Measuring PI3K Lipid Kinase Activity

Huntington’s disease (HD) is a progressive neurodegenerative disease caused by a glutamine repeat expansion in mutant huntingtin (mHtt). Despite the known genetic cause of HD, the pathophysiology of this disease remains to be elucidated. Inositol polyphosphate multikinase (IPMK) is an enzyme that displays soluble inositol phosphate kinase activity, lipid kinase activity, and various noncatalytic interactions. We report a severe loss of IPMK in the striatum of HD patients and in several cellular and animal models of the disease. This depletion reflects mHtt-induced impairment of COUP-TF-interacting protein 2 (Ctip2), a striatal-enriched transcription factor for IPMK, as well as alterations in IPMK protein stability. IPMK overexpression reverses the metabolic activity deficit in a cell model of HD. IPMK depletion appears to mediate neural dysfunction, because intrastriatal delivery of IPMK abates the progression of motor abnormalities and rescues striatal pathology in transgenic murine models of HD.

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Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110α and p110β catalytic subunits of class-Ia phosphoinositide 3-kinases

Biochemical Journal ◽

10.1042/bj3500353 ◽

2000 ◽

Vol 350 (2) ◽

pp. 353-359 ◽

Cited By ~ 30

Author(s):

Carolyn A. BEETON ◽

Edwin M. CHANCE ◽

Lazaros C. FOUKAS ◽

Peter R. SHEPHERD

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Membrane Lipid ◽

Kinetic Properties ◽

Protein Kinase Activity ◽

Lipid Kinase ◽

Functional Roles ◽

Wide Range ◽

High Concentrations

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110α and p110β isoforms. The lipid-kinase activity did not display Michaelis–Menten kinetics but modelling the kinetic data demonstrated that p110α has a higher Vmax and a 25-fold higher Km for PtdIns than p110β. A similar situation occurs with PtdIns(4,5)P2, because at low concentration of PtdIns(4,5)P2 p110β is a better PtdIns(4,5)P2 kinase than p110α, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110β functions better in areas of membranes containing low levels of substrate whereas p110α would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110β phosphorylated p85 to a lower degree than did p110α. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110α and p110β. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110α has a higher Km (550µM) than p110β (Km 8µM). Similarly, the relative Vmax towards peptide substrate of p110α was three times higher than that of p110β. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110α and p110β in vivo.

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Early Activation of Sphingosine Kinase in Mast Cells and Recruitment to FcεRI Are Mediated by Its Interaction with Lyn Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.24.19.8765-8777.2004 ◽

2004 ◽

Vol 24 (19) ◽

pp. 8765-8777 ◽

Cited By ~ 52

Author(s):

Nicole Urtz ◽

Ana Olivera ◽

Elisa Bofill-Cardona ◽

Robert Csonga ◽

Andreas Billich ◽

...

Keyword(s):

Mast Cells ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Immunoglobulin E ◽

Sphingosine Kinase ◽

Lipid Kinase ◽

Lyn Kinase ◽

Sphingosine 1 Phosphate ◽

High Affinity Receptor

ABSTRACT Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca2+, released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-γ and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcεRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcεRI triggering, enhanced sphingosine kinase activity was associated with FcεRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn−/ − mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcεRI proximal event.

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A Mammalian Ortholog of Saccharomyces cerevisiae Vac14 That Associates with and Up-Regulates PIKfyve Phosphoinositide 5-Kinase Activity

Molecular and Cellular Biology ◽

10.1128/mcb.24.23.10437-10447.2004 ◽

2004 ◽

Vol 24 (23) ◽

pp. 10437-10447 ◽

Cited By ~ 48

Author(s):

Diego Sbrissa ◽

Ognian C. Ikonomov ◽

Jana Strakova ◽

Rajeswari Dondapati ◽

Krzysztof Mlak ◽

...

Keyword(s):

Mammalian Cells ◽

Kinase Activity ◽

Ectopic Expression ◽

Multivesicular Body ◽

Hek293 Cells ◽

Lipid Kinase ◽

Morphological Alterations ◽

Protein Levels ◽

Interfering Rna

ABSTRACT Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P2, produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P2 synthesis and endomembrane homeostasis in mammalian cells.

