scholarly journals Inositol polyphosphate metabolism and inositol lipids in a green alga, Chlamydomonas eugametos

1992 ◽  
Vol 281 (1) ◽  
pp. 261-266 ◽  
Author(s):  
R F Irvine ◽  
A J Letcher ◽  
L R Stephens ◽  
A Musgrave
Keyword(s):  
Kinase Activity ◽  
Early Time ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Physiological Conditions ◽  
Chlamydomonas Eugametos ◽  
Inositol Lipids ◽  
Mammalian Counterpart ◽  
Polyphosphate Metabolism

Swimming suspensions of Chlamydomonas eugametos were pelleted and homogenized, and the metabolism of inositol polyphosphates by cellular homogenates or supernatants was investigated. Ins(1,4,5)P3 was dephosphorylated under physiological conditions to yield a single InsP2, Ins(1,4]2. In the presence of ATP it was phosphorylated to give Ins(1,3,4,5)P3 as the only InsP4. The Ins(1,4,5)P3 3-kinase activity was predominantly soluble, was not detectably affected by calmodulin or Ca2+, and had a Km for Ins(1,4,5)P3 of 50 microM (two orders of magnitude higher than its mammalian counterpart). Ins(1,3,4,5)P4 was dephosphorylated by the cellular supernatants to Ins(1,3,4)P3 and Ins(1,4,5)P3, and could be phosphorylated to Ins(1,3,4,5,6)P4. No Ins(1,3,4)P3 6-kinase activity could be detected, and experiments with [3H]Ins(1,4,[32P]5)P3 revealed that Ins(1,3,4,5,6)P5 is formed from Ins(1,4,5)P3 with little loss of the 5-phosphate, i.e. the predominant route of synthesis is probably by a direct 6-phosphorylation of Ins(1,3,4,5)P4. Similar experiments with an (NH4)2SO4 fraction of turkey erythrocyte cytosol gave essentially the same result, i.e. direct phosphorylation of Ins(1,3,4,5)P4 in the 6 position is the predominant route of synthesis of InsP5 from that InsP4 in vitro. No InsP6 formation was detected in any of these experiments, but labelling of intact C. eugametos with [3H]inositol revealed that the cells do synthesize InsP6. The lipids of C. eugametos cells contain PtdIns, PtdIns(4)P and PtdIns(4,5)P2 [Irvine, Letcher, Lander, Drøbak, Dawson & Musgrave (1989) Plant Physiol. 64, 888-892]. Further examination of 32P-labelled lipids revealed that about 20% of the PtdInsP was the PtdIns(3)P isomer, and about 1% or less of the PtdInsP2 was the PtdIns(3,4)P2 isomer. The overall inositide metabolism of C. eugametos resembles that of a mammalian cell more closely than it does that of a plant cell or slime mould, and this suggests firstly that the known metabolism of inositol polyphosphates arose at an early time in eukaryotic evolution, and secondly that Chlamydomonas might prove a useful organism for genetic and comparative studies of inositide enzymology.

scholarly journals Multiple isomers of inositol pentakisphosphate in Epstein-Barr-virus- transformed (T5-1) B-lymphocytes. Identification of inositol 1,3,4,5,6-pentakisphosphate, d-inositol 1,2,4,5,6-pentakisphosphate and l-inositol 1,2,4,5,6-pentakisphosphate

1991 ◽  
Vol 280 (2) ◽  
pp. 323-329 ◽  
Author(s):  
F M McConnell ◽  
L R Stephens ◽  
S B Shears
Keyword(s):  
Inositol Polyphosphate ◽  
Permeabilized Cells ◽  
Barr Virus ◽  
Epstein Barr ◽  
Polyphosphate Metabolism ◽  
And Function ◽  
Lymphocyte Cell ◽  
Lymphocyte Cell Line

Substantial amounts of three [3H]InsP5 isomers were detected in [3H]inositol-labelled human lymphoblastoid (T5-1) cells. Their structures were determined by h.p.l.c. [Phillippy & Bland (1988) Anal. Biochem. 175, 162-166], and by utilizing a stereospecific D-inositol 1,2,4,5,6-pentakisphosphate 3-kinase from Dictyostelium discoideum [Stephens & Irvine (1990) Nature (London) 346, 580-583]. The structures were: inositol 1,3,4,5,6-pentakisphosphate, D-inositol 1,2,4,5,6-pentakisphosphate and L-inositol 1,2,4,5,6-pentakisphosphate. The relative proportions of these isomers (approx. 73:14:14 respectively) were unaffected by cross-linking anti-IgD receptors. The T5-1 cells also contained InsP6 and three Ins P4s, which were identified as the 1,3,4,5, 1,3,4,6 and 3,4,5,6 isomers. In incubations with permeabilized T5-1 cells, both 1,3,4,6 and 3,4,5,6 isomers of InsP4 were phosphorylated solely to Ins(1,3,4,5,6)P5. Permeabilized cells also dephosphorylated InsP6, even in the presence of a large excess of glucose 6-phosphate to saturate non-specific phosphatases. In the latter experiments the following isomers of InsP5 accumulated: D- and/or L-Ins(1,2,3,4,5)P5, plus D- and/or L-Ins(1,2,4,5,6)P5. This demonstration that multiple isomers of InsP5 may be formed in vivo and in vitro by a transformed lymphocyte cell line adds a new level of complexity to the study of inositol polyphosphate metabolism and function.

scholarly journals MINPP1 prevents intracellular accumulation of the cation chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

2020 ◽  
Author(s):  
Ekin Ucuncu ◽  
Karthyayani Rajamani ◽  
Miranda S.C. Wilson ◽  
Daniel Medina-Cano ◽  
Nami Altin ◽  
...  
Keyword(s):  
Critical Role ◽  
Distinct Type ◽  
Cell Physiology ◽  
Inositol Polyphosphate ◽  
Loss Of Function ◽  
Pontocerebellar Hypoplasia ◽  
Inositol Polyphosphates ◽  
Induced Pluripotent ◽  
Basal Ganglia Involvement ◽  
Polyphosphate Metabolism

ABSTRACTInositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the multiple inositol polyphosphate phosphatase 1 gene (MINPP1). Patients were found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We found that patient-derived and genome edited MINPP1-/- induced pluripotent stem cells (iPSCs) are not able to differentiate efficiently into neurons. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakiphosphate (IP6), detected in HEK293, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP6 level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP6-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis.

scholarly journals Crystallographic and kinetic analyses of human IPMK reveal disordered domains modulate ATP binding and kinase activity

2018 ◽  
Author(s):  
Corey D. Seacrist ◽  
Raymond D. Blind
Keyword(s):  
Protein Interactions ◽  
Kinase Activity ◽  
Kinetic Studies ◽  
Fold Increase ◽  
Atp Binding ◽  
Inositol Polyphosphate ◽  
Ampk Signaling ◽  
N Terminus ◽  
Kinetic Mechanisms

ABSTRACTInositol polyphosphate multikinase (IPMK) is a member of the IPK-superfamily of kinases, catalyzing phosphorylation of several soluble inositols and the signaling phospholipid PI(4,5)P2(PIP2). IPMK also has critical non-catalytic roles in p53, mTOR/Raptor, TRAF6 and AMPK signaling mediated partly by two disordered domains. Although IPMK non-catalytic functions are well established, it is less clear if the disordered domains are important for IPMK kinase activity or ATP binding. Here, kinetic and structural analyses of an engineered human IPMK lacking all disordered domains (ΔIPMK) are presented. Although theKMfor PIP2is identical between ΔIPMK and wild type, ΔIPMK has a 1.8-fold increase inkcatfor PIP2, indicating the native IPMK disordered domains decrease IPMK activityin vitro. The 2.5 Å crystal structure of ΔIPMK is reported, confirming the conserved ATP-grasp fold. A comparison with other IPK-superfamily structures revealed a putative “ATP-clamp” in the disordered N-terminus, we predicted would stabilize ATP binding. Consistent with this observation, removal of the ATP clamp sequence increases theKMfor ATP 4.9-fold, indicating the N-terminus enhances ATP binding to IPMK. Together, these structural and kinetic studies suggest in addition to mediating protein-protein interactions, the disordered domains of IPMK impart modulatory capacity to IPMK kinase activity through multiple kinetic mechanisms.

scholarly journals Reduced inositol polyphosphate accumulation and inositol supply induced by lithium in stimulated cerebral cortex slices

1990 ◽  
Vol 267 (3) ◽  
pp. 781-786 ◽  
Author(s):  
E D Kennedy ◽  
R A J Challiss ◽  
C I Ragan ◽  
S R Nahorski
Keyword(s):  
Cerebral Cortex ◽  
Second Messengers ◽  
Kinetic Studies ◽  
Phosphoinositide Metabolism ◽  
Maximal Effect ◽  
Inositol Polyphosphate ◽  
Inositol Polyphosphates ◽  
Cortex Slices ◽  
Relationship Of

The ability of lithium to interfere with phosphoinositide metabolism in rat cerebral cortex slices has been examined by monitoring the accumulation of CMP-phosphatidate (CMP-PtdOH) and the reduction in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels. A small accumulation of [14C]CMP-PtdOH was seen in slices prelabelled with [14C]cytidine and stimulated with carbachol (1 mM) or Li+ (1 mM). However, simultaneous addition of both agents for 30 min produced a 22-fold accumulation, with Li+ producing a half-maximal effect at a concentration of 0.61 +/- 0.19 mM. Kinetic studies revealed that the effects of carbachol and Li+ on CMP-PtdOH accumulation occurred with no initial lag apparent under these conditions and that preincubation with myo-inositol (10 or 30 mM) dramatically attenuated CMP-PtdOH accumulation. myo-Inositol could also attenuate the rate of accumulation of CMP-PtdOH when added 20 min after carbachol and Li+; these effects were not observed when equimolar concentrations of scyllo-inositol were added. Use of specific radioreceptor assays allowed the mass accumulations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 to be monitored. Following a lag of 5-10 min, Li+ resulted in a marked reduction in the accumulation of both inositol polyphosphates resulting from muscarinic-cholinergic stimulation. Preincubation of cerebral cortex slices with myo- (but not scyllo-) inositol delayed, but did not prevent, the reduction in the accumulation of Ins(1,4,5)P3 or Ins(1,3,4,5)P4. The results suggest that cerebral cortex, at least in vitro, is very sensitive to myo-inositol depletion under conditions of muscarinic receptor stimulation. The relationship of such depletion to the generation of inositol polyphosphate second messengers is discussed.

