A direct fluorometric activity assay for lipid kinases and phosphatases

Abstract

Lipid kinases and phosphatases play key roles in cell signaling and regulation, are implicated in many human diseases, and are thus attractive targets for drug development. Currently, no direct in vitro activity assay is available for these important enzymes, which hampers mechanistic studies as well as high-throughput screening of small molecule modulators. Here, we report a highly sensitive and quantitative assay employing a ratiometric fluorescence sensor that directly and specifically monitors the real-time concentration change of a single lipid species. Because of its modular design, the assay system can be applied to a wide variety of lipid kinases and phosphatases, including class I phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). When applied to PI3K, the assay provided detailed mechanistic information about the product inhibition and substrate acyl-chain selectivity of PI3K and enabled rapid evaluation of small molecule inhibitors. We also used this assay to quantitatively determine the substrate specificity of PTEN, providing new insight into its physiological function. In summary, we have developed a fluorescence-based real-time assay for PI3K and PTEN that we anticipate could be adapted to measure the activities of other lipid kinases and phosphatases with high sensitivity and accuracy.

PI(3,5)P2Controls the Signaling Activity of Class I PI3K

“…To test if the observed inhibition of Class I PI3K by PI(3,5)P 2 is caused by direct PI(3,5)P 2 -p85 interaction and to understand how PI(3,5)P 2 that is mainly found in endolysosomes can inhibit the action of Class I PI3K at PM, we performed both enzyme kinetic and membrane binding studies of Class I PI3Kα (equimolecular p110 α + p85α) using model membranes with defined lipid compositions. For enzyme kinetics of PI3Kα, we employed a recently developed in vitro activity assay that allows direct real-time monitoring of the reaction and accurate quantitative analysis of kinetic parameters (Sun et al, 2020). The assay system consists of POPC/POPS/PI(4,5)P 2 (77:20:3) LUVs, a ratiometric PI(4,5)P 2 sensor, PI3Kα, a pY-containing peptide derived from PDGFβ (pY2), and ATP.…”

Oncogenic mutations ofPIK3CAlead to increased membrane recruitment driven by reorientation of the ABD, p85 and C-terminus

“…Due to this complication, we characterised the lipid kinase activity using an assay that measures the consumption of PIP 2 through altered recruitment of a high specificity PIP 2 fluorescent sensor consisting of ENTH domain (Fig. 4B) 40 . Lipid kinase assays on PIP 2 /PS/PE vesicles showed that all mutants were significantly activated over wild type p110α both in the absence and presence of pY, with ~10-50-fold increased activity (Fig.…”

“…Lipid kinase assays were done using a slightly modified procedure from 40 . The kinase buffer contains 20 mM HEPES pH 7.5, 100 mM NaCl, 7 mM MgCl2, 50 µM CHAPS and 2 mM TCEP and the quench buffer contains 20 mM HEPES pH 7.5 and 40 mM EDTA.…”

“…Lipid kinase assays were done using a slightly modified procedure from 40 nm. This data was normalised against a 0-100% ADP window made using conditions containing either 100 μM ATP/ADP with vesicles and kinase buffer.…”

Non-catalytic role of phosphoinositide 3-kinase in mesenchymal cell migration through non-canonical induction of p85β/AP-2-mediated endocytosis

“…Such a seemingly meaningless regulation may be explained by the kinetic difference. Upon stimulation, PI (3,4,5)P3 production can initiate within milli-seconds to seconds timescale 75 , while clathrin-mediated endocytosis occurs more gradually (tens of seconds to a few minutes) 76 . The temporal difference creates an autonomous delayed negative feedback loop, which is one of the signature characteristics necessary for self-organized signal transduction often proposed in directed cell migration 77 .…”