scholarly journals Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases

1986 ◽  
Vol 240 (2) ◽  
pp. 341-348 ◽  
Author(s):  
A Imai ◽  
M J Rebecchi ◽  
M C Gershengorn
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Product Inhibition ◽  
Differential Regulation ◽  
Gh3 Cells ◽  
Maximal Effect ◽  
Phosphatidylinositol Kinase ◽  
Rat Pituitary ◽  
Phosphoinositide Kinase ◽  
Phosphatidylinositol 4

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.

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 Phosphatidylinositol 4,5-bisphosphate is regenerated by speeding of the PI 4-kinase pathway during long PLC activation

2020 ◽  
Vol 152 (12) ◽  
Author(s):  
Jongyun Myeong ◽  
Lizbeth de la Cruz ◽  
Seung-Ryoung Jung ◽  
Jun-Hee Yeon ◽  
Byung-Chang Suh ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Lipid Kinase ◽  
Agonist Action ◽  
Lipid Species ◽  
Phosphoinositide Cycle ◽  
M1 Muscarinic ◽  
Phosphatidylinositol 4 ◽  
Cellular Compartments ◽  
Regulatory Controls

The dynamic metabolism of membrane phosphoinositide lipids involves several cellular compartments including the ER, Golgi, and plasma membrane. There are cycles of phosphorylation and dephosphorylation and of synthesis, transfer, and breakdown. The simplified phosphoinositide cycle comprises synthesis of phosphatidylinositol in the ER, transport, and phosphorylation in the Golgi and plasma membranes to generate phosphatidylinositol 4,5-bisphosphate, followed by receptor-stimulated hydrolysis in the plasma membrane and return of the components to the ER for reassembly. Using probes for specific lipid species, we have followed and analyzed the kinetics of several of these events during stimulation of M1 muscarinic receptors coupled to the G-protein Gq. We show that during long continued agonist action, polyphosphorylated inositol lipids are initially depleted but then regenerate while agonist is still present. Experiments and kinetic modeling reveal that the regeneration results from gradual but massive up-regulation of PI 4-kinase pathways rather than from desensitization of receptors. Golgi pools of phosphatidylinositol 4-phosphate and the lipid kinase PI4KIIIα (PI4KA) contribute to this homeostatic regeneration. This powerful acceleration, which may be at the level of enzyme activity or of precursor and product delivery, reveals strong regulatory controls in the phosphoinositide cycle.

scholarly journals Assembly of the PtdIns 4-kinase Stt4 complex at the plasma membrane requires Ypp1 and Efr3

2008 ◽  
Vol 183 (6) ◽  
pp. 1061-1074 ◽  
Author(s):  
Dan Baird ◽  
Chris Stefan ◽  
Anjon Audhya ◽  
Sabine Weys ◽  
Scott D. Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Essential Gene ◽  
Additional Component ◽  
Lipid Kinase ◽  
Phosphoinositide Kinase ◽  
Multiple Copies ◽  
The Stability ◽  
Phosphatidylinositol 4

The phosphoinositide phosphatidylinositol 4-phosphate (PtdIns4P) is an essential signaling lipid that regulates secretion and polarization of the actin cytoskeleton. In Saccharomyces cerevisiae, the PtdIns 4-kinase Stt4 catalyzes the synthesis of PtdIns4P at the plasma membrane (PM). In this paper, we identify and characterize two novel regulatory components of the Stt4 kinase complex, Ypp1 and Efr3. The essential gene YPP1 encodes a conserved protein that colocalizes with Stt4 at cortical punctate structures and regulates the stability of this lipid kinase. Accordingly, Ypp1 interacts with distinct regions on Stt4 that are necessary for the assembly and recruitment of multiple copies of the kinase into phosphoinositide kinase (PIK) patches. We identify the membrane protein Efr3 as an additional component of Stt4 PIK patches. Efr3 is essential for assembly of both Ypp1 and Stt4 at PIK patches. We conclude that Ypp1 and Efr3 are required for the formation and architecture of Stt4 PIK patches and ultimately PM-based PtdIns4P signaling.

