The challenge of identifying substrates of SCF ubiquitin ligases

In this thesis, we describe the development of a number of affinity purification methods aimed at identifying substrates of E3 ubiquitin ligases and focus on the study of two novel substrates, namely PDZGEF1 and Tiam1, guanine nucleotide exchange factors of the small GTPases Rap and Rac, respectively. In Chapter 1, we give an overview of the ubiