Whereas the genomic response to TCDD has been analysed in several large-scale studies
at the level of the transcriptome and the proteome both in vitro and in vivo, a systematic
study of the proteins involved in the early nongenomic action of TCDD has not been performed previously. Therefore, the present study was conducted with the aim to identify TCDD-induced alterations in protein phosphorylation occurring early after the onset of TCDD exposure in order to shed light on the alterations in signal transduction potentially involved in the early, nongenomic effects of TCDD.
In the present study, the rat hepatoma cell line 5L was used as a model
system. This cell line is a descendant of the H-4-II-E cell line established by
[Pitot et al., 1964] from the Reuber H35 rat hepatoma [Reuber, 1961]. 5L cells are
epithelial-like cells which express both the AhR and the ARNT protein and which
are highly responsive to TCDD-induced gene activation. They have been previ-
ously used in a variety of studies dealing with the role of AhR in cell cycle
regulation and TCDD toxicity [G¨ottlicher et al., 1990], [G¨ottlicher and Wiebel, 1991],
[Wiebel et al., 1991], [Weiss et al., 1996], [Ge and Elferink, 1998], [Reiners et al., 1999], [Elferink et al., 2001], [Levine-Fridman et al., 2004]. With respect to nongenomic actions of TCDD in this cell line, Weiss et al. (2005) observed that TCDD exposure of the cells is associated with the rapid phosphorylation of the p38 mitogen-activated protein kinase (MAPK) in an AhR-dependent, but, importantly, transcription-independent manner. p38 activation was observed as early as 1 h after addition of TCDD and resulted in subsequent
transcriptional induction of the proto-oncogene c-jun. Phosphorylation of p38 was not
1.3. THE AIM OF THE PRESENT STUDY
between AhR and MAPK signalling. The signalling pathway resulting in p38 phosphory- lation and the kinase involved remained unclear.
In the present quantitative phosphoproteomic analysis of the effects of TCDD in 5L cells, short exposure periods of 0.5, 1 and 2 hours were used, i.e. treatments that were un- likely to result in significant alterations in the abundance of proteins with TCDD-regulated
expression which requires some time due to its dependence on de novo transcription and
translation. The study was performed with the expectation to uncover proteins previ- ously unknown to exhibit rapid alterations in phosphorylation as a consequence of TCDD exposure and which might give clues to pathways involved in nongenomic signalling pre- ceding alterations in gene expression. Moreover, it was anticipated to identify proteins with altered phosphorylation as a consequence of their involvement in the regulation of transcription induced by the DRE-bound liganded AhR/ARNT heterodimer. And, finally, the study was expected to provide novel information on the global phosphoproteome of rat cells with respect to the identification of novel phosphorylation sites of phosphoproteins and the discovery of proteins previously not known to be phosphorylated.
Chapter 2
Material and methods
2.1
Materials
RPMI 1640 without arginine and lysine, PBS and dialysed fetal calf serum were from Gibco-
Invitrogen (Carlsbad, CA). U-13C
6-L-lysine hydrochloride and U-13C6 15N4-L-arginine hy-
drochloride were from Invitrogen (Carlsbad, CA, USA). U-15N4-L-arginine hydrochloride
was from Cambridge Isotope Laboratories (Andover, MA, USA) and TCDD (purity >
99%) in dimethyl sulfoxide was from ¨Okometric (Bayreuth, Germany). Benzonase, KN-
92, KN-93 and W-7 were from Merck (Darmstadt, Germany). Arginine, lysine, Tris,
MgCl2, NaCl, phosphatase inhibitor cocktails 1 and 2, acetonitrile, formic acid, ethanol,
acetone, PHOS-SelectT M Iron Affinity Gel, 2-APB, BAPTA-AM, besylate, Diltiazem hy-
drochloride, Nifedipine, sodium orthovanadate and Triton X-100 were from Sigma-Aldrich (Taufkirchen, Germany). ”Complete” protease inhibitor cocktail tablets were from Roche (Mannheim, Germany). Sequencing grade modified trypsin was from Promega (Madison, WI, USA). Lys-C was from Wako (Richmond, VA, USA). Hydrogen peroxide was pur-
chased from J.T. Baker (Deventer, Netherlands). TSKGel Amide-80 5µm HILIC 2 mm
column was from Tosoh Bioscience (Stuttgart, Germany). Poros Oligo R3 RP material was from PerSeptive Biosystems (Framingham, MA, USA). GELoader tips were from Ep- pendorff (Hamburg, Germany). Glycolic acid and trifluoroacetic acid were from Fluka (St. Louis, MO, USA). 3M Empore C8 disks were from 3M Bioanalytical Technologies (St. Paul, MN, USA). Syringe for HPLC loading was from SGE (Victoria, Australia). The water was from an Elga Purelab Ultra (Bucks, UK). Titanium dioxide beads were
CHAPTER 2. MATERIAL AND METHODS
(Ammerbuch-Entringen, Germany). PicoTipTMemitter needles were from New Objective,
DNU - Nowak Umweltanalysen (Berlin, Germany).
2.1.1
Software
The mass spectrometry raw files generated by the LTQ Orbitrap XL mass spectrometer were inspected with the software Xcalibur 2.1 (Thermo Fisher Scientific, Bremen, Ger- many). MALDI spectra were analysed using the program flexAnalysis (Bruker Daltonics, Bremen, Germany). MSQuant (University of Southern Denmark, Odense, Denmark) was used for the quantitation and phosphosite localization scoring of the identified phospho- peptides. The program ”R” (Version 2.9.1), a statistical data analysis software freely available under: http://cran.r-project.org, was used for the statistical analysis. For this work, the following R-Packages were dowloaded and used: limma, q-value, BioIDmapper and biomaRt.
Protein Center (http://www.proxeon.com; Proxeon, Odense, Denmark) is a consoli- dated protein database. The software was used for the annotation of proteins to databases like Pfam or Gene Ontology to support the interpretation of the experimental data.