Metabolic evidence for PtdIns(4,5)P2-directed phospholipase C in permeabilized plant protoplasts

Comparison of the sequences of the genes encoding phospholipase C (PLC) which have been cloned to date in plants with their mammalian counterparts suggests that plant PLC is similar to PLCδ of mammalian cells. The physiological role and mechanism of activation of PLCδ is unclear. It has recently been shown that Ins(1,4,5)P3 may not solely be the product of PtdIns(4,5)P2-directed PLC activity. Enzyme activities capable of producing Ins(1,4,5)P3 from endogenous inositol phosphates are present in Dictyostelium and also in rat liver. Significantly it has not been directly determined whether Ins(1,4,5)P3 present in higher plants is the product of a PtdIns(4,5)P2-directed PLC activity. Therefore we have developed an experimental strategy for the identification of d-Ins(1,4,5)P3 in higher plants. By the use of a short-term non-equilibrium labelling strategy in permeabilized plant protoplasts, coupled to the use of a ‘metabolic trap‘ to prevent degradation of [32P]Ins(1,4,5)P3, we were able to determine the distribution of 32P in individual phosphate esters of Ins(1,4,5)P3. The [32]Ins(1,4,5)P3 identified showed the same distribution of label in individual phosphate esters as that of [32P]PtdIns(4,5)P2 isolated from the same tissue. We thus provide in vivo evidence for the action of a PtdIns(4,5)P2-directed PLC activity in plant cells which is responsible for the production of Ins(1,4,5)P3 observed here. This observation does not, however, exclude the possibility that in other cells or under different conditions Ins(1,4,5)P3 can be generated by alternative routes.

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Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2018-2025.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2018-2025

Author(s):

L Sultzman ◽

C Ellis ◽

L L Lin ◽

T Pawson ◽

J Knopf

Keyword(s):

Growth Factor ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Cell Surface Receptor ◽

Platelet Derived Growth Factor ◽

Surface Receptor ◽

Phospholipase C Gamma

Upon binding to its cell surface receptor, platelet-derived growth factor (PDGF) causes the tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and stimulates the production of diacylglycerol and inositol 1,4,5-triphosphate. We showed that following stimulation by PDGF, rat-2 cells overexpressing PLC-gamma 1 display an increase in the levels of both tyrosine-phosphorylated PLC-gamma 1 and inositol phosphates compared with the parental rat-2 cells. This increased responsiveness to PDGF is a direct effect of PLC-gamma 1 overexpression, as a cell line expressing similar levels of an enzymatically inactive point mutant of PLC-gamma 1, PLC-gamma 1 335Q, did not show elevated inositol phosphate production in response to PDGF. Hematopoietic cells express PLC-gamma 2, a PLC isoform that is closely related to PLC-gamma 1. When rat-2 cells overexpressing PLC-gamma 2 were treated with PDGF, an increase in both the tyrosine phosphorylation and the in vivo activity of PLC-gamma 2 was observed. Aluminum fluoride (AIF4-), a universal activator of PLC linked to G-proteins, did not produce an increase in the levels of inositol phosphates in either of the overexpressing cell lines compared with parental rat-2 cells, demonstrating that PLC-gamma isoforms respond specifically to a receptor with tyrosine kinase activity.

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A Shigella type 3 effector protein co-opts host inositol pyrophosphates for activity

10.1101/2021.09.01.458261 ◽

2021 ◽

Author(s):

Thomas E. Wood ◽

Jessica M. Yoon ◽

Heather D. Eshleman ◽

Daniel J. Slade ◽

Cammie F. Lesser ◽

...

Keyword(s):

