Name: monitoring hippo signaling pathway activity using a luciferase-based large tumor suppressor (lats) biosensor
Uploaded: 2018-09-13T15:00:00
Description: Watch this Scientific Journal Video about Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor at JoVE.com

This work was supported by grants from the Canadian Institute of Health Research (CIHR#119325, 148629) and the Canadian Breast Cancer Foundation (CBCF) to XY. TA is supported by the Vanier Canada Graduate Scholarship and the Ontario International Graduate Scholarship. HJJVR is supported by a Queen Elizabeth II Graduate Scholarship in Science and Technology.

1. Investigation of a Putative Regulator of Hippo Signaling Using the LATS-BS
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	<li>Plating and transfection of cells
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		<li>Preparation of cell culture
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			<li>Warm 1x PBS, DMEM containing 10% FBS and 1% penicillin/streptomycin, and 0.25% trypsin-EDTA to 37 &#176;C in a water bath for approximately 30 min.</li>
			<li>Thoroughly clean a biosafety cabinet with 70% ethanol.</li>
			<li>Place tissue culture dishes, Pasteur pipettes, serological pipettes, and pipette tips into the tissue cu...

<ol>
	<li>Justice, R. W., Zilian, O., Woods, D. F., Noll, M., Bryant, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+tumor+suppressor+gene+warts+encodes+a+homolog+of+human+myotonic+dystrophy+kinase+and+is+required+for+the+control+of+cell+shape+and+proliferation.">The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation.</a> Genes &amp; Development. 9 (5), 534-546 (1995).</li><li>Xu, T., Wang, W., Zhang, S., Stewart, R. 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While the Hippo pathway plays critical roles in various biological processes, and dysregulation of the Hippo pathway leads to cancer6, how the Hippo pathway is regulated in response to various stimuli is not completely understood. In addition, there has been no quantitative and real-time system to assess the activity of core Hippo components. Recently, we developed and validated a novel biosensor for measuring LATS kinase activity in the Hippo pathway24. This biosensor can ...

The Hippo signaling pathway was first identified in Drosophila as a novel regulator of cell growth and animal size1,2. Since its initial discovery, mounting evidence has convincingly shown that the Hippo pathway plays critical roles in the development (e.g., early embryonic development, organ size control, and three-dimensional [3-D] morphology), tumorigenesis (e.g., tumor development, metastasis, angiogenesis, immune evasion, genomic instability, stress response, and drug resistance), and tissue homeostasis (e.g., stem cell renewal and differentiation and tissue regeneration after injury)3,4,5,6,7,8,9,10. Hippo signaling is frequently dysregulated in various cancers7,8,9,10,11,12. Therefore, elucidating functions of the Hippo pathway in cancer biology and therapeutics and regenerative medicine has become one of the hottest areas in biomedical research.
In brief, in the Hippo pathway, upon activation by upstream regulators (e.g., cell-cell contact, nutrient stress, and extracellular matrix [ECM]), MST1/2 (MST; mammalian homologs of Drosophila Hippo) serine/threonine (S/T) kinases phosphorylate/activate adaptor proteins hMOB1 and WW45, as well as LATS1/2 (LATS) kinases which, subsequently, phosphorylate transcriptional co-activator YAP and its paralog TAZ at conserved HX(H/R/K)XX(S/T) (H, histidine; R, arginine; K, lysine; S, serine; T, threonine; X, any amino acids) motifs, including YAP-S127 and TAZ-S8911,12. S127-phosphorylated YAP (YAP-pS127) and S89-phosphorylated TAZ (TAZ-pS89) are degraded by ubiquitination or bind to cytoplasmic protein 14-3-3 and are prevented from interacting with TEAD1-4 transcription factors in the nucleus to transactivate downstream genes involved in the cell proliferation and apoptosis (Figure 1). Despite the tremendous interest in the Hippo pathway, few tools for measuring Hippo signaling exist and those that do have been historically limited to reporters of YAP/TAZ/TEAD transcriptional output. Indeed, until very recently, there were no tools for measuring the dynamics and activity of the Hippo signaling components in a quantitative, real-time, high-throughput, and non-invasive manner both in vitro and in vivo.
Given our emerging understanding of the role of protein-protein interactions in physiology and pathology, there is great interest in the development of tools that can be used to study these interactions in a quantitative and real-time manner13,14,15,16. Indeed, there has been significant progress in the development of bio-analytical strategies, including the yeast two-hybrid (Y2H)17, the surface plasmon resonance (SPR)18, and F&#246;rster resonance energy transfer (FRET)19 assays, to evaluate protein-protein interactions. However, these approaches carry the limitation of requiring significant optimization of the reporter orientation, such that many constructs must be tested to find an efficient one. Further, these approaches also have a relatively low signal-to-noise ratio, such that discerning a true positive signaling can be challenging.
Protein complementation assays were developed to overcome these limitations. The first generation of protein complementation assays was based on split multicolor fluorescence proteins and could not solve the aforementioned limitations20. Multicolor fluorescence proteins consist of only one domain, making it difficult to split them into two separate stable fragments with low affinity and background noise21. Subsequently, firefly luciferase was identified as a new candidate for use in developing split protein complementation assays. In this approach, firefly luciferase is split into two fragments (N-terminal and C-terminal luciferase [NLuc and CLuc]) with each fragment fused to a target protein of interest. If the NLuc and CLuc are brought into proximity upon the interaction of the two target proteins, luciferase activity is reconstituted and bioluminescent light is generated in the presence of luciferin substrate and ATP22. In 2001, by performing a combinatorial screening using a library containing NLuc and CLuc fragments cut at various sites and attached to proteins with different linkers, Paulmurugan and Gambhir at Stanford University developed an optimized split-firefly luciferase fragment-assisted complementation system for protein-protein interactions23. In this system, firefly luciferase is cut at amino acid (aa) 398 to form NLuc and CLuc, which are attached to two proteins of interest using a flexible linker of eight glycine residues and two serine residues.
Using a similar approach, we recently developed a new LATS-BS by fusing NLuc to 15 aa of YAP surrounding the LATS phosphorylation site at S127 (YAP15) and CLuc with 14-3-3. The full-length YAP protein was not used, to avoid confounding signals by post-translational modifications of YAP (e.g., phosphorylation at other sites and ubiquitination) by other upstream regulators. The LATS-BS presented here can non-invasively monitor Hippo signaling activity both in vitro in living cells and in vivo in mice20,24 (Figure 2). Here, we describe a detailed protocol for measuring LATS kinase activity in vitro using the LATS-BS. First, we show how the LATS-BS can be used to investigate the effect of an overexpressed protein on LATS activity. Then, we show how the biosensor can be used to monitor the activity of the Hippo pathway after treatment with agents regulating the Hippo pathway. This protocol could be used to identify and characterize signaling pathways or stimuli regulating LATS kinase activity.

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			<td height="42" style="height:42px;width:267px;">Trypsin-EDTA&#160;</td>
			<td style="width:121px;">Life Technologies</td>
			<td style="width:91px;">2520056</td>
			<td style="width:213px;">0.25%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:91px;">F1051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DMEM</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:91px;">D6429-500ml</td>
			<td>DMEM with high glucose&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Penicillin Streptomycin</td>
			<td style="width:121px;">Life Technologies</td>
			<td style="width:91px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PolyJet In Vitro DNA Transfection Reagent&#160;</td>
			<td style="width:121px;">Signagen</td>
			<td style="width:91px;">SL100688.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">5x Passive lysis buffer</td>
			<td style="width:121px;">Promega</td>
			<td style="width:91px;">E194A</td>
			<td>30 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dual-Glo&#174; Luciferase Assay System</td>
			<td style="width:121px;">Promega</td>
			<td style="width:91px;">E2940</td>
			<td>100 ml kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">20/20 luminometer</td>
			<td style="width:121px;">Turner Biosystems</td>
			<td style="width:91px;">998-2036</td>
			<td>Single tube reader luminometer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">GloMax&#174;Navigator with dual injectors</td>
			<td style="width:121px;">Promega</td>
			<td style="width:91px;">GM2010</td>
			<td>96-well plates reader luminometer</td>
		</tr>
	</tbody>
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monitoring hippo signaling pathway activity using a luciferase-based large tumor suppressor (lats) biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

The LATS-BS was cotransfected with different genes to evaluate their effect on LATS activity (Figure 3). In this experiment, Renilla was used as an internal control. The transient expression of YAP-5SA, a constitutive active form of YAP, causes increasing levels of YAP transcriptional targets and a subsequent increase in LATS kinase activity through an established feedback pathway25. MST, the upstream activator of LATS in the ...