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Phosphatidylinositol 3-kinase: Inhibition of intrinsic protein-serine kinase activity by phosphoinositides, and of lipid kinase activity by Mn2+

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/0167-4889(95)00032-n ◽

1995 ◽

Vol 1267 (2-3) ◽

pp. 139-144 ◽

Cited By ~ 6

Author(s):

Ved P.S. Chauhan ◽

Surya S. Singh ◽

Abha Chauhan ◽

Hans Brockerhoff

Keyword(s):

Kinase Activity ◽

Phosphatidylinositol 3 Kinase ◽

Serine Kinase ◽

Kinase Inhibition ◽

Lipid Kinase ◽

Intrinsic Protein ◽

Phosphatidylinositol 3

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p110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity

Molecular and Cellular Biology ◽

10.1128/mcb.00471-15 ◽

2015 ◽

Vol 35 (19) ◽

pp. 3258-3273 ◽

Cited By ~ 3

Author(s):

Aditi Chaudhari ◽

Daniel Krumlinde ◽

Annika Lundqvist ◽

Levent M. Akyürek ◽

Sashidhar Bandaru ◽

...

Keyword(s):

Kinase Activity ◽

Human Cancer ◽

Hot Spot ◽

Metabolic Reprogramming ◽

Whole Body ◽

Lipid Kinase ◽

Glycogen Accumulation ◽

Adenoviral Gene Transfer ◽

Hepatic Expression

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110αin vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity.

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Loss of PI3 kinase association improves the sensitivity of secondary mutation of KIT to Imatinib

Cell & Bioscience ◽

10.1186/s13578-020-0377-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Guangrong Zhu ◽

Jun Shi ◽

Shaoting Zhang ◽

Yue Guo ◽

Ling Huang ◽

...

Keyword(s):

Drug Resistance ◽

Targeted Therapy ◽

Kinase Activity ◽

Cell Transformation ◽

Pi3 Kinase ◽

Driver Mutations ◽

Secondary Mutation ◽

Lipid Kinase

Abstract Background KIT mutations are the predominant driver mutations in gastrointestinal stromal tumors (GISTs), and targeted therapy against KIT has improved treatment outcome dramatically. However, gaining secondary mutation of KIT confers drug resistance of GISTs leading to treatment failure. Results In this study, we found that secondary mutation of KIT dramatically increases the ligand-independent activation of the receptor and their resistance to the often used KIT inhibitor Imatinib in the treatment of GISTs. PI3 kinase plays essential roles in the cell transformation mediated by the primary mutation of KIT. We found that loss of PI3 kinase association, but not the inhibition of the lipid kinase activity of PI3 kinase, inhibits the ligand-independent activation of secondary mutations of KIT, and increases their sensitivity to Imatinib, and loss of PI3 kinase association inhibits secondary mutations of KIT mediated cell survival and proliferation in vitro. The in vivo assay further showed that the growth of tumors carrying secondary mutations of KIT is more sensitive to Imatinib when PI3 kinase association is blocked while inhibition of the lipid kinase activity of PI3 kinase cannot inhibit tumor growth, indicating that PI3 kinase is important for the drug resistance of secondary mutation of KIT independent of the lipid kinase activity of PI3 kinase. Conclusions Our results suggested that PI3 kinase is necessary for the ligand-independent activation of secondary mutations of KIT, and loss of PI3 kinase association improves the sensitivity of secondary mutations to the targeted therapy independent of the lipid kinase activity of PI3 kinase.

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

Biochemical Journal ◽

10.1042/bj20070821 ◽

2007 ◽

Vol 409 (2) ◽

pp. 501-509 ◽

Cited By ~ 29

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.

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

Biochemical Journal ◽

10.1042/bj3200643 ◽

1996 ◽

Vol 320 (2) ◽

pp. 643-649 ◽

Cited By ~ 36

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.

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