Phosphatidylinositol 5 Phosphate 4-Kinase Regulates Plasma-Membrane PIP3 Turnover and Insulin Signaling

Sharma, Sanjeev; Mathre, Swarna; Ramya, Visvanathan; Shinde, Dhananjay; Raghu, Padinjat

doi:10.1016/j.celrep.2019.04.084

Cited by 36 publications

(47 citation statements)

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“…We also assayed PI5P levels from Drosophila S2R+ cells that had been depleted of dPIP4.K using with two different dsRNAs [recently reported in [23]]. Under our experimental conditions, treatment with either dsRNA resulted in complete depletion and dPIP4K protein could not be detected by western blot analysis (Figure 4E).…”

Section: Resultsmentioning

confidence: 83%

“…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%

“…The organic phase obtained after lipid extraction was directly subjected to derivatization using 2 M TMS-diazomethane (Acros AC385330050), with all necessary cautions as mentioned in Sharma et al, 2019 [23]. After this, 50 γl TMS-diazomethane was added to each tube and vortexed gently for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…The reaction was neutralized using 10 γl of glacial acetic acid. This was followed by two post derivatization washes as described in Sharma et al, 2019 [23]. To this final extract, 90% (v/v) methanol was added to this and dried for ∼2 hours in a centrifugal vacuum concentrator at 300 rpm.…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Ghosh

Sharma

Shinde

et al. 2019

Preprint

Self Cite

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“…The type 2 phosphatidylinositol-5-phosphate 4-kinases (PI5P4Ks), composed of a family of 3 isoforms (α, ß and g), catalyze the formation of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P 2 ) by phosphorylating phosphatidylinositol 5phosphate (PI-5-P) at the 4 position of the inositol ring. These kinases play a role in a variety of disease states, including cancer, insulin signaling, and oxidative stress (Carricaburu et al, 2003;Jones et al, 2006;Keune et al, 2012;Emerling et al, 2013;Jones et al, 2013;Jude et al, 2015;Sharma et al, 2019;Wang et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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

Noch

Yim

Milner

et al. 2020

Preprint

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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 5kinases, encoded by PIP5K1A, PIP5K1B and PIP5K1C, and type 2 phosphatidylinositol-5phosphate 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 PI5P4Kß, encoded by these genes. In mice, we demonstrate that PI5P4Kα is expressed in adulthood, whereas PI5P4Kß is expressed early in development. PI5P4Kα localizes to white matter tracts, especially the corpus callosum, and at a low level in neurons, while PI5P4Kß is expressed in neuronal populations, especially hippocampus and cortex. Dual labeling studies demonstrate that PI5P4Kα co-localizes with the oligodendrocyte marker, Olig2, whereas PI5P4Kß co-localizes with the neuronal marker, NeuN. Immunohistochemical subcellular distribution studies demonstrate that PI5P4Kα and PI5P4Kß are expressed in the early endosome system. 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. Immunohistochemical analysis of macaque and human brain tissue demonstrate a conserved pattern for PI5P4Kα and PI5P4Kß. These results highlight the diverse cell-autonomous expression of PI5P4Kα and PI5P4Kß and support further exploration into their role in synaptic function in the brain.

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Expanding role of PI5P4Ks in cancer: A promising druggable target

2021

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Cancer cells are challenged by a myriad of microenvironmental stresses, and it is their ability to efficiently adapt to the constantly changing nutrient, energy, oxidative, and/or immune landscape that allows them to survive and proliferate. Such adaptations, however, result in distinct vulnerabilities that are attractive therapeutic targets. Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are a family of druggable stress-regulated phosphoinositide kinases that become conditionally essential as a metabolic adaptation, paving the way to targeting cancer cell dependencies. Further, PI5P4Ks have a synthetic lethal interaction with the tumor suppressor p53, the loss of which is one of the most prevalent genetic drivers of malignant transformation. PI5P4K's emergence as a crucial axis in the expanding landscape of phosphoinositide signaling in cancer has already stimulated the development of specific inhibitors. Thus, a better understanding of the biology of the PI5P4Ks will allow for targeted and effective therapeutic interventions. Here, we attempt to summarize the mounting roles of the PI5P4Ks in cancer, including evidence that targeting them is a therapeutic vulnerability and promising nextin-line treatment for multiple cancer subtypes.

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Phosphatidylinositol 5 Phosphate 4-Kinase Regulates Plasma-Membrane PIP3 Turnover and Insulin Signaling

Abstract: Highlights d Drosophila PIP4K mutant larvae have increased PIP 3 levels in cells d Cells show enhanced sensitivity to insulin in the absence of PIP4K d PIP4K regulates enzymes involved in PIP 3 turnover at the plasma membrane d Loss of PIP4K suppresses insulin resistance phenotypes

Cited by 36 publications

References 59 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

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

Expanding role of PI5P4Ks in cancer: A promising druggable target

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