Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases

Choi, Sungyeol; Hedman, Andrew C.; Sayedyahossein, Samar; Thapa, Narendra; Sacks, David B.; Anderson, Richard A.

doi:10.1038/ncb3441

Cited by 115 publications

(196 citation statements)

References 79 publications

(110 reference statements)

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“…The cells were grown at 37 C until an OD600 of 1.0 was reached, at which point they were induced by 0.3 mmol/L isopropyl b-D-thiogalactoside at 16 C for 18 hours. The cells were then harvested and resuspended in buffer containing 50 mmol/L HEPES (pH 7.5), 1 mol/L NaCl, 2 mmol/L DTT, 20 mmol/L MgCl 2 , 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mg/mL lysozyme.…”

Section: Virtual Screeningmentioning

confidence: 99%

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PI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor of PI4KIIα, Inhibits the Growth of Breast Cancer Cells

Gao

Zhao

et al. 2017

Cancer Research

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While phosphatidylinositol 4-kinase (PI4KIIa) has been identified as a potential target for antitumor therapy, the clinical applications of PI4KIIa are limited by a lack of specific inhibitors. Here we report the first small-molecule inhibitor (SMI) of human PI4KIIa. Docking-based and ligand-based virtual screening strategies were first employed to identify promising hits, followed by two rounds of kinase activity inhibition validation. 2-(3-(4-Chlorobenzoyl)thioureido)-4-ethyl-5-methylthiophene-3-carboxamide (PI-273) exhibited the greatest inhibitory effect on PI4KIIa kinase activity (IC 50 ¼ 0.47 mmol/L) and suppressed cell proliferation. Surface plasmon resonance and thermal shift assays indicated that PI-273 interacted directly with PI4KIIa. Kinetic analysis identified PI-273 as a reversible competitive inhibitor with respect to the substrate phosphatidylinositol (PI), which contrasted with most other PI kinase inhibitors that bind the ATP binding site. PI-273 reduced PI4P content, cell viability, and AKT signaling in wild-type MCF-7 cells, but not in PI4KIIa knockout MCF-7 cells, indicating that PI-273 is highly selective for PI4KIIa. Mutant analysis revealed a role of palmitoylation insertion in the selectivity of PI-273 for PI4KIIa. In addition, PI-273 treatment retarded cell proliferation by blocking cells in G 2 -M, inducing cell apoptosis and suppressing colony-forming ability. Importantly, PI-273 significantly inhibited MCF-7 cell-induced breast tumor growth without toxicity. PI-273 is the first substratecompetitive, subtype-specific inhibitor of PI4KIIa, the use of which will facilitate evaluations of PI4KIIa as a cancer therapeutic target. Cancer Res; 77(22); 6253-66. Ó2017 AACR.

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Section: Virtual Screeningmentioning

confidence: 99%

“…For measurement of PI4P in tumor tissues, lipids extracted from equal weights (approximately 10 mg) of xenograft tumors were analyzed for PI4P using the PI4P Mass ELISA Kit described above and normalized by the tissue weights. Statistical analysis was performed with 6 paired cases (37).…”

Section: Thermal Shift Assay and Cellular Thermal Shift Assaymentioning

confidence: 99%

PI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor of PI4KIIα, Inhibits the Growth of Breast Cancer Cells

Gao

Zhao

et al. 2017

Cancer Research

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“…For example, in the Ras-ERK pathway, Raf, MEK, and ERK interact with IQGAP1, which physically links the kinases in close proximity to facilitate their sequential phosphorylation (Ren et al, 2007). Recently, a scaffold role for IQGAP1 in the PI3K-Akt pathway has been revealed (Choi et al, 2016). In response to receptor activation, PI4KIIIa, PIPKIa, and PI3K are assembled by IQGAP1 and this results in sequential generation of phosphatidylinositol-3,4,5-trisphosphate (PI3,4,5P 3 ) from phosphatidylinositol.…”

mentioning

confidence: 99%

And Akt‐ion! IQGAP1 in control of signaling pathways

Choi

Anderson

2017

The EMBO Journal

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“…Moreover the insulin-induced phosphorylation of PKB was reduced in liver and muscle tissue of IQGAP1 knock out animals compared to wt [116]. In different cell lines, IQGAP1 was shown to interact with IR via its IQ domains, and the PTB domain of IRS1 to interact with the RGCT domain of IQGAP1 [117].…”

