PIP4Ks Suppress Insulin Signaling through a Catalytic-Independent Mechanism

Wang, Diana G.; Paddock, Marcia N.; Lundquist, Mark R.; Sun, Janet; Mashadova, Oksana; Amadiume, Solomon; Bumpus, Timothy W.; Hodakoski, Cindy; Hopkins, Benjamin D.; Fine, Matthew; Hill, Amanda A.; Yang, Tong; Baskin, Jeremy M.; Dow, Lukas E.; Cantley, Lewis C.

doi:10.1016/j.celrep.2019.04.070

Cited by 39 publications

(48 citation statements)

References 48 publications

(76 reference statements)

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“…Results from such samples using our assay could help decipher signalling mechanisms in human tumours and help inform decision making in clinical oncology. Finally, PIP4K a key enzyme in the regulation of PI5P levels has been recently implicated in the control of insulin receptor signalling and Type II diabetes [23,35,36]. The mechanisms by which PIP4K regulates insulin signalling remain unclear but the use of our assay on human samples of small size and high value could help in the study of the role of this enzyme and its control of PI5P levels in human type II diabetes settings.…”

Section: Discussionmentioning

confidence: 99%

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

et al. 2019

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Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In the present study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use heavy oxygen-labelled ATP (18O-ATP) to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.

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Section: Discussionmentioning

confidence: 99%

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Results from such samples using our assay could help decipher signalling mechanisms in human tumours and help inform decision making in clinical oncology. Finally, PIP4K a key enzyme in the regulation of PI5P levels has been recently implicated in the control of insulin receptor signalling and Type II diabetes [23,34,35]. The mechanisms by which PIP4K regulates insulin signalling remain unclear but the use of our assay on human samples of small size and high value could help in the study of the role of this enzyme and its control of PI5P levels in human type II diabetes settings.…”

Section: Discussionmentioning

confidence: 99%

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

Ghosh

Sharma

Shinde

et al. 2019

Preprint

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Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In this study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use 18O-ATP to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from both Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity levels from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.

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“…It is also noteworthy that several genetic susceptibility loci in PI5P4Kγ have been linked to autoimmune diseases, including rheumatoid arthritis and type 1 diabetes mellitus, and that PI5P4Kγdeficient mice exhibit a hyperimmune phenotype (Barton et al, 2008;Fung et al, 2009;Shim et al, 2016 (Shim et al, 2016). Such upregulation of S6-Kinase activity may be explained by the role of PI5P4Kγ in inhibiting PI4P5K activity and downstream PI 3-kinase activity (Wang et al, 2019). Therefore, PI5P4Kα and PI5P4Kβ kinase activity The localization of PI5P4Kα and PI5P4Kβ to the perisynaptic region continues to support the role of the PI5P4Ks in intracellular PI-4,5-P 2 pathways (Clarke et al, 2009).…”

Section: Regional and Cell Type-specific Expression Of Pi5p4kα And mentioning

confidence: 99%

Distribution and localization of phosphatidylinositol 5‐phosphate, 4‐kinase alpha and beta in the brain

Noch

Yim

Milner

et al. 2020

J of Comparative Neurology

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Phosphatidylinositol-4,5-bisphosphate (PI-4,5-P 2) is critical for synaptic vesicle docking and fusion and generation of the second messengers, diacylglycerol and inositol-1,4,5-trisphosphate. PI-4,5-P 2 can be generated by two families of kinases: type 1 phosphatidylinositol-4-phosphate 5-kinases, encoded by PIP5K1A, PIP5K1B and PIP5K1C, and type 2 phosphatidylinositol-5-phosphate 4-kinases, encoded by PIP4K2A, PIP4K2B, and PIP4K2C. While the roles of the type 1 enzymes in brain function have been extensively studied, the roles of the type 2 enzymes are poorly understood. Using selective antibodies validated by genetic deletion of pip4k2a or pip4k2b in mouse brain, we characterized the location of the enzymes, PI5P4Kα and PI5P4Kβ, encoded by these genes. In mice, we demonstrate that PI5P4Kα is expressed in adulthood, whereas PI5P4Kβ is expressed early in development. PI5P4Kα localizes to white matter tracts, especially the corpus callosum, and at a low level in neurons, while PI5P4Kβ is expressed in neuronal populations, especially hippocampus and cortex. Dual labeling studies demonstrate that PI5P4Kα co-localizes with the oligodendrocyte marker, Olig2, whereas PI5P4Kβ co-localizes with the neuronal marker, NeuN. Ultrastructural analysis demonstrates that both kinases are contained in axon terminals and dendritic spines adjacent to the synaptic membrane, which support a potential role in synaptic transmission. Immunoperoxidase analysis of macaque and human brain tissue demonstrate a conserved pattern for PI5P4Kα and PI5P4Kβ. These results highlight the diverse cell-autonomous expression of PI5P4Kα and PI5P4Kβ and support further exploration into their role in synaptic function in the brain.

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PIP4Ks Suppress Insulin Signaling through a Catalytic-Independent Mechanism

Cited by 39 publications

References 48 publications

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues

Distribution and localization of phosphatidylinositol 5‐phosphate, 4‐kinase alpha and beta in the brain

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