Mammalian Cells ◽

Inositol Phosphates ◽

Bacterial Pathogen ◽

Cysteine Proteases ◽

Effector Proteins ◽

Host Cells ◽

Expression Library ◽

Inositol Pyrophosphates ◽

Type 3

Shigella spp. cause diarrhea by invading human intestinal epithelial cells. Effector proteins delivered into target host cells by the Shigella type 3 secretion system modulate host signaling pathways and processes in a manner that promotes infection. The effector OspB activates mTOR, the central cellular regulator of growth and metabolism, and potentiates the inhibition of mTOR by rapamycin. The net effect of OspB on cell monolayers is cell proliferation at infectious foci. To gain insights into the mechanism by which OspB potentiates rapamycin inhibition of mTOR, we employ in silico analyses to identify putative catalytic residues of OspB and show that a conserved cysteine-histidine dyad is required for this activity of OspB. In a screen of an over-expression library in Saccharomyces cerevisiae, we identify a dependency of OspB activity on inositol pyrophosphates, a class of eukaryotic secondary messengers that are distinct from the inositol phosphates known to act as cofactors for bacterial cysteine proteases. We show that inositol pyrophosphates are required for OspB activity not only in yeast, but also in mammalian cells - the first demonstration of inositol pyrophosphates being required for virulence of a bacterial pathogen in vivo.

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Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

Bioscience Reports ◽

10.1007/bf01119588 ◽

1985 ◽

Vol 5 (3) ◽

pp. 189-204 ◽

Cited By ~ 36

Author(s):

E. S. Canellakis ◽

D. A. Kyriakidis ◽

C. A. Rinehart ◽

S.-C. Huang ◽

C. Panagiotidis ◽

...

Keyword(s):

Ornithine Decarboxylase ◽

Mammalian Cells ◽

Ribosomal Proteins ◽

Physiological Role ◽

Arginine Decarboxylase ◽

Cell Of Origin ◽

Polyamine Biosynthesis ◽

Recent Developments

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and of Escherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. coli antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 × 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli, in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S20/L26 and L34, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.

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Jun Dimerization Protein 2 Functions as a Progesterone Receptor N-Terminal Domain Coactivator

Molecular and Cellular Biology ◽

10.1128/mcb.22.15.5451-5466.2002 ◽

2002 ◽

Vol 22 (15) ◽

pp. 5451-5466 ◽

Cited By ~ 75

Author(s):

Suzanne E. Wardell ◽

Viroj Boonyaratanakornkit ◽

James S. Adelman ◽

Ami Aronheim ◽

Dean P. Edwards

Keyword(s):

Progesterone Receptor ◽

Protein Interactions ◽

Mammalian Cells ◽

Leucine Zipper ◽

Physiological Role ◽

Target Cells ◽

Basic Leucine Zipper ◽

Interacting Protein

ABSTRACT The progesterone receptor (PR) contains two transcription activation function (AF) domains, constitutive AF-1 in the N terminus and AF-2 in the C terminus. AF-2 activity is mediated by a hormone-dependent interaction with a family of steroid receptor coactivators (SRCs). SRC-1 can also stimulate AF-1 activity through a secondary domain that interacts simultaneously with the primary AF-2 interaction site. Other protein interactions and mechanisms that mediate AF-1 activity are not well defined. By interaction cloning, we identified an AP-1 family member, Jun dimerization protein 2 (JDP-2), as a novel PR-interacting protein. JDP-2 was first defined as a c-Jun interacting protein that functions as an AP-1 repressor. PR and JDP-2 interact directly in vitro through the DNA binding domain (DBD) of PR and the basic leucine zipper (bZIP) region of JDP-2. The two proteins also physically associate in mammalian cells, as detected by coimmunoprecipitation, and are recruited in vivo to a progesterone-inducible target gene promoter, as detected by a chromatin immunoprecipitation (ChIP) assay. In cell transfection assays, JDP-2 substantially increased hormone-dependent PR-mediated transactivation and worked primarily by stimulating AF-1 activity. JDP-2 is a substantially stronger coactivator of AF-1 than SRC-1 and stimulates AF-1 independent of SRC-1 pathways. The PR DBD is necessary but not sufficient for JDP-2 stimulation of PR activity; the DBD and AF-1 are required together. JDP-2 lacks an intrinsic activation domain and makes direct protein interactions with other coactivators, including CBP and p300 CBP-associated factor (pCAF), but not with SRCs. These results indicate that JDP-2 stimulates AF-1 activity by the novel mechanism of docking to the DBD and recruiting or stabilizing N-terminal PR interactions with other general coactivators. JDP-2 has preferential activity on PR among the nuclear receptors tested and is expressed in progesterone target cells and tissues, suggesting that it has a physiological role in PR function.

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Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2.