Watch this Scientific Journal Video about Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor at JoVE.com

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

My lab has been studying the role of Hippo pathway in breast and lung cancer, and recently, we have developed a novel biosensor to monitor Hippo signaling activity. This method can be used to answer key questions about cellular signaling networks, such as how the Hippo pathway responds to factors and how it interacts with other signaling pathways. The main advantage of this technique over existing methods is its great sensitivity and also the large number of the samples can be processed quickly and simultaneously.This technique has diverse applications in basic and translational research to investigate regulators of the Hippo signaling pathway both in vitro and in vivo. To begin, mini-prep LATS-BS plasmids fresh from DH5-Alpha bacteria liquid culture. An hour prior to transfection, aspirate the medium from the plate, and add 500 microliters of growth medium to each well.One important point is that LATS biosensor must be prepared fresh in order to get maximum expression and activity. Also, it is recommended to always use an upstream regulator of the Hippo pathway, such as MSD, as a positive control when performing luciferase assay. After this, return the cells to the incubator.Next, prepare solution A, a DNA solution, and solution B, a diluted polymer-based transfection reagent, as described in the text protocol. After this, immediately add solution B to solution A, and gently pipette up and down to mix. Incubate the sample at room temperature for 15 minutes to allow the transfection complexes to form.Then add the total volume of transfection complexes drop-wise to each well of the plate with gentle swirling. Incubate the cells overnight at 37 degrees Celsius. The next day, remove the transfection complex-containing media and replace it with fresh complete media.Then culture the cells at 37 degrees Celsius for one more day. Dilute an appropriate amount of 5x Passive lysis buffer with distilled water. Then, completely aspirate the media from the plate.Add one milliliter of PBS to each well, and swirl the plate gently to remove detached cells and residual growth media. Then aspirate the PBS completely. Next, add 250 microliters of 1xPLB to each well.Place the culture plate on a rocking platform for 15 minutes to ensure even coverage of the cell monolayer with PLB. After this, transfer the lysate to a 1.5 milliliter microcentrifuge tube. Remove the LAR-2 from 80 degrees Celsius storage, and equilibrate it to room temperature.Then, prepare Renilla detection reagent for an appropriate quantity of assays. Add the Renilla detection reagent to 50x substrate and its buffer. Next, dispense 10 microliters of each cell lysate into 1.5 milliliter tubes.Then, program a luminometer to perform a two second pre-measurement delay, followed by a 10 second measurement period. Carefully, transfer 20 microliters of LAR-2 reagent into one tube, and quickly pipette up and down to mix. Then, immediately place the tube in the luminometer and initiate the reading.After the reading, remove the sample tube from the luminometer, add 20 microliters of Renilla reagent, and pipette up and down to mix. Then, place the sample in the luminometer and initiate the next reading. First, culture, detach, count, and plate HEK293 Ad-cells as described in the text protocol.Then, one hour before transfection, aspirate the media from the plate and replace it with five milliliters of fresh complete growth media. Finally, add diluted solution B to solution A, and pipette up and down to mix. Then, incubate the sample at room temperature for 15 minutes before adding the total volume of the transfection complexes to the plate, while swirling gently.Incubate the cells overnight at 37 degrees Celsius. The next day, trypsinize and count the cells. Plate 10, 000 to 20, 000 cells into each well of a 96-well plate in a total volume of 100 microliters of media per well.After this, incubate the cells for one more day. One to four hours prior to performing the luciferase assay, add agents potentially regulating the Hippo pathway, at a final concentration of 10 micromolar per well. Next, aspirate the media completely from the cells and wash the cells in each well with 100 microliters of PBS.Then, add 20 microliters of PLB to each well. Place the culture plate on a rocking platform for 15 minutes. Then, transfer 20 microliters of LAR-2 reagent to each well of the plate, and pipette up and down to mix.Immediately after this, place the plate into a plate-reading luminometer and initiate the reading. In the first part of this protocol, the LATS-BS was cotransfected with different genes to evaluate their effect on LATS kinase activity. In the second protocol, LATS-BS was transfected into HEK293-Ad, and the cells were passed into 96-well plates.40 hours after transfection, the cells were treated with different known small molecule regulators of Hippo signaling, followed by a luciferase assay. When interpreting the results of these assays, it should be noted that proteins and drug treatments may activate feedback pathways that influence biosensor activity. Further experiments will be necessary to confirm a relationship between the candidate and the Hippo signaling pathway.Following this procedure, other methods like quantitative Real-Time PCR, or Western Blot, can be used to answer additional questions about LATS kinase activity. After its development, this technique paved the way for the researchers in the field of cancer biology to explore the dynamics and activity of LATS kinase and Hippo signaling pathway in many biological and biochemical processes. Don't forget that drugs for screening can be extremely hazardous, and appropriate precautions should always be taken while performing this procedure.

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Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...