scholarly journals Cloning and characterization of a 92 kDa soluble phosphatidylinositol 4-kinase

1996 ◽  
Vol 320 (2) ◽  
pp. 643-649 ◽  
Author(s):  
Tamotsu NAKAGAWA ◽  
Kaoru GOTO ◽  
Hisatake KONDO
Keyword(s):  
Kinase Activity ◽  
Catalytic Domain ◽  
Adult Brain ◽  
Epitope Tag ◽  
Lipid Kinase ◽  
Calculated Molecular Mass ◽  
Shared Identity ◽  
Brain Cdna Library ◽  
Lipid Kinases ◽  
Phosphatidylinositol 4

A phosphatidylinositol (PtdIns) 4-kinase cDNA cloned from a rat brain cDNA library encoded a protein of 816 amino acids with a calculated molecular mass of 91654 Da. This molecule contained a lipid-kinase-unique domain and a presumed lipid/protein kinase homology domain that are found in other PtdIns 4-kinases and PtdIns 3-kinases. Furthermore, this kinase molecule had 43.3% shared identity with the presumed catalytic domain of yeast PtdIns 4-kinase, PtdInsK1, and the two molecules had a region of similarity that is not conserved in other lipid kinases. By examining PtdIns kinase activity in transfected COS-7 cells using epitope tag immunoprecipitation as well as conventional methods, the product PtdIns phosphate was identified as phosphatidylinositol 4-phosphate (PtdIns4P), but not phosphatidylinositol 3-phosphate (PtdIns3P). The PtdIns 4-kinase activity was recovered predominantly from the soluble fraction and the activity was markedly enhanced in the presence of Triton X-100 and was relatively insensitive to inhibition by adenosine. In addition, the PtdIns 4-kinase activity was completely inhibited in the presence of 10 µM wortmannin. When examined by epitope tag immunocytochemistry, the immunoreactivity for the PtdIns 4-kinase molecule was dominantly aggregated in a cytoplasmic region juxtaposed to the nuclei and was faintly but widely dispersed in the cytoplasm. By in situ hybridization analysis, the mRNA for PtdIns 4-kinase was expressed ubiquitously and was detected in most neurons throughout the grey matter of the brain, with higher expression intensity found in fetal than in adult brain.

Molecular determinants of activation and membrane targeting of phosphoinositol 4-kinase IIβ

2007 ◽  
Vol 409 (2) ◽  
pp. 501-509 ◽  
Author(s):  
Gwanghyun Jung ◽  
Jing Wang ◽  
Pawel Wlodarski ◽  
Barbara Barylko ◽  
Derk D. Binns ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Integral Membrane Protein ◽  
Type Ii ◽  
Lipid Kinase ◽  
Catalytic Domains ◽  
Specific Behaviour ◽  
Molecular Determinants

Mammalian cells contain two isoforms of the type II PI4K (phosphoinositol 4-kinase), PI4KIIα and β. These 55 kDa proteins have highly diverse N-terminal regions (approximately residues 1–90) but conserved catalytic domains (approximately from residue 91 to the C-termini). Nearly the entire pool of PI4KIIα behaves as an integral membrane protein, in spite of a lack of a transmembrane domain. This integral association with membranes is due to palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domain. Although the CCPCC motif is conserved in PI4KIIβ, only 50% of PI4KIIβ is membrane-associated, and approximately half of this pool is only peripherally attached to the membranes. Growth factor stimulation or overexpression of a constitutively active Rac mutant induces the translocation of a portion of cytosolic PI4KIIβ to plasma membrane ruffles and stimulates its activity. Here, we demonstrate that membrane-associated PI4KIIβ undergoes two modifications, palmitoylation and phosphorylation. The cytosolic pool of PI4KIIβ is not palmitoylated and has much lower lipid kinase activity than the membrane-associated kinase. Although only membrane-associated PI4KIIβ is phosphorylated in the unique N-terminal region, this modification apparently does not influence its membrane binding or activity. A series of truncation mutants and α/β chimaeras were generated to identify regions responsible for the isoform-specific behaviour of the kinases. Surprisingly, the C-terminal approx. 160 residues, and not the diverse N-terminal regions, contain the sites that are most important in determining the different solubilities, palmitoylation states and stimulus-dependent redistributions of PI4KIIα and β.

Regulation and recruitment of phosphatidylinositol 4-kinase on immature secretory granules is independent of ADP-ribosylation factor 1

2002 ◽  
Vol 363 (2) ◽  
pp. 289-295 ◽  
Author(s):  
Christina PANARETOU ◽  
Sharon A. TOOZE
Keyword(s):  
Kinase Activity ◽  
Secretory Granules ◽  
Specific Inhibitor ◽  
Small Gtpases ◽  
Type Ii ◽  
Adp Ribosylation ◽  
Lipid Kinases ◽  
Phosphatidylinositol 4 ◽  
Golgi Network ◽  
Adp Ribosylation Factor

