scholarly journals Identification and characterization of differentially active pools of type IIα phosphatidylinositol 4-kinase activity in unstimulated A431 cells

2003 ◽  
Vol 376 (2) ◽  
pp. 497-503 ◽  
Author(s):  
Mark G. WAUGH ◽  
Shane MINOGUE ◽  
Deena BLUMENKRANTZ ◽  
J. Simon ANDERSON ◽  
J. Justin HSUAN
Keyword(s):  
Kinase Activity ◽  
Cell Signalling ◽  
Cell Types ◽  
Intrinsic Activity ◽  
A431 Cells ◽  
Cell Functions ◽  
Subcellular Membrane ◽  
Phosphatidylinositol Kinase ◽  
Wide Range ◽  
Phosphatidylinositol 4

The seven known polyphosphoinositides have been implicated in a wide range of regulated and constitutive cell functions, including cell-surface signalling, vesicle trafficking and cytoskeletal reorganization. In order to understand the spatial and temporal control of these diverse cell functions it is necessary to characterize the subcellular distribution of a wide variety of polyphosphoinositide synthesis and signalling events. The predominant phosphatidylinositol kinase activity in many mammalian cell types involves the synthesis of the signalling precursor, phosphatidylinositol 4-phosphate, in a reaction catalysed by the recently cloned PI4KIIα (type IIα phosphatidylinositol 4-kinase). However the regulation of this enzyme and the cellular distribution of its product in different organelles are very poorly understood. This report identifies the existence, in unstimulated cells, of two major subcellular membrane fractions, which contain PI4KIIα possessing different levels of intrinsic activity. Separation of these membranes from each other and from contaminating activities was achieved by density gradient ultracentrifugation at pH 11 in a specific detergent mixture in which both membrane fractions, but not other membranes, were insoluble. Kinetic comparison of the purified membrane fractions revealed a 4-fold difference in Km for phosphatidylinositol and a 3.5-fold difference in Vmax, thereby indicating a different mechanism of regulation to that described previously for agonist-stimulated cells. These marked differences in basal activity and the occurrence of this isozyme in multiple organelles emphasize the need to investigate cell signalling via PI4KIIα at the level of individual organelles rather than whole-cell lysates.

Diphosphoinositide metabolism in bovine adrenal medulla

1976 ◽  
Vol 54 (8) ◽  
pp. 746-753 ◽  
Author(s):  
Y. A. Lefebvre ◽  
D. A. White ◽  
J. N. Hawthorne
Keyword(s):  
Adrenal Medulla ◽  
Calcium Binding ◽  
Microsomal Fraction ◽  
Kinase Activity ◽  
Hydrolytic Activity ◽  
Vesicle Membrane ◽  
Bovine Adrenal Medulla ◽  
Phosphatidylinositol Kinase ◽  
Membrane Bound ◽  
Phosphatidylinositol 4

(1) A phosphatidylinositol kinase (EC 2.7.1.67) ofa chromaffin vesicle membrane preparation isolated from bovine adrenal medulla was characterized. Its activity towards endogenous and exogenous phosphatidylinositol was very similar to the kinase activity of the microsomal fraction prepared from the same tissue.(2) Phosphomonoesterase (EC 3.1.3.36) and diesterase activity hydrolysing membrane bound phosphatidylinositol 4-phosphate was located mainly in the microsomal fraction. No hydrolytic activity was present in the vesicle membrane.(3) Phosphorylation of chromaffin vesicle membrane phosphatidylinositol did not increase calcium-binding by the membranes.

scholarly journals Detection of protein kinase activity specifically activated at metaphase-anaphase transition.

1996 ◽  
Vol 132 (4) ◽  
pp. 635-641 ◽  
Author(s):  
M Sekimata ◽  
K Tsujimura ◽  
J Tanaka ◽  
Y Takeuchi ◽  
N Inagaki ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Glioma Cells ◽  
Cell Types ◽  
Protein Kinase Activity ◽  
Cleavage Furrow ◽  
Wide Range ◽  
Interphase Cells ◽  
Cycle Dependent ◽  
Mitotic Cells

We have previously reported that Ser13 and Ser34 on glial fibrillary acidic protein (GFAP) in the cleavage furrow of glioma cells are phosphorylated during late mitotic phase (Matsuoka, Y., K. Nishizawa, T. Yano, M. Shibata, S. Ando, T. Takahashi, and M. Inagaki. 1992, EMBO (Eur. Mol. Biol. Organ.) J. 11:2895-2902). This observation implies a possibility that there is a protein kinase specifically activated at metaphase-anaphase transition. To further analyze the cell cycle-dependent GFAP phosphorylation, we prepared monoclonal antibodies KT13 and KT34 which recognize the phosphorylation of GFAP at Ser13 and Ser34, respectively. Immunocytochemical studies with KT13 and KT34 revealed that the GFAP phosphorylation in the cleavage furrow during late mitotic phase occurred not only in glioma cells but also in human SW-13 and mouse Ltk- cells in which GFAP was ectopically expressed, thus the phosphorylation can be monitored in a wide range of cell types. Furthermore, we detected kinase activity which phosphorylates GFAP at Ser13 and Ser34 in the lysates of late mitotic cells but not in those of interphase cells or early mitotic cells. These results suggest that there exists a protein kinase which is specifically activated at the transition of metaphase to anaphase not only in GFAP-expressing cells but also in cells without GFAP.

