FRET analysis of protein tyrosine kinase c-Src activation mediated via aryl hydrocarbon receptor

https://doi.org/10.1016/j.bbagen.2010.11.007Get rights and content

Abstract

Background

Activation of the protein tyrosine kinase c-Src (c-Src kinase) induced by the exposure to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown in various cell types. Most previous works used Western blot analysis to detect the phosphorylation on the Tyr416 residue, which activates c-Src kinase.

Methods

Here we compared the results of c-Src tyrosine phosphorylation via aryl hydrocarbon receptor (AhR)-dependent mechanisms from Western blot analysis with fluorescent resonance energy transfer (FRET) assay detecting c-Src activation after treatment with TCDD to activate AhR in two different human cell types.

Results

Western blot analyses show time-dependent phosphorylation of c-Src by TCDD in HepG2 and MCF-10A cells. Data from FRET assay visualized and quantified the activation of c-Src kinase induced by TCDD in living cells of both cell types. The FRET efficiency decreased by 20%, 5 min after TCDD treatment and continued decreasing until the end of the experiment, 25 min after TCDD treatment. PP2, a c-Src specific inhibitor, suppressed both TCDD- and epidermal growth factor- (EGF) induced c-Src activation. In contrast, the AhR antagonist 3′-methoxy-4′nitroflavone (MNF) blocked only TCDD- but not EGF-induced activation of c-Src.

Conclusions

The current study shows that the early activation of c-Src via EGF and AhR signaling pathways can be visualized in living cells using the FRET assay which is in line with Western blot analysis.

General Significance

The FRET assay provides a useful tool to visualize and quantify c-Src kinase activation via AhR in living cells.

Research Highlights

► Activation of c-Src kinase by TCDD in living cells. ► FRET assay to detect early and AhR-dependent activation of c-Src kinase. ► Regulatory role of AhR and cPLA2 during activation of c-Src in living cells.

Introduction

c-Src kinase is the first discovered non-receptor protein tyrosine kinase. c-Src plays an important role in cell differentiation, proliferation, survival, cell adhesion, cell morphology, and motility [1], [2]. c-Src kinase is expressed ubiquitously and its activity is tightly regulated. Phosphorylation on the Tyr416 residue of c-Src kinase, which is located in the activation loop, promotes its kinase activity.

The rapid activation of c-Src kinase after TCDD treatment has been first reported in our lab in several different cell lines [3], [4], [5], [6]. Most previous work to detect the activation of c-Src kinase used a specific antibody against phospho-c-Src (Tyr416) in Western blot analysis. However, in most cases, the results are only semi-quantified, which do not offer spatial resolution to resolve the kinetics and cannot be visualized. Fluorescence resonance energy transfer (FRET) has been proven to be a powerful tool to visualize and quantify the signaling cascades in living cells with high spatiotemporal resolutions. A FRET-based biosensor has been developed in Dr. Tsien's lab [7]. A plasmid encoding a c-Src kinase substrate peptide conjugated to YFP (yellow fluorescence protein) and CFP (cyan fluorescence protein) was generated and transfected into cells. When c-Src kinase inside the cell is activated, the tyrosine residue on the substrate peptide is phosphorylated and the conformational change of the substrate peptide increases the distance and changes the relative orientation between the YFP and CFP to generate a change of the fluorescence resonance energy transfer (FRET) effect. The yellow emission (527 nm) from YFP decreases and the cyan emission (476 nm) from CFP increases. As a result, the ratio of yellow/cyan emission decreases more drastically. The FRET-based biosensors provide a complementary approach to traditional biochemical assays for the analysis of the c-Src kinase activity. The FRET assay has been shown to be a very useful method to detect and visualize the activation of c-Src kinase in living cells. The measurement is non-invasive and can be repeated with the same cells, allowing for determination of the time-course of the kinase activation in live cells. Here we used this FRET-based biosensor to study the activation of c-Src kinase after TCDD exposure in two cell lines, the human hepatoma cell line HepG2 and the human mammary epithelial cell line MCF10A. The results show a rapid c-Src kinase activation in a Fret-based assay, which was confirmed in Western blot analysis of the phosphorylated form of Src in both cell lines tested. The effect of TCDD on c-Src activation required the activation of the aryl hydrocarbon receptor (AhR). TCDD is an environmental pollutant and the prototypical ligand of the transcription factor AhR. After ligand binding the AhR dimerizes with the aryl hydrocarbon receptor nuclear transporter (ARNT), and binds on dioxin responsive elements (DRE) and regulates the expression of a diversity of genes, which is well established for the induction of cytochrome P4501A1 (CYP1A1) in response to several environmental pollutants such as polyhalogenated aromatic hydrocarbons [8]. Furthermore, recent studies show that the activation of AhR by TCDD also induces several proinflammatory genes like interleukin 8, IL-1β, CCL1 as well as cyclooxygenase 2 (COX-2) [9], [10], [11], [12]. COX-2, also known as prostaglandin H synthase 2 (PGHS-2), is the key enzyme in prostaglandin biosynthesis, and acts both as a dioxygenase and as a peroxidase. In contrast to the isoform COX-1, COX-2 is inducible by drugs and environmental toxicants [13]. The induction of COX-2 by TCDD has been shown to involve the activation of c-Src and to involve binding of the CAAT enhancer binding protein (C/EBP)β on the COX-2 promoter [12], [14], [15]. The activation of the AhR by omeprazole for instance, has been shown to require a signal transduction pathway that involves c-Src kinase and that this activation pathway is different from the mechanism exerted by high-affinity ligands [16], [17]. In summary, the c-Src kinase activity seems to be a critical component in AhR signaling and gene regulation especially in the non-classical AhR-mediated pathway.