scholarly journals Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P2

2009 ◽  
Vol 422 (1) ◽  
pp. 23-35 ◽  
Author(s):  
Gerald R. V. Hammond ◽  
Giampietro Schiavo ◽  
Robin F. Irvine
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Plasma Membranes ◽  
Membrane Lipid ◽  
Present Evidence ◽  
Immunocytochemical Detection ◽  
Plasma Membrane Lipid ◽  
Cytoplasmic Vesicles ◽  
Immunocytochemical Techniques ◽  
Phosphatidylinositol 4

PtdIns4P is the major precursor for the synthesis of the multifunctional plasma membrane lipid, PtdIns(4,5)P2. Yet PtdIns4P also functions as a regulatory lipid in its own right, particularly at the Golgi apparatus. In the present study we define specific conditions that enable preservation of several organellar membranes for the immunocytochemical detection of PtdIns4P. We report distinct pools of this lipid in both Golgi and plasma membranes, which are synthesized by different PI4K (phosphatidylinositol 4-kinase) activities, and also the presence of PtdIns4P in cytoplasmic vesicles, which are not readily identifiable as PI4K containing trafficking intermediates. In addition, we present evidence that the majority of PtdIns4P resides in the plasma membrane, where it is metabolically distinct from the steady-state plasma membrane pool of PtdIns(4,5)P2.

scholarly journals Relationship between phosphoinositide kinase activities and protein tyrosine phosphorylation in plasma membranes from A431 cells

1990 ◽  
Vol 272 (3) ◽  
pp. 665-670 ◽  
Author(s):  
B Payrastre ◽  
M Plantavid ◽  
M Breton ◽  
E Chambaz ◽  
H Chap
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Phosphorylation ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
Protein Tyrosine Phosphorylation ◽  
A431 Cells ◽  
Phosphoinositide Kinase ◽  
Epidermal Growth ◽  
First Time ◽  
Dose Dependent

Production of PtdIns(4)P and PtdIns(4,5)P2 by plasma-membrane preparations from A431 cells was selectively stimulated in a dose-dependent manner by epidermal growth factor (EGF) in the presence of Na3VO4. Na3VO4 itself mimicked this effect, which was overcome after treatment by a specific phosphotyrosyl phosphatase isolated from A431 cells. PtdIns and PtdIns(4)P kinase activities were present in phosphotyrosyl-proteins isolated from EGF- and/or Na3VO4-stimulated A431 cells by immunoaffinity using an anti-phosphotyrosine antibody. These data suggest for the first time an EGF-dependent regulation of PtdIns 4-kinase and PtdIns(4)P 5-kinase activities by a mechanism involving a tyrosine-phosphorylation process.

scholarly journals Effects of glucagon and Ca2+ on the metabolism of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in isolated rat hepatocytes and plasma membranes

1987 ◽  
Vol 241 (3) ◽  
pp. 835-845 ◽  
Author(s):  
D E Whipps ◽  
A E Armston ◽  
H J Pryor ◽  
A P Halestrap
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Mitochondrial Fraction ◽  
Ruthenium Red ◽  
Isolated Rat Hepatocytes ◽  
Guanine Nucleotide Regulatory Protein ◽  
Phosphatidylinositol 4

Rat hepatocytes whose phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) had been labelled for 60 min with 32P were treated with glucagon for 10 min or phenylephrine for 2 min. Glucagon caused a 20% increase in PIP but no change in PIP2 whereas phenylephrine caused a similar increase in PIP but a 15% decrease in PIP2. Addition of both hormones together for 10 min produced a 40% increase in PIP. A crude liver mitochondrial fraction incubated with [32P]Pi and ADP incorporated label into PIP, PIP2 and phosphatidic acid. The PIP2 was shown to be in contaminating plasma membranes and PIP in both lysosomal and plasma-membrane contamination. A minor but definitely mitochondrial phospholipid, more polar than PIP2, was shown to be labelled with 32P both in vitro and in hepatocytes. The rate of 32P incorporation into PIP was faster in mitochondrial/plasma-membrane preparations from rats treated with glucagon or if 3 microM-Ca2+ and Ruthenium Red were present in the incubation buffer. Loss of 32P from membranes labelled in vitro was shown to be accompanied by formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate, and was faster in preparations from glucagon-treated rats or in the presence of 3 microM-Ca2+. It is concluded that glucagon stimulates both PIP2 phosphodiesterase and phosphatidylinositol kinase activities, as does the presence of 3 microM-Ca2+. The resulting formation of IP3 may be responsible for the observed release of intracellular Ca2+ stores. The roles of a guanine nucleotide regulatory protein and phosphorylation in mediating these effects are discussed.

