3-related kinase)–p53–p21 signalling pathway and is thus a key pathway involved in p53 activation upon mild hypothermia. In addition, we show that although p38MAPK (p38 mitogen-activated protein kinase) is also involved in activation of p53 upon mild hypothermia, this is probably the result of activation of p38MAPK by ATR. Furthermore, we show that cold-induced changes in cell membrane lipid composition are correlated with the activation of the ATR–p53–p21 pathway. Therefore we provide the first mechanistic detail of cell sensing and signalling upon mild hypothermia in mammalian cells leading to p53 and p21 activation, which is known to lead to cell cycle arrest.

scholarly journals Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes

2001 ◽  
Vol 154 (4) ◽  
pp. 707-718 ◽  
Author(s):  
Leana M. Topper ◽  
Holger Bastians ◽  
Joan V. Ruderman ◽  
Gary J. Gorbsky
Keyword(s):  
Mammalian Cells ◽  
Proteasome Inhibitors ◽  
Metaphase Plate ◽  
Cytoplasmic Dynein ◽  
Mitogen Activated Protein Kinase ◽  
Protein Levels ◽  
Mitogen Activated Protein ◽  
Associated Proteins ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E–associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

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