Heterotrimeric G-proteins, as well as small GTPases of the Rho and ADP-ribosylation factor (ARF) family, are implicated in the regulation of lipid kinases, including PtdIns 4-kinases and PtdIns(4)P 5-kinases. Here, we describe a PtdIns 4-kinase activity on immature secretory granules (ISGs), regulated secretory organelles formed from the trans-Golgi network (TGN), and investigate the regulation of PtdIns4P levels on these membranes. Over 50% of the PtdIns 4-kinase activity on ISGs is inhibited by both a low concentration of adenosine and the monoclonal antibody 4C5G, a specific inhibitor of the type II PtdIns 4-kinase. Treatment of ISGs with mastoparan 7 (M7) stimulates the type II PtdIns 4-kinase via pertussis-toxin-sensitive Gi/G0 proteins, which, in contrast with previous results obtained with chromaffin granules [Gasman, Chasserot-Golaz, Hubert, Aunis and Bader (1998) J. Biol. Chem. 273, 16913–16920], does not require Rho A, B or C. M7 treatment also leads to an inhibition in the recruitment of ARF to ISG membranes: this inhibition is not dependent on Gi/G0 activation, and is not linked to the stimulation of PtdIns 4-kinase observed with M7. PtdIns 4-kinase activity on ISGs is not regulated by myristoylated ARF1—GTP, in contrast with results obtained with Golgi membranes [Godi, Pertile, Meyers, Marra, Di Tullio, Iurisci, Luini, Corda and De Matteis (1999) Nat. Cell Biol. 1, 280–287; Jones, Morris, Morgan, Kondo, Irvine and Cockcroft (2000) J. Biol. Chem. 275, 13962–13170], whereas ARF1—GTP does regulate the production of PtdIns(4,5)P2. Our results suggest that the regulation of PtdIns 4-kinase on the ISGs differs in comparison with that on the TGN, and might be related to a specific requirement of ISG maturation.

Phosphoinositide 3-kinase: the protein kinase that time forgot

2004 ◽  
Vol 32 (2) ◽  
pp. 330-331 ◽  
Author(s):  
L.C. Foukas ◽  
P.R. Shepherd
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Class I ◽  
Protein Kinase Activity ◽  
Lipid Kinase ◽  
Vast Amount ◽  
Lipid Kinases ◽  
Phosphoinositide 3 Kinase ◽  
In Cells

Class I phosphoinositide 3-kinases were originally characterized as lipid kinases, although more than 10 years ago they were also found to phosphorylate protein serine residues. However, while there is a vast amount of data on the function of this lipid kinase activity, relatively little is known about the function of the protein kinase activity. We discuss the evidence that suggests that the protein kinase activity of phosphoinositide 3-kinases mediates important signalling functions in cells.

scholarly journals Enzyme activity effects of N-terminal His-tag attached to catalytic sub-unit of phosphoinositide-3-kinase

2013 ◽  
Vol 33 (6) ◽  
Author(s):  
James M. J. Dickson ◽  
Woo-Jeong Lee ◽  
Peter R. Shepherd ◽  
Christina M. Buchanan
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Cell Signalling ◽  
Protein Kinase Activity ◽  
Lipid Kinase ◽  
His Tag ◽  
The Impact

NTT (N-terminal tags) on the catalytic (p110) sub-unit of PI 3-K (phosphoinositol 3-kinase) have previously been shown to increase cell signalling and oncogenic transformation. Here we test the impact of an NT (N-terminal) His-tag on in vitro lipid and protein kinase activity of all class-1 PI 3-K isoforms and two representative oncogenic mutant forms (E545K and H1047R), in order to elucidate the mechanisms behind this elevated signalling and transformation observed in vivo. Our results show that an NT His-tag has no impact on lipid kinase activity as measured by enzyme titration, kinetics and inhibitor susceptibility. Conversely, the NT His-tag did result in a differential effect on protein kinase activity, further potentiating the elevated protein kinase activity of both the helical domain and catalytic domain oncogenic mutants with relation to p110 phosphorylation. All other isoforms also showed elevated p110 phosphorylation (although not statistically significant). We conclude that the previously reported increase in cell signalling and oncogenic-like transformation in response to p110 NTT is not mediated via an increase in the lipid kinase activity of PI 3-K, but may be mediated by increased p110 autophosphorylation and/or other, as yet unidentified, intracellular protein/protein interactions. We further observe that tagged recombinant protein is suitable for use in in vitro lipid kinase screens to identify PI 3-K inhibitors; however, we recommend that in vivo (including intracellular) experiments and investigations into the protein kinase activity of PI 3-K should be conducted with untagged constructs.

scholarly journals Development of a High-Throughput Screening Assay to Identify Inhibitors of the Lipid Kinase PIP5K1C

2014 ◽  
Vol 20 (5) ◽  
pp. 655-662 ◽  
Author(s):  
Brittany D. Wright ◽  
Catherine Simpson ◽  
Michael Stashko ◽  
Dmitri Kireev ◽  
Emily A. Hull-Ryde ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mobility Shift ◽  
Screening Assay ◽  
Lipid Kinase ◽  
Cellular Processes ◽  
High Throughput Screening Assay ◽  
Lipid Kinases ◽  
Mobility Shift Assay ◽  
Phosphatidylinositol 4