scholarly journals MINPP1 prevents intracellular accumulation of the chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ekin Ucuncu ◽  
Karthyayani Rajamani ◽  
Miranda S. C. Wilson ◽  
Daniel Medina-Cano ◽  
Nami Altin ◽  
...  
Keyword(s):  
Critical Role ◽  
Distinct Type ◽  
Hek293 Cells ◽  
Cell Physiology ◽  
Inositol Polyphosphate ◽  
Loss Of Function ◽  
Pontocerebellar Hypoplasia ◽  
Inositol Hexakisphosphate ◽  
Inositol Polyphosphates ◽  
Polyphosphate Metabolism

AbstractInositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the multiple inositol-polyphosphate phosphatase 1 gene (MINPP1). Patients are found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We find that patient-derived and genome edited MINPP1−/− induced stem cells exhibit an inefficient neuronal differentiation combined with an increased cell death. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakisphosphate (IP6), detected in HEK293 cells, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP6 level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP6-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis.

Myo-inositol Polyphosphate Metabolism and Signaling in Arabidopsis

2013 ◽  
Vol 48 (1) ◽  
pp. 94-106
Author(s):  
Wu Li ◽  
Wang Ruozhong ◽  
Xu Wenzhong
Keyword(s):  
Inositol Polyphosphate ◽  
Polyphosphate Metabolism

Activation of bovine oocytes by protein synthesis inhibitors: new findings on the role of MPF/MAPKs†

2021 ◽  
Author(s):  
Cecilia Valencia ◽  
Felipe Alonso Pérez ◽  
Carola Matus ◽  
Ricardo Felmer ◽  
María Elena Arias
Keyword(s):  
Protein Synthesis ◽  
Kinase Activity ◽  
Cyclin B1 ◽  
Protein Synthesis Inhibitors ◽  
Vitro Fertilization ◽  
New Findings ◽  
Bovine Oocytes ◽  
Dependent Pathway

Abstract The present study evaluated the mechanism by which protein synthesis inhibitors activate bovine oocytes. The aim was to analyze the dynamics of MPF and MAPKs. MII oocytes were activated with ionomycin (Io), ionomycin+anisomycin (ANY) and ionomycin+cycloheximide (CHX) and by in vitro fertilization (IVF). The expression of cyclin B1, p-CDK1, p-ERK1/2, p-JNK, and p-P38 were evaluated by immunodetection and the kinase activity of ERK1/2 was measured by enzyme assay. Evaluations at 1, 4, and 15 hours postactivation (hpa) showed that the expression of cyclin B1 was not modified by the treatments. ANY inactivated MPF by p-CDK1Thr14-Tyr15 at 4 hpa (P < 0.05), CHX increased pre-MPF (p-CDK1Thr161 and p-CDK1Thr14-Tyr15) at 1 hpa and IVF increased p-CDK1Thr14-Tyr15 at 17 hours postfertilization (hpf) (P < 0.05). ANY and CHX reduced the levels of p-ERK1/2 at 4 hpa (P < 0.05) and its activity at 4 and 1 hpa, respectively (P < 0.05). Meanwhile, IVF increased p-ERK1/2 at 6 hpf (P < 0.05); however, its kinase activity decreased at 6 hpf (P < 0.05). p-JNK in ANY, CHX, and IVF oocytes decreased at 4 hpa (P < 0.05). p-P38 was only observed at 1 hpa, with no differences between treatments. In conclusion, activation of bovine oocytes by ANY, CHX, and IVF inactivates MPF by CDK1-dependent specific phosphorylation without cyclin B1 degradation. ANY or CHX promoted this inactivation, which seemed to be more delayed in the physiological activation (IVF). Both inhibitors modulated MPF activity via an ERK1/2-independent pathway, whereas IVF activated the bovine oocytes via an ERK1/2-dependent pathway. Finally, ANY does not activate the JNK and P38 kinase pathways.

scholarly journals Plant Physiology, 2021 Functional redox links between lumen thiol oxidoreductase1 and serine/threonine-protein kinase STN7

2021 ◽  
Author(s):  
Jianghao Wu ◽  
Liwei Rong ◽  
Weijun Lin ◽  
Lingxi Kong ◽  
Dengjie Wei ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Disulfide Bonds ◽  
Kinase Activity ◽  
Light Harvesting ◽  
Photosynthetic Electron Transport ◽  
State Transitions ◽  
Light Harvesting Complex ◽  
Light Harvesting Complex Ii

Abstract In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1.

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