scholarly journals Inositol phospholipid arachidonic acid metabolism in GH3 pituitary cells

1986 ◽  
Vol 236 (1) ◽  
pp. 235-242 ◽  
Author(s):  
D T Dudley ◽  
A A Spector
Keyword(s):  
Arachidonic Acid ◽  
Molecular Species ◽  
Arachidonic Acid Metabolism ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Trh Stimulation ◽  
Inositol Phospholipids ◽  
Rat Pituitary ◽  
Arachidonic Acids ◽  
Phosphatidylinositol 4

Inositol phospholipids in cultured GH3 cells, a prolactin secreting, thyrotropin-releasing hormone (TRH) sensitive rat pituitary cell line, exhibit a preferential selectivity for incorporating arachidonic acid. Fatty acid composition data show that all inositol phospholipids are enriched in stearic and arachidonic acids to a much greater degree than other cellular phospholipids. Incubation of GH3 cells with radioactive stearate, oleate, arachidonate, eicosapentaenoate or docosahexaenoate also showed that much more stearate and arachidonate were incorporated into inositol phospholipids. In short term incubations with tracer amounts of radioactive arachidonate, incorporation was initially into phosphatidylinositol (PtdIns), with phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] being labelled at later times. During longer incubations, all of the inositol phospholipids reach equilibrium at about 10 h, and the resulting specific activities of the three fractions were similar. These findings suggest that arachidonate is incorporated initially into PtdIns and that PtdIns is then phosphorylated. There was no release of either arachidonate or eicosanoid products when GH3 cells were incubated with TRH. However, TRH stimulation of 32P-labelled GH3 cells resulted in rapid breakdown of PtdIns(4,5)P2 and PtdIns4P, with concomitant increases in [32P]phosphatidic acid and [32P]PtdIns. When the [32P]PtdIns was further analysed by argentation chromatography to separate PtdIns molecular species, it was found that tetraenoic (stearate/arachidonate) species accounted for 80% of the stimulated labelling. The selectivity for arachidonate incorporation into inositol phospholipids coupled with turnover of the arachidonate-containing molecular species suggests that inositol phospholipids containing arachidonic acid or the diacylglycerol resulting therefrom may play a vital cellular role in GH3 cells. This role may involve the operation of the PtdIns cycle itself rather than a stimulated release of arachidonate for eicosanoid formation.

scholarly journals Oncogenic KRAS is dependent upon an EFR3A-PI4KA signaling axis for potent tumorigenic activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hema Adhikari ◽  
Walaa E. Kattan ◽  
Shivesh Kumar ◽  
Pei Zhou ◽  
John F. Hancock ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Therapeutic Potential ◽  
Effector Proteins ◽  
Adapter Protein ◽  
Oncogenic Signaling ◽  
Human Cancers ◽  
Phosphatidylinositol Kinase ◽  
Signaling Axis ◽  
Tumorigenic Activity ◽  
Phosphatidylinositol 4

AbstractThe HRAS, NRAS, and KRAS genes are collectively mutated in a fifth of all human cancers. These mutations render RAS GTP-bound and active, constitutively binding effector proteins to promote signaling conducive to tumorigenic growth. To further elucidate how RAS oncoproteins signal, we mined RAS interactomes for potential vulnerabilities. Here we identify EFR3A, an adapter protein for the phosphatidylinositol kinase PI4KA, to preferentially bind oncogenic KRAS. Disrupting EFR3A or PI4KA reduces phosphatidylinositol-4-phosphate, phosphatidylserine, and KRAS levels at the plasma membrane, as well as oncogenic signaling and tumorigenesis, phenotypes rescued by tethering PI4KA to the plasma membrane. Finally, we show that a selective PI4KA inhibitor augments the antineoplastic activity of the KRASG12C inhibitor sotorasib, suggesting a clinical path to exploit this pathway. In sum, we have discovered a distinct KRAS signaling axis with actionable therapeutic potential for the treatment of KRAS-mutant cancers.

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