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

Summary

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.

Abstract

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.

Introduction

The Hippo signaling pathway was first identified in Drosophila as a novel regulator of cell growth and animal size^1,2. 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 cancers^{7,8,9,10,11,12}. 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-S89^11,12. 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 manner^13,14,15,16. 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 Förster 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 mice^20,24 (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.

Protocol

1. Investigation of a Putative Regulator of Hippo Signaling Using the LATS-BS

1. Plating and transfection of cells
   1. Preparation of cell culture
      1. Warm 1x PBS, DMEM containing 10% FBS and 1% penicillin/streptomycin, and 0.25% trypsin-EDTA to 37 °C in a water bath for approximately 30 min.
      2. Thoroughly clean a biosafety cabinet with 70% ethanol.
      3. Place tissue culture dishes, Pasteur pipettes, serological pipettes, and pipette tips into the tissue culture hood as required.
      4. Take out 1x PBS, the media, and the trypsin-EDTA from the water bath, clean them with 70% ethanol, and place them in the hood.
   2. Maintenance and plating of the cells for transfection
      1. Choose a cell line with a high transfection efficiency (e.g., HEK293) for the experiment. Check for the expression of Hippo pathway components in this cell line by western blot to make sure Hippo signaling is active.
      2. Grow HEK293 cells in DMEM in 10 cm Petri dishes in an incubator at 37 °C and 5% CO₂. Monitor the cells under a microscope every day. Passage the cells as described below when 80–90% confluency is observed.
      3. Wash the cells by adding 5 mL of 1x PBS into a plate, gently swirling the plate, and aspirating the media completely.
      4. Add 1 mL of trypsin-EDTA to the plate. Swirl the plate to ensure that the trypsin evenly covers the cells.
      5. Incubate the cells at 37 °C in a CO₂ incubator for 5 min or until all the cells are detached.
      6. Add 4 mL of media (with 10% FBS and 1% penicillin/streptomycin) to neutralize the trypsin, resuspend the cells by pipetting up and down several times, and dispense one-tenth (0.5 mL) of the cells into new 100 mm plates (for a passing ratio of 1:10) containing 9.5 mL of complete media.
      7. For LATS-BS transfection, count the cells using a hemocytometer and plate 2 x 10⁵ cells into each well of a 12-well plate 24 h before transfection.
         NOTE: Using higher cell numbers may increase the background bioluminescent signal as Hippo signaling activity is regulated by cell confluency. Using lower cell numbers may increase cell sensitivity to transfection reagents and increase cell death²⁴.
   3. Transfection of the LATS-BS alone or together with the candidate gene
      1. Miniprep LATS-BS plasmids (NLuc-YAP15-pcDNA3.1-Hygro and 14-3-3-CLuc-pcDNA3.1-Hygro) fresh from DH5a bacteria liquid culture (necessary to achieve an optimum LATS-BS expression).
      2. 1 h before transfection, aspirate the medium from the plate, add 500 µL of complete growth medium to each well of the 12-well plates and return the cells to the incubator.
         NOTE: The following protocol describes transfection methods using a commercially available polymer-based transfection reagent. Other transfection reagents may also be suitable for transfection; however, we have not yet tested them.
      3. Make solutions A and B as described in Table 1. For N transfections, make (N + 0.5) x solution B premix.
      4. Immediately add the diluted polymer-based transfection reagent (solution B) to DNA (solution A). Pipette up and down gently to mix.
      5. Incubate the sample at room temperature for 15 min to allow transfection complexes to form.
      6. Add the total volume (76 µL) of transfection complexes dropwise to each well of the 12-well plate with gentle swirling.
      7. Incubate the cells overnight (18–24 h) at 37 °C.
         Note: For cells that are sensitive to polymer-based transfection reagents, the incubation time can be reduced to 5 h.
      8. The day after transfection, remove the transfection-complex-containing media and replace it with fresh complete media. Culture the cells at 37 °C for another day to perform luciferase assay.
2. Luciferase assay
   NOTE: The Luciferase Reporter Assay System detecting firefly and Renilla together in one assay is used for the luciferase assays. If Renilla was not used as an internal control, perform one-step luciferase assay by adding LARII without adding the Renilla detection reagent.
   1. Preparation of the cell lysates
      1. Dilute 5x passive lysis buffer (PLB) with distilled water as follows:
      2. Total volume of 1x PLB required = N x (50 μL of 5x PLB + 200 μL of dH2O) = N x 250 μL of 1x PLB per well of the 12-well plates (N = number of samples).
      3. Aspirate the media completely. Add 1 mL of 1x PBS into each well. Swirl the plate gently to remove detached cells and residual growth media. Aspirate the 1x PBS completely. Make sure no residual 1x PBS remains prior to the addition of 1x PLB.
      4. Add 250 μL of 1x PLB/well.
      5. Place the culture plates on a rocking platform with gentle rocking to ensure complete and even coverage of the cell monolayer with 1x PLB. Rock the culture plates at room temperature for 15 min to allow the cells to lyse.
      6. Transfer the lysate to a 1.5 mL microcentrifuge tube.
   2. Measuring the bioluminescent signal
      1. Take out the LARII (20 μL/sample) from -80 °C and equilibrate it to room temperature.
      2. Prepare Renilla detection reagent for N assays: add (N + 1) x 0.4 μL of 50x Substrate to (N + 1) x 19.6 μL of its Buffer.
      3. Predispense 10 μL of each cell lysate to 1.5-mL tubes.
      4. Program a luminometer to perform a 2-s premeasurement delay, followed by a 10-s measurement period for each dual luciferase assay.
         NOTE: The first and second measurements will be corresponding to the LATS-BS signal and the Renilla internal control signal, respectively. Any luminometer compatible with a single or dual luciferase can be used.
      5. Carefully transfer 20 μL of LARII reagent into one tube and quickly pipette up and down 3x to mix it.
      6. Immediately place the tube in the luminometer and initiate the reading.
      7. Remove the sample tube from the luminometer, immediately add 20 μL of Renilla Reagent, and mix by pipetting up and down 3x. Bring the sample in the luminometer and initiate the next reading. Record the measurements.
      8. Discard the reaction tube and proceed to the next sample.

