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 regeneratio....

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 ....

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

List of materials used in this article

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