Exploring signal transduction networks using mass spectrometry-based proteomics

Abstract

Mass spectrometry (MS)-based proteomics can be used to answer a diversity of biological questions. In this thesis, we describe the application of several MS-based proteomics approaches to get insight into several aspects of signal transduction. In Chapter 2, quantitative global phosphoproteomics are employed to study phosphorylation events that occur upon activation of the Rap guanine nucleotide exchange factor (GEF) exchange protein directly activated by cAMP (Epac) in human umbilical vein endothelial cells (HUVECs). We identify nearly 18,000 phosphorylation sites of which 220 are shown to be regulated upon Epac-Rap activation. Bioinformatical analyses reveal that these regulated phosphosites reside in proteins regulating the actin cytoskeleton, cell-cell junctions and cell adhesion, which are processes that have previously been linked to Rap. In the Addendum to Chapter 2, we describe a preceding effort to study global phosphorylation events upon Epac activation, using a generated HeLa cell line stably expressing Epac1. A total of 6,750 phosphorylation sites are identified and 77 of these displayed regulation upon Epac1 activation. Further investigation of one of the regulated phosphoproteins, the Rac1 effector CD2-associated protein (CD2AP), reveals the requirement of an intact microtubule network in Epac-mediated endothelial barrier function. Chapter 3 focuses more specifically on Epac1 itself and we describe several MS-based approaches that are utilized to investigate Epac1-containing protein complexes as well as phosphorylation of Epac1. Besides identifying the known Epac1 interactor Ran binding protein 2 (RanBP2), we find that a remarkable amount of cytoskeleton-associated proteins interact with Epac1. In addition, we identify several phosphorylation sites in Epac1. We employ a tandem affinity purification (TAP) strategy using MCF7 human breast cancer cells to identify proteins that interact with TSPY-like 5 (TSPYL5, also known as KIAA1750) in Chapter 4. We identify ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease (HAUSP)) as a specific TSPYL5 interactor and demonstrate that this interaction eventually leads to reduced protein levels of the p53 tumor suppressor, which is the cellular target of USP7. Finally, Chapter 5 describes that phosphorylation of multiple sites in the transactivation domain of the transcription factor FoxM1 by CyclinA/cdk complexes, results in relief of inhibition by the N-terminal autorepressor domain during the G2 phase of the cell cycle. At least one of the responsible sites is identified by a targeted MS-based phosphoproteomics approach using on-line titanium dioxide (TiO2) phosphopeptide enrichment. In addition, a number of previously unreported phosphorylation sites on FoxM1 are identified. The work described in this thesis exemplifies that MS-based proteomics can be exploited to examine a wide variety of cellular and biological processes. As this technology is continuously advancing, biologically relevant data can be obtained in an increasingly fast, sensitive and accurate manner. This will significantly contribute to our knowledge of the inner workings of a cell, tissue or an entire organism, which can subsequently be applied in studies on aberrant cellular behavior in the many human diseases, ultimately providing suitable treatments.