Publications

• High-throughput, cell-free, liposome-based approach for assessing in vitro activity of lipid kinases.

  Demian D.J., Clugston S.L., Foster M.M., Rameh L., Sarkes D., Townson S.A., Yang L., Zhang M., Charlton M.E.

  Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate i ... >> More

  Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [gamma-32P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testing of large numbers of small molecules (100 s to 1 MM+). The authors present the development and implementation of a fully synthetic, liposome-based assay for assessing in vitro activity of phosphatidylinositol-5-phosphate-4-kinase isoforms (PIP4KIIbeta and alpha) in 2 commonly used homogeneous technologies. They have validated these assays through compound testing in both traditional TLC and radioactive filterplate approaches as well as binding validation using isothermic calorimetry. A directed library representing known kinase pharmacophores was screened against type IIbeta phosphatidylinositol-phosphate kinase (PIPK) to identify small-molecule inhibitors. This assay system can be applied to other types and isoforms of PIPKs as well as a variety of other lipid kinase targets. << Less

  J Biomol Screen 14:838-844(2009) [PubMed] [EuropePMC]

• The Lipid Kinase PI5P4Kbeta Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis.

  Sumita K., Lo Y.H., Takeuchi K., Senda M., Kofuji S., Ikeda Y., Terakawa J., Sasaki M., Yoshino H., Majd N., Zheng Y., Kahoud E.R., Yokota T., Emerling B.M., Asara J.M., Ishida T., Locasale J.W., Daikoku T., Anastasiou D., Senda T., Sasaki A.T.

  While cellular GTP concentration dramatically changes in response to an organism's cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kβ, a phosphoinositide kinase that ... >> More

  While cellular GTP concentration dramatically changes in response to an organism's cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kβ, a phosphoinositide kinase that regulates PI(5)P levels, detects GTP concentration and converts them into lipid second messenger signaling. Biochemical analyses show that PI5P4Kβ preferentially utilizes GTP, rather than ATP, for PI(5)P phosphorylation, and its activity reflects changes in direct proportion to the physiological GTP concentration. Structural and biological analyses reveal that the GTP-sensing activity of PI5P4Kβ is critical for metabolic adaptation and tumorigenesis. These results demonstrate that PI5P4Kβ is the missing GTP sensor and that GTP concentration functions as a metabolic cue via PI5P4Kβ. The critical role of the GTP-sensing activity of PI5P4Kβ in cancer signifies this lipid kinase as a cancer therapeutic target. << Less

  Mol. Cell 61:187-198(2016) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.