Section snippets

Reagents and antibodies

TCDD (> 99% purity) was obtained from Dow Chemicals Co. (Midland, MI). Epidermal Growth Factor (EGF) was purchased from Sigma-Aldrich Co. (St. Louis, MO). 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and methylarachidonyl fluorophosphonate (MAFP, a cPLA2 inhibitor) were purchased from Calbiochem (San Diego, CA). The AhR antagonist 3′-methoxy-4′-nitroflavone (MNF) was a kind gift from Dr. Josef Abel (University of Duesseldorf, Germany). Rabbit polyclonal anti-c-Src and

Enhanced phosphorylation on the Tyr416 residue of c-Src kinase after TCDD treatment in HepG2 and MCF10A cells

The activation of c-Src kinase after TCDD treatment was first verified in HepG2 and MCF10A cells through the Western blot using a specific antibody capable of reacting with a phosphorylated (Tyr 416), and therefore an activated form of c-Src kinase. In HepG2 cells, the c-Src kinase was found to be activated as early as 5 min after TCDD treatment, manifested by the enhanced phosphorylation on the the Tyr416 residues (Fig. 1A). The activation of c-Src kinase became significant 15 min after TCDD

Discussion

c-Src kinase is expressed ubiquitously and its activity is tightly regulated. There are two phosphorylation sites in c-Src kinase, which are critical in regulating its activity. One site is the Tyr 527 residue. Under basal conditions in vivo, 90–95% of c-Src kinase is phosphorylated at Tyr 527 [19]. The phosphorylation on this site makes it bind to its own SH2 domain and this intramolecular association stabilizes the inactive form of the enzyme. On the other hand, the phosphorylation on another

Acknowledgements

We like to thank Dr. Roger Tsien (University of California, San Diego, CA) for providing us with critical c-Src reporter plasmid. Funding was provided by the National Institute of Environmental Health grant R21ES15846 and Grant-in-Aid from the American Heart Association (C.F.A.V.), and National Eye Institute grant REY016754A and AHA grant 0665201Y (J.Z.). J.Z. is a Hai Tian scholar at Dalian University of Technology.

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    1

    Equal contribution.

    2

    Present address: Lab of Biomedical Optics, College of Physics and Optoelectronic Engineering, Dalian University of Technology, Dalian 116023, China.

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