scholarly journals Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat β-cell tumour

1984 ◽  
Vol 219 (2) ◽  
pp. 471-480 ◽  
Author(s):  
N E Tooke ◽  
C N Hales ◽  
J C Hutton
Keyword(s):  
Plasma Membrane ◽  
Beta Cell ◽  
Secretory Granule ◽  
Plasma Membranes ◽  
Kinase Activity ◽  
Secretory Granules ◽  
Cell Tumour ◽  
Density Gradients ◽  
Phosphatidylinositol Kinase ◽  
Phosphatidylinositol 4

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.

scholarly journals Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition

2020 ◽  
Author(s):  
Yi Shen ◽  
Francesco Simone Ruggeri ◽  
Daniele Vigolo ◽  
Ayaka Kamada ◽  
Seema Qamar ◽  
...  
Keyword(s):  
Phase Transition ◽  
Liquid Phase ◽  
Rna Binding ◽  
Cell Signalling ◽  
Physical Factors ◽  
Transition Control ◽  
Cell Functions ◽  
Fused In Sarcoma ◽  
Wide Range ◽  
Liquid To Solid Transition

A wide range of systems containing proteins have been shown to undergo liquid-liquid phase separation (LLPS) forming membraneless compartments, such as processing bodies1, germ granules2, stress granules3 and Cajal bodies4. The condensates resulting from this phase transition control essential cell functions, including mRNA regulation, cytoplasm structuring, cell signalling and embryogenesis1–4. RNA-binding Fused in Sarcoma (FUS) protein is one of the most studied systems in this context, due to its important role in neurodegenerative diseases5–7. It has recently been discovered that FUS condensates can undergo an irreversible phase transition which results in fibrous aggregate formation6. Gelation of protein condensates is generally associated with pathology. One case where liquid-to-solid transition (LST) of liquid-liquid phase separated proteins is functional, however, is that of silk spinning8,9, which is largely driven by shear, but it is not known what factors control the pathological gelation of functional condensates. Here we show that four proteins and one peptide system not related to silk, and with no function associated with fibre formation, have a strong propensity to undergo LST when exposed to even low levels of mechanical shear comparable to those found inside a living cell, once present in their liquid-liquid phase separated forms. Using microfluidics to control the application of mechanical shear, we generated fibres from single protein condensates and characterized their structures and material properties as a function of shear stress. Our results inform on the molecular grammar underlying protein LST and highlight generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. Taken together, these observations suggest that the shear plays an important role in the irreversible phase transition of liquid-liquid phase separated droplets, shed light on the role of physical factors in driving this transition in protein aggregation related diseases, and open a new route towards artificial shear responsive biomaterials.

scholarly journals First ComprehensiveIn SilicoAnalysis of the Functional and Structural Consequences of SNPs in HumanGalNAc-T1Gene

2014 ◽  
Vol 2014 ◽  
pp. 1-15 ◽  
Author(s):  
Hussein Sheikh Ali Mohamoud ◽  
Muhammad Ramzan Manwar Hussain ◽  
Ashraf A. El-Harouni ◽  
Noor Ahmad Shaik ◽  
Zaheer Ulhaq Qasmi ◽  
...  
Keyword(s):  
Disease Susceptibility ◽  
Cell Signalling ◽  
Gene Mutations ◽  
Cell Types ◽  
Biological Tissues ◽  
Coding Regions ◽  
Wide Range ◽  
Glycosylation Pathway ◽  
And Function ◽  
Regulatory Snps

GalNAc-T1, a key candidate of GalNac-transferases genes family that is involved in mucin-typeO-linked glycosylation pathway, is expressed in most biological tissues and cell types. Despite the reported association ofGalNAc-T1gene mutations with human disease susceptibility, the comprehensive computational analysis of coding, noncoding and regulatory SNPs, and their functional impacts on protein level, still remains unknown. Therefore, sequence- and structure-based computational tools were employed to screen the entire listed coding SNPs ofGalNAc-T1gene in order to identify and characterize them. Our concordantin silicoanalysis by SIFT, PolyPhen-2, PANTHER-cSNP, and SNPeffect tools, identified the potential nsSNPs (S143P, G258V, and Y414D variants) from 18 nsSNPs ofGalNAc-T1. Additionally, 2 regulatory SNPs (rs72964406 and #x26; rs34304568) were also identified inGalNAc-T1by using FastSNP tool. Using multiple computational approaches, we have systematically classified the functional mutations in regulatory and coding regions that can modify expression and function ofGalNAc-T1enzyme. These genetic variants can further assist in better understanding the wide range of disease susceptibility associated with the mucin-based cell signalling and pathogenic binding, and may help to develop novel therapeutic elements for associated diseases.

