Biophysical methods for the characterization of PTEN/lipid bilayer interactions

Harishchandra, Rakesh Kumar; Neumann, Brittany M.; Gericke, Arne; Ross, Alonzo H.

doi:10.1016/j.ymeth.2015.02.004

Cited by 11 publications

(13 citation statements)

References 122 publications

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“…Notably, the simulations suggest that binding of the C2 domains is relatively insensitive to a change from, e.g., PIP 2 to PIP 3 , at least in the CG simulations, despite PIP 3 being the substrate for PTEN. This is broadly consistent with experimental studies, as reviewed by, e.g., Harishchandra et al (2015), which indicate comparable binding affinities for PIP 2 and PIP 3 . Perhaps surprisingly, given in vitro experimental data suggesting PTEN binding is not altered by the presence/absence of cholesterol (Das et al, 2003), the inclusion of 25% cholesterol speeds up binding of the C2 domain.…”

Section: A Protocol For Determining and Analyzing C2-domainbinding Modessupporting

confidence: 92%

Binding of Ca2+-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study

Larsen

Sansom

2021

Structure

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C2 domains facilitate protein interactions with lipid bilayers in either a Ca 2+ -dependent or -independent manner. We used molecular dynamics (MD) simulations to explore six Ca 2+ -independent C2 domains, from KIBRA, PI3KC2a, RIM2, PTEN, SHIP2, and Smurf2. In coarse-grained MD simulations these C2 domains formed transient interactions with zwitterionic bilayers, compared with longer-lived interactions with anionic bilayers containing phosphatidylinositol bisphosphate (PIP 2 ). Type I C2 domains bound non-canonically via the front, back, or side of the b sandwich, whereas type II C2 domains bound canonically, via the top loops. C2 domains interacted strongly with membranes containing PIP 2 , causing bound anionic lipids to cluster around the protein. Binding modes were refined via atomistic simulations. For PTEN and SHIP2, CG simulations of their phosphatase plus C2 domains with PIP 2 -containing bilayers were also performed, and the roles of the two domains in membrane localization compared. These studies establish a simulation protocol for membrane-recognition proteins.

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Section: A Protocol For Determining and Analyzing C2-domainbinding Modessupporting

confidence: 92%

Binding of Ca2+-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study

Larsen

Sansom

2021

Structure

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“…PIP Strips were used for the determination of the lipid‐binding property of PTEN, which reflects its association with anionic lip ids of the plasma membrane (36). The binding of phosphorylated PTEN on lipids located on the PIP Strip was significantly higher than that of nonphosphorylated PTEN (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…PTEN was incubated with FAK as described above and then the PTEN—lipid interaction was determined by using PIP Strips (36) which are hydrophobic membranes spotted with physiologically relevant lipids (Echelon Biosciences Inc.). The association of PTEN with lipids on the strips was detected by probing with an anti‐PTEN antibody, according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Focal adhesion kinase phosphorylates the phosphatase and tensin homolog deleted on chromosome 10 under the control of p110δ phosphoinositide‐3 kinase

2015

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The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is regulated by various mechanisms that are not fully understood. This includes regulation by Tyr phosphorylation by a mechanism that remains elusive. Here, we show that focal adhesion kinase (FAK) phosphorylates PTEN in vitro, in cell-free systems and in cells. Furthermore, by mass spectrometry, we identified Tyr336 on PTEN as being phosphorylated by FAK. Tyr336 phosphorylation increased phosphatase activity, protein-lipid interaction, and protein stability of PTEN. In cells, including primary mouse macrophages and human cancer cell lines, FAK was found to be negatively regulated by p110d phosphoinositide-3 kinase (PI3K), whereas the activation of FAK was positively regulated by RhoA-associated kinase (ROCK). Indeed, the phosphorylation of FAK was unexpectedly increased in macrophages derived from mice expressing kinase-dead p110d. Pharmacologic inactivation of RhoA/ROCK reduced the phosphorylation of FAK to normal levels in cells with genetically inactivated p110d. Likewise, pharmacologic inactivation of FAK reduced the phosphorylation of PTEN in cells expressing kinase-dead p110d and restored the functional defects of p110d inactivation, including Akt phosphorylation and cell proliferation. This work identifies FAK as a target of p110d PI3K that links RhoA with PTEN and establishes for the first time that PTEN is a substrate of FAK-mediated Tyr phosphorylation.-Tzenaki, N., Aivaliotis, M., Papakonstanti, E. A. Focal adhesion kinase phosphorylates the phosphatase and tensin homolog deleted on chromosome 10 under the control of p110d phosphoinositide-3 kinase. FASEB J. 29, 4840-4852 (2015). www.fasebj.org

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“…While most of the PTEN contained within cells is localized in the cytoplasm, PTEN’s best characterized enzymatic action occurs at the inner leaflet of the plasma membrane, where the substrate PI(3,4,5)P3 is localized [ 7 ]. In addition to the substrate, the membrane binding of PTEN involves (at least) two additional lipid types.…”

Section: Introductionmentioning

confidence: 99%

“…This problem can be solved by working with single lipid bilayer membranes deposited onto a solid support [ 26 ]. Solid supported membranes, in either solely physisorbed [ 7 ] or partially tethered form [ 27 ], have previously been used to study the membrane binding behavior of PTEN. This approach allows the application of surface sensitive techniques, including surface plasmon resonance spectroscopy [ 27 ] and total internal reflection microscopy imaging [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

Kinetics of PTEN-mediated PI(3,4,5)P3 hydrolysis on solid supported membranes

Deb

Ferreira

et al. 2018

PLoS ONE

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Phosphatidylinositides play important roles in cellular signaling and migration. Phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) is an important phosphatidylinositide because it acts as a secondary messenger to trigger cell movement and proliferation. A high level of PI(3,4,5)P3 at the plasma membrane is known to contribute to tumorigenesis. One key enzyme that regulates PI(3,4,5)P3 levels at the plasma membrane is phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which dephosphorylates PI(3,4,5)P3 through hydrolysis to form phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). It has been reported that PI(4,5)P2 is involved in positive feedback in the PI(3,4,5)P3 hydrolysis by PTEN. However, how PI(3,4,5)P3 dephosphorylation by PTEN is regulated, is still under debate. How other PI(3,4,5)P3-binding proteins affect the dephosphorylation kinetics catalyzed by PTEN also remains unclear. Here, we develop a fluorescent-protein biosensor approach to study how PI(3,4,5)P3 dephosphorylation is regulated by PTEN as well as its membrane-mediated feedback mechanisms. Our observation of sigmoidal kinetics of the PI(3,4,5)P3 hydrolysis reaction supports the notion of autocatalysis in PTEN function. We developed a kinetic model to describe the observed reaction kinetics, which allowed us to i) distinguish between membrane-recruitment and allosteric activation of PTEN by PI(4,5)P2, ii) account for the influence of the biosensor on the observed reaction kinetics, and iii) demonstrate that all of these mechanisms contribute to the kinetics of PTEN-mediated catalysis.

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Biophysical methods for the characterization of PTEN/lipid bilayer interactions

Cited by 11 publications

References 122 publications

Binding of Ca2+-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study

Binding of Ca2+-independent C2 domains to lipid membranes: A multi-scale molecular dynamics study

Focal adhesion kinase phosphorylates the phosphatase and tensin homolog deleted on chromosome 10 under the control of p110δ phosphoinositide‐3 kinase

Kinetics of PTEN-mediated PI(3,4,5)P3 hydrolysis on solid supported membranes

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