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2018 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2018-2025 ◽

Cited By ~ 59

Author(s):

L Sultzman ◽

C Ellis ◽

L L Lin ◽

T Pawson ◽

J Knopf

Keyword(s):

Growth Factor ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Cell Surface Receptor ◽

Platelet Derived Growth Factor ◽

Surface Receptor ◽

Phospholipase C Gamma

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A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways

The Journal of Cell Biology ◽

10.1083/jcb.124.6.935 ◽

1994 ◽

Vol 124 (6) ◽

pp. 935-947 ◽

Cited By ~ 45

Author(s):

K Mensa-Wilmot ◽

JH LeBowitz ◽

KP Chang ◽

A al-Qahtani ◽

BS McGwire ◽

...

Keyword(s):

Phospholipase C ◽

Mammalian Cells ◽

Leishmania Major ◽

Protozoan Parasite ◽

T Lymphoma ◽

Major Surface ◽

Parasite Leishmania ◽

Negative Phenotype

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.

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ITPK1 mediates the lipid-independent synthesis of inositol phosphates controlled by metabolism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1911431116 ◽

2019 ◽

Vol 116 (49) ◽

pp. 24551-24561 ◽

Cited By ~ 18

Author(s):

Yann Desfougères ◽

Miranda S. C. Wilson ◽

Debabrata Laha ◽

Gregory J. Miller ◽

Adolfo Saiardi

Keyword(s):

Phospholipase C ◽

Mammalian Cells ◽

Inositol Phosphates ◽

Phosphate Starvation ◽

Dependent Manner ◽

Social Amoeba ◽

Functional Roles ◽

Inositol Tetrakisphosphate ◽

Independent Synthesis ◽

Multiple Signaling

Inositol phosphates (IPs) comprise a network of phosphorylated molecules that play multiple signaling roles in eukaryotes. IPs synthesis is believed to originate with IP3 generated from PIP2 by phospholipase C (PLC). Here, we report that in mammalian cells PLC-generated IPs are rapidly recycled to inositol, and uncover the enzymology behind an alternative “soluble” route to synthesis of IPs. Inositol tetrakisphosphate 1-kinase 1 (ITPK1)—found in Asgard archaea, social amoeba, plants, and animals—phosphorylates I(3)P1 originating from glucose-6-phosphate, and I(1)P1 generated from sphingolipids, to enable synthesis of IP6. We also found using PAGE mass assay that metabolic blockage by phosphate starvation surprisingly increased IP6 levels in a ITPK1-dependent manner, establishing a route to IP6 controlled by cellular metabolic status, that is not detectable by traditional [3H]-inositol labeling. The presence of ITPK1 in archaeal clades thought to define eukaryogenesis indicates that IPs had functional roles before the appearance of the eukaryote.

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Hspa4l-Deficient Mice Display Increased Incidence of Male Infertility and Hydronephrosis Development

Molecular and Cellular Biology ◽

10.1128/mcb.01332-06 ◽

2006 ◽

Vol 26 (21) ◽

pp. 8099-8108 ◽

Cited By ~ 41

Author(s):

Torsten Held ◽

Ilona Paprotta ◽

Janchiv Khulan ◽

Bernhardt Hemmerlein ◽

Lutz Binder ◽

...

Keyword(s):

Germ Cells ◽

Sperm Count ◽

Physiological Role ◽

Heat Shock Gene ◽

Seminiferous Tubules ◽

Genes Encoding ◽

Null Mutant Mice ◽

Pachytene Spermatocytes ◽

Deficient Mice

ABSTRACT The Hspa4l gene, also known as Apg1 or Osp94, belongs to the HSP110 heat shock gene family, which includes three genes encoding highly conserved proteins. This study shows that Hspa4l is expressed ubiquitously and predominantly in the testis. The protein is highly expressed in spermatogenic cells, from late pachytene spermatocytes to postmeiotic spermatids. In the kidney, the protein is restricted to cortical segments of distal tubules. To study the physiological role of this gene in vivo, we generated mice deficient in Hspa4l by gene targeting. Hspa4l-deficient mice were born at expected ratios and appeared healthy. However, approximately 42% of Hspa4l −/− male mice suffered from fertility defects. Whereas the seminiferous tubules of Hspa4l −/− testes contained all stages of germ cells, the number of mature sperm in the epididymis and sperm motility were drastically reduced. The reduction of the sperm count was due to the elimination of a significant number of developing germ cells via apoptosis. No defects in fertility were observed in female mutants. In addition, 12% of null mutant mice developed hydronephrosis. Concentrations of plasma and urine electrolytes in Hspa4l −/− mice were similar to wild-type values, suggesting that the renal function was not impaired. However, Hspa4l −/− animals were preferentially susceptible to osmotic stress. These results provide evidence that Hspa4l is required for normal spermatogenesis and suggest that Hspa4l plays a role in osmotolerance.

