Monitoring Inositol-Specific Phospholipase C Activity Using a Phospholipid FlashPlate®

Mullinax, Thomas R.; Henrich, Georgette; Kasila, Patricia; Ahern, David G.; Wenske, Elizabeth A; Hou, Cuifen; Argentieri, Dennis; Bembenek, Michael E.

doi:10.1177/108705719900400309

Cited by 14 publications

(8 citation statements)

References 6 publications

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“…18 The assay complements a recent report for monitoring the inositol phosphate production following cellular receptor activation. 25 Thus, together these formats should provide important tools for understanding the molecular mechanisms of inhibition following receptor signaling events through the PLC-mediated pathway.…”

supporting

confidence: 54%

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Development of a High-Throughput Assay for Two Inositol-Specific Phospholipase Cs Using a Scintillation Proximity Format

Bembenek¹,

Jain²,

Prack³

et al. 2003

ASSAY and Drug Development Technologies

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Inositol-specific PLCs comprise a family of enzymes that utilize phosphoinositide substrates, e.g., PIP(2), to generate intracellular second messengers for the regulation of cellular responses. In the past, monitoring this reaction has been difficult due to the need for radiolabeled substrates, separation of the reaction products by organic-phase extraction, and finally radiometric measurements of the segregated products. In this report, we have studied the enzymatic characteristics of two novel PLCs that were derived from functional genomic analyses using a phospholipid-modified solid scintillating support. This method allows for the hydrophobic capture of the [(3)H]phosphoinositide substrate on a well defined scintillation surface and the homogenous measurement of the enzymatic hydrolysis of the substrate by proximity effects. Our results show that the assay format is robust and well suited for this class of lipid-metabolizing enzymes.

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supporting

confidence: 54%

“…18 A comparison of the reactions upon addition of either recombinantly expressed PLC-16835 or PLC-32544 was performed next. As can be seen in Fig.…”

mentioning

confidence: 99%

Development of a High-Throughput Assay for Two Inositol-Specific Phospholipase Cs Using a Scintillation Proximity Format

Bembenek¹,

Jain²,

Prack³

et al. 2003

ASSAY and Drug Development Technologies

Self Cite

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“…The labelled cells were then plated onto Matrigel and immediately visualized by fluorescent real-time video microscopy (dual-colour Nikon Elipse Microscope, Hamamatsu ORCA-ER camera, Simple PCI software) at excitation wavelengths of 380 nm (green: low/basal intracellular calcium) and 340 nm (red: high/released intracellular calcium) over a 30-minute period (emission 510 nM). For phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ] hydrolysis assay, previously described methodology (Mullinax et al, 1999) was used. The phosphorylation assay and purification of GSTtagged proteins was as previously described (Rodriguez et al, 2001).…”

Section: Analysis Of Cell Morphology and Motility On Matrigelmentioning

confidence: 99%

PLCγ1 is essential for early events in integrin signalling required for cell motility

Jones

Peak

Brader

et al. 2005

Journal of Cell Science

101

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Cell motility is a critical event in many processes and is underlined by complex signalling interactions. Although many components have been implicated in different forms of cell migration, identification of early key mediators of these events has proved difficult. One potential signalling intermediate, PLCγ1, has previously been implicated in growth-factor-mediated chemotaxis but its position and roles in more-complex motility events remain poorly understood. This study links PLCγ1 to early, integrin-regulated changes leading to cell motility. The key role of PLCγ1 was supported by findings that specific depletion of PLCγ1 by small interfering (si)RNA, or by pharmacological inhibition, or the absence of this isoform in PLCγ1–/– cells resulted in the failure to form cell protrusions and undergo cell spreading and elongation in response to integrin engagement. This integrin-PLCγ1 pathway was shown to underlie motility processes involved in morphogenesis of endothelial cells on basement membranes and invasion of cancer cells into such three-dimensional matrices. By combining cellular and biochemical approaches, we have further characterized this signalling pathway. Upstream of PLCγ1 activity, β1 integrin and Src kinase are demonstrated to be essential for phosphorylation of PLCγ1, formation of protein complexes and accumulation of intracellular calcium. Cancer cell invasion and the early morphological changes associated with cell motility were abolished by inhibition of β1 integrin or Src. Our findings establish PLCγ1 as a key player in integrin-mediated cell motility processes and identify other critical components of the signalling pathway involved in establishing a motile phenotype. This suggests a more general role for PLCγ1 in cell motility, functioning as a mediator of both growth factor and integrin-initiated signals.