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes, including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Because small-molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel, high-throughput, microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay uses fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated adenosine triphosphate Michaelis constant (Km) of 15 µM, performed with z’ values >0.7, and was used to screen a kinase-focused library of ~4700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be adapted easily to screen additional lipid kinases.

scholarly journals Expression and Purification of PI3 Kinase α and Development of an ATP Depletion and an AlphaScreen PI3 Kinase Activity Assay

2008 ◽  
Vol 13 (10) ◽  
pp. 1035-1040 ◽  
Author(s):  
Brigitte Boldyreff ◽  
Tine L. Rasmussen ◽  
Hans H. Jensen ◽  
Alexandre Cloutier ◽  
Lucille Beaudet ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Catalytic Domain ◽  
Kinase Inhibitor ◽  
Pi3 Kinase ◽  
Atp Depletion ◽  
Nickel Affinity Chromatography ◽  
Phosphatidylinositol 4 ◽  
Kinase Activity Assay

Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110α catalytic domain with an N-terminal His-tag and the p85α regulatory domain in Sf9 insect cells. The complex consisting of p110α and p85α was purified by nickel affinity chromatography. The authors established an adenosine triphosphate (ATP) depletion assay to measure the activity of p110α/p85α. The assay was optimized by testing different lipids as substrates, as well as various kinase and lipid concentrations. Furthermore, they analyzed autophosphorylation of p110α/p85α and determined the IC50 for wortmannin, a known PI3 kinase inhibitor. The IC50 for wortmannin was determined to be 7 nM. From a selection of substrates, phosphatidylinositol-4, 5-biphosphate turned out to be the best substrate at a concentration of 50 μM. p110α/p85α underwent autophosphorylation most prominently at the p85α subunit. However, in the presence of lipid substrate, the autophosphorylation was negligible. In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors. ( Journal of Biomolecular Screening 2008:1035-1040)

scholarly journals Septin function tunes lipid kinase activity and phosphatidylinositol 4,5 bisphosphate turnover during G-protein coupled PLC signaling in vivo

2021 ◽  
Author(s):  
Aastha Kumari ◽  
Avishek Ghosh ◽  
Sourav Kolay ◽  
RAGHU PADINJAT
Keyword(s):  
Plasma Membrane ◽  
Kinase Activity ◽  
Electrical Response ◽  
Receptor Activation ◽  
Sensory Transduction ◽  
Photon Absorption ◽  
Lipid Kinase ◽  
Phosphatidylinositol 4

The hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane by receptor activated phospholipase C (PLC) activity is a conserved mechanism of signal transduction. Given the low abundance of PI(4,5)P2 at the plasma membrane, its hydrolysis needs to be coupled to lipid resynthesis to ensure continued PLC activity during receptor activation. However, the mechanism by which PI(4,5)P2 depletion during signalling is coupled to its resynthesis remains unknown. PI(4,5)P2 synthesis is catalyzed by lipid kinase activity and the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K) is the final step in this process. In Drosophila photoreceptors, sensory transduction of photon absorption is transduced into PLC activity leading to an electrical response to light. During this process, PI(4,5)P2 is resynthesized by a PIP5K activity but the mechanism by which the activity of this enzyme is coupled to PLC signalling is not known. In this study, we identify a unique protein isoform of dPIP5K, dPIP5KL that is both necessary and sufficient to mediate PI(4,5)P2 synthesis during phototransduction. The activity of dPIP5KL in vitro is enhanced by depletion of PNUT, a non-redundant subunit of the septin family of GTP binding proteins and in vivo, depletion of pnut rescues the effect of dPIP5KL depletion on the light response and PI(4,5)P2 resynthesis during PLC signalling. Lastly we find that depletion of Septin Interacting Protein 1 (Sip1),previously shown to bind PNUT, phenocopies the effect of dPIP5KL depletion in vivo. Thus, our work defines a septin 7 and Sip1 mediated mechanism through which PIP5K activity is coupled to ongoing PLC mediated PI(4,5)P2 depletion.

scholarly journals Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C

2000 ◽  
Vol 352 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Valérie DEWASTE ◽  
Valérie POUILLON ◽  
Colette MOREAU ◽  
Stephen SHEARS ◽  
Kazunaga TAKAZAWA ◽  
...  
Keyword(s):  
Northern Blot Analysis ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Inhibitory Effect ◽  
Enzymic Activity ◽  
Cloning And Expression ◽  
Human Tissues ◽  
Calculated Molecular Mass ◽  
Kinase C ◽  
Inositol 1,4,5 Trisphosphate

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P3 to Ins(1,3,4,5)P4. cDNAs encoding two isoenzymes of Ins(1,4,5)P3 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P3 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75.207kDa and a Km for Ins(1,4,5)P3 of 6µM. Northern-blot analysis showed the presence of a transcript of approx. 3.9kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5αF′ bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P3 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin–Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P3 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin–Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin.

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