scholarly journals Identification of Key Phospholipids That Bind and Activate Atypical PKCs

Biomedicines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 45
Author(s):  
Suresh Velnati ◽  
Sara Centonze ◽  
Federico Girivetto ◽  
Daniela Capello ◽  
Ricardo M. Biondi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Kinase Activity ◽  
Membrane Lipids ◽  
Atypical Protein Kinase C ◽  
Atypical Protein Kinase ◽  
Kinase C ◽  
Specific Regulation ◽  
Phosphatidylinositol 4

PKCζ and PKCι/λ form the atypical protein kinase C subgroup, characterised by a lack of regulation by calcium and the neutral lipid diacylglycerol. To better understand the regulation of these kinases, we systematically explored their interactions with various purified phospholipids using the lipid overlay assays, followed by kinase activity assays to evaluate the lipid effects on their enzymatic activity. We observed that both PKCζ and PKCι interact with phosphatidic acid and phosphatidylserine. Conversely, PKCι is unique in binding also to phosphatidylinositol-monophosphates (e.g., phosphatidylinositol 3-phosphate, 4-phosphate, and 5-phosphate). Moreover, we observed that phosphatidylinositol 4-phosphate specifically activates PKCι, while both isoforms are responsive to phosphatidic acid and phosphatidylserine. Overall, our results suggest that atypical Protein kinase C (PKC) localisation and activity are regulated by membrane lipids distinct from those involved in conventional PKCs and unveil a specific regulation of PKCι by phosphatidylinositol-monophosphates.

scholarly journals Tuning the electronic structure of Ag-Pd alloys to enhance performance for alkaline oxygen reduction

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
José A. Zamora Zeledón ◽  
Michaela Burke Stevens ◽  
G. T. Kasun Kalhara Gunasooriya ◽  
Alessandro Gallo ◽  
Alan T. Landers ◽  
...  
Keyword(s):  
Metal Binding ◽  
Precious Metal ◽  
Metal Content ◽  
Density Functional ◽  
Binding Energies ◽  
Intrinsic Activity ◽  
Energy Technologies ◽  
Highly Active ◽  
Wide Range ◽  
Precious Metal Content

AbstractAlloying is a powerful tool that can improve the electrocatalytic performance and viability of diverse electrochemical renewable energy technologies. Herein, we enhance the activity of Pd-based electrocatalysts via Ag-Pd alloying while simultaneously lowering precious metal content in a broad-range compositional study focusing on highly comparable Ag-Pd thin films synthesized systematically via electron-beam physical vapor co-deposition. Cyclic voltammetry in 0.1 M KOH shows enhancements across a wide range of alloys; even slight alloying with Ag (e.g. Ag0.1Pd0.9) leads to intrinsic activity enhancements up to 5-fold at 0.9 V vs. RHE compared to pure Pd. Based on density functional theory and x-ray absorption, we hypothesize that these enhancements arise mainly from ligand effects that optimize adsorbate–metal binding energies with enhanced Ag-Pd hybridization. This work shows the versatility of coupled experimental-theoretical methods in designing materials with specific and tunable properties and aids the development of highly active electrocatalysts with decreased precious-metal content.

scholarly journals The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration

2020 ◽  
Author(s):  
Paymaan Jafar-nejad ◽  
Berit Powers ◽  
Armand Soriano ◽  
Hien Zhao ◽  
Daniel A Norris ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Muscular Atrophy ◽  
Neurological Diseases ◽  
Cell Types ◽  
Rnase H ◽  
Central Administration ◽  
Spinal Motor Neurons ◽  
New Class ◽  
Wide Range ◽  
Therapeutic Development

Abstract Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.

scholarly journals Energetics of mesoscale cell turbulence in two-dimensional monolayers

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shao-Zhen Lin ◽  
Wu-Yang Zhang ◽  
Dapeng Bi ◽  
Bo Li ◽  
Xi-Qiao Feng
Keyword(s):  
Kinetic Energy ◽  
Tumor Metastasis ◽  
Cell Types ◽  
Boltzmann Distribution ◽  
Scaling Exponent ◽  
Biological Processes ◽  
Two Dimensional ◽  
Cell Monolayers ◽  
Wide Range ◽  
Epithelial Cell Monolayers

AbstractInvestigation of energy mechanisms at the collective cell scale is a challenge for understanding various biological processes, such as embryonic development and tumor metastasis. Here we investigate the energetics of self-sustained mesoscale turbulence in confluent two-dimensional (2D) cell monolayers. We find that the kinetic energy and enstrophy of collective cell flows in both epithelial and non-epithelial cell monolayers collapse to a family of probability density functions, which follow the q-Gaussian distribution rather than the Maxwell–Boltzmann distribution. The enstrophy scales linearly with the kinetic energy as the monolayer matures. The energy spectra exhibit a power-decaying law at large wavenumbers, with a scaling exponent markedly different from that in the classical 2D Kolmogorov–Kraichnan turbulence. These energetic features are demonstrated to be common for all cell types on various substrates with a wide range of stiffness. This study provides unique clues to understand active natures of cell population and tissues.

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