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“…Indeed this methodology has a low to very low throughput, is not versatile, and requires large amounts of reagents. To overcome these issues and characterize GTP-binding proteins of the Ras superfamily in a systematic manner, we developed a robust assay platform using two well adopted high throughput technologies, AlphaScreen TM (9) and FlashPlate® (10). AlphaScreen is a bead-based non-radioactive and homogenous proximity assay used to measure interaction between biological binding partners.…”

mentioning

confidence: 99%

Biochemical Clustering of Monomeric GTPases of the Ras Superfamily

Caruso

Jenna

Beaulne³

et al. 2005

Molecular & Cellular Proteomics

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To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen Ras GTPases are small GTP-binding proteins involved in diverse cellular processes such as apoptosis, cell proliferation/differentiation, cytoskeleton reorganization, and membrane trafficking. These proteins cycle between an inactive GDP-bound form and an active GTP-bound form. Under their GTP-bound form their active conformation allows them to interact with effector molecules and to generate specific biological responses (1). Two intrinsic enzymatic activities regulate the balance between GTP-bound and GDP-bound conformations: 1) GDP/GTP exchange and 2) GTP hydrolysis activities (2). Whereas the intrinsic enzymatic activities have been characterized for a few of these proteins, such studies were carried out in non-systematic manners and by different research groups (3-6).The analysis of full genome sequences has allowed large scale biology experimental approaches consisting of systematic characterization of entire families of proteins instead of investigating them individually. Developing such "functional proteomic" approaches requires the design of high throughput and versatile experimental procedures (7,8). For the study of small G proteins, conventional filtration assays, although powerful at small scale, are not appropriate for systematic studies. Indeed this methodology has a low to very low throughput, is not versatile, and requires large amounts of reagents. To overcome these issues and characterize GTP-binding proteins of the Ras superfamily in a systematic manner, we developed a robust assay platform using two well adopted high throughput technologies, AlphaScreen TM (9) and FlashPlate® (10). AlphaScreen is a bead-based non-radioactive and homogenous proximity assay used to measure interaction between biological binding partners. The principle of this technology relies on the use of a Donor bead and an Acceptor bead that generate a light signal when brought into proximity (Ͻ200 nm). Upon laser excitation at 680 nm, the Donor beads, containing a photosensitizer, will generate short lived singlet oxygen that can diffuse only a short distance before returning to the ground state. The Acceptor beads, containing chemiluminescers and fluorophores, will react with this singlet oxygen and will emit an amplified light signal measurable at ϳ600 nm. AlphaScreen (PerkinElmer) provides highly versatile, sensitive, and homogeneous assays that allow us to perform studies at a higher throughput and at a lower cost.FlashPlates are white polystyrene microplates in which the interior of wells is coated with a thin layer of polystyrenebased scintillation reagent. The principle of this homogeneous radiometric technology is based on proximity betwe...

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Monitoring Inositol-Specific Phospholipase C Activity Using a Phospholipid FlashPlate®

Cited by 14 publications

References 6 publications

Development of a High-Throughput Assay for Two Inositol-Specific Phospholipase Cs Using a Scintillation Proximity Format

Development of a High-Throughput Assay for Two Inositol-Specific Phospholipase Cs Using a Scintillation Proximity Format

PLCγ1 is essential for early events in integrin signalling required for cell motility

Biochemical Clustering of Monomeric GTPases of the Ras Superfamily

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