2. A screen to Identify Hippo Pathway Regulators Using the LATS-BS

1. Cell culture and transfections
   1. Culture HEK293AD cells as described in section 1.1.
      NOTE: HEK293AD is more adherent than other HEK293 cell lines, which makes it a good cell line for use in screens.
   2. Detach cells as described in section 1.1.2. Count and plate 2 x 10⁶ cells in 100 mm plates 24 h before transfection.
   3. 1 h before transfection, aspirate the media from the plate and replace them with 5 mL of fresh complete growth media.
   4. Make solutions A and B as described in Table 2.
   5. Immediately add the diluted polymer-based transfection reagent (solution B) to DNA (solution A). Pipette up and down to mix.
   6. Incubate the sample at room temperature for 15 min to allow transfection complexes to form.
      1. Add the total volume (500 µL) of transfection complexes dropwise to cells with gentle swirling.
      2. Incubate the cells overnight at 37 °C.
      3. The next day, trypsinize and count the cells. Plate 1 x 10⁴ to 2 x 10⁴ cells into each well of a 96-well plate in a total volume of 100 µL of media per well. Incubate for another day.
   7. The next day, add agents/small molecules regulating the Hippo pathway at a final concentration of 10 µM to each well, 1–4 h before performing the luciferase assay.
      Note: For most small molecules/agents, a 4 h treatment at 10 µM does not affect cell viability. If a lower concentration is used, treatment may be extended for a longer period of time.
2. Luciferase assay
   1. In vitro luciferase assay
      1. Aspirate the media from the cells completely. Wash the cells with 100 µL of 1x PBS in each well. Aspirate completely.
      2. Add 20 µL of 1x PLB (prepared as described in step 1.2.1.1) into each well of the 96-well plates.
      3. Place the culture plates on a rocking platform with gentle rocking at room temperature for 15 min.
      4. Carefully transfer 20 µL of LARII reagent into each well and pipette up and down 3x to mix.
      5. Immediately place the plate in a plate-reading luminometer and initiate the reading.
   2. Living cell luciferase assay
      1. Make 11x D-luciferin stock as follows: dissolve 1.5 mg of D-luciferin powder in 1 mL of normal culture media.
      2. Transfer 10 µL of 11x D-luciferin into each well and mix it gently with the media which is already in the well.
      3. Replace the cells into the incubator for 10 min.
      4. Place the plate in the luminometer and initiate the reading.
         Note: If the signal intensity is not strong enough at this concentration of D-luciferin, another 10 µL of the D-luciferin stock may be added to the cells.

Results

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 ...

Discussion

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 ...

Materials

Name	Company	Catalog Number	Comments
Trypsin-EDTA	Life Technologies	2520056	0.25%
Fetal Bovine Serum (FBS)	Sigma	F1051
DMEM	Sigma	D6429-500ml	DMEM with high glucose
Penicillin Streptomycin	Life Technologies	15140122
PolyJet In Vitro DNA Transfection Reagent	Signagen	SL100688.5
5x Passive lysis buffer	Promega	E194A	30 ml
Dual-Glo® Luciferase Assay System	Promega	E2940	100 ml kit
20/20 luminometer	Turner Biosystems	998-2036	Single tube reader luminometer
GloMax®Navigator with dual injectors	Promega	GM2010	96-well plates reader luminometer