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Downregulation of mitochondrial biogenesis by virus infection triggers antiviral responses by cyclic GMP-AMP synthase

PLoS Pathogens ◽

10.1371/journal.ppat.1009841 ◽

2021 ◽

Vol 17 (10) ◽

pp. e1009841

Author(s):

Hiroki Sato ◽

Miho Hoshi ◽

Fusako Ikeda ◽

Tomoko Fujiyuki ◽

Misako Yoneda ◽

...

Keyword(s):

Mitochondrial Biogenesis ◽

Cyclic Gmp ◽

Mammalian Cells ◽

Measles Virus ◽

Rna Viruses ◽

Mitochondrial Morphology ◽

Mitochondrial Stress ◽

Antiviral Responses ◽

Genes Encoding

In general, in mammalian cells, cytosolic DNA viruses are sensed by cyclic GMP-AMP synthase (cGAS), and RNA viruses are recognized by retinoic acid-inducible gene I (RIG-I)-like receptors, triggering a series of downstream innate antiviral signaling steps in the host. We previously reported that measles virus (MeV), which possesses an RNA genome, induces rapid antiviral responses, followed by comprehensive downregulation of host gene expression in epithelial cells. Interestingly, gene ontology analysis indicated that genes encoding mitochondrial proteins are enriched among the list of downregulated genes. To evaluate mitochondrial stress after MeV infection, we first observed the mitochondrial morphology of infected cells and found that significantly elongated mitochondrial networks with a hyperfused phenotype were formed. In addition, an increased amount of mitochondrial DNA (mtDNA) in the cytosol was detected during progression of infection. Based on these results, we show that cytosolic mtDNA released from hyperfused mitochondria during MeV infection is captured by cGAS and causes consequent priming of the DNA sensing pathway in addition to canonical RNA sensing. We also ascertained the contribution of cGAS to the in vivo pathogenicity of MeV. In addition, we found that other viruses that induce downregulation of mitochondrial biogenesis as seen for MeV cause similar mitochondrial hyperfusion and cytosolic mtDNA-priming antiviral responses. These findings indicate that the mtDNA-activated cGAS pathway is critical for full innate control of certain viruses, including RNA viruses that cause mitochondrial stress.

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Talented family: Inositol phosphates

The Biochemist ◽

10.1042/bio03105026 ◽

2009 ◽

Vol 31 (5) ◽

pp. 26-29 ◽

Cited By ~ 1

Author(s):

Robin Irvine

Keyword(s):

Phospholipase C ◽

Higher Plants ◽

Inositol Phosphates ◽

Inositol Phosphate ◽

Animal Cells ◽

Slime Moulds ◽

Starting Point ◽

Inositol Lipids

So, what's a review on inositol phosphates doing in a nice lipid-themed place like this? (given how watersoluble and un-lipiddy they are). But they are, at least in animal cells, inextricably linked with inositol lipids. The link is the phospholipase C (PI-PLC) reaction, which splits phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] into diacylglycerol and Ins(1,4,5)P3, of which the latter mobilizes intracellular Ca2+ (this well-known inositol phosphate will not be discussed further). Over the last few years, a consensus has emerged that the PI-PLC reaction is also the starting point of all inositol phosphate synthesis in animal cells (Figure 1). Some organisms, especially InsP6-obsessives such as higher plants (who make lots of InsP6 to use as a phosphate-storage compound) and slime moulds (who make even more InsP6 even more quickly, for reasons known only to slime moulds) can also phosphorylate inositol sequentially up to InsP6 without any lipid intermediate. But we animals apparently have to start with Ins(1,4,5)P3 generated from PtdIns(4,5)P2 to make all our inositol phosphates.

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