Section: Figure 6 Fluorescence Microscopic Images Demonstrating the mentioning

confidence: 93%

“…Using various cancer cell-lines it was shown that upon agonist stimulation IQGAP1 binds all the phosphoinositide kinases needed for sequential phosphorylation of phosphatidylinositol to PIP3, including PI3K, as well as the downstream substrates of PI3K: PKB and PDK1 [116].…”

Section: Figure 6 Fluorescence Microscopic Images Demonstrating the mentioning

confidence: 99%

Insulin Signalling in Human Adipocytes and its Interplay with beta-Adrenergic Control of Lipolysis

Karlsson¹

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Cover: Fluorescence microscopic image demonstrating the association of caveolin-1 and IQGAP1 (paper II). In a proximity ligation assay each red dot represents a single IQGAP1 -caveolin-1 complex stained with Hoechst, nucleus is stained with DAPI. © Cecilia Jönsson 2018Published articles in this thesis have been reprinted with the permission of respective copyright holders. Printed by LiU-Tryck, Linköping 2018 During the course of the research underlying this thesis, Cecilia Jönsson was enrolled in Forum Scientium, a multidisciplinary doctoral program at Linköping University, Sweden AbstractThe prevalence of obesity has over the last 40 years nearly tripled and obesity is one of the major risk factors of developing type 2 diabetes. Type 2 diabetes was formerly called adultonset diabetes but today, probably due to the rise in childhood obesity, it is also seen in children and adolescents. Type 2 diabetes is diagnosed when the body no longer can control the glucose levels in the blood. This is due to an insulin resistant state in the insulin responding tissues, liver, adipose and muscle and insufficient production of insulin in the pancreas. However, in spite of extensive research the mechanisms behind insulin resistance is still not known.The adipose tissue is believed to play a major role in the development of whole body insulin resistance. Adipocytes are the most important sites for storage of the high energy containing triacylglycerols. Insulin stimulation causes the adipocyte to increase the uptake of glucose and to reduce lipolysis: the hydrolysis of triacylglycerol and release of glycerol and fatty acids. The insulin signalling network is complex with numerous proteins involved. These signalling proteins not only transmit the insulin signal but also create negative and positive feedbackloops and induce cross talk between different parts of the network and with the signalling of other hormones. One important positive feedback in insulin signalling is the mTORC1 mediated feedback to phosphorylation of IRS1 at serine 307. In paper I we found that in human adipocytes this feedback is not likely catalysed by the assumed kinase S6K1. However we find an immunoprecipitate of mTOR to contain a ser307 phosphorylating kinase.Scaffolding proteins serve as docking sites for several proteins to promote protein-protein interactions that facilitate signal transduction. In paper II we demonstrate the existence of the scaffolding protein IQGAP1 in human adipocytes and that the expression of IQGAP1 is downregulated in type 2 diabetes. We reveal that IQGAP1 co-localises with caveolae, invaginations of the plasma membrane where the insulin receptor is situated, and that this interaction is increased upon insulin stimulation.In paper III we focus on the control of lipolysis, and sought to understand the interplay between insulin and beta-adrenergic stimulation. We demonstrated that the re-esterification of fatty acids is downregulated in type 2 diabetes causing an increased release of fatty acids from the cells. We showed th...

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Agonist-stimulated phosphatidylinositol-3,4,5-trisphosphate generation by scaffolded phosphoinositide kinases

Cited by 115 publications

References 79 publications

PI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor of PI4KIIα, Inhibits the Growth of Breast Cancer Cells

PI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor of PI4KIIα, Inhibits the Growth of Breast Cancer Cells

And Akt‐ion! IQGAP1 in control of signaling pathways

Insulin Signalling in Human Adipocytes and its Interplay with beta-Adrenergic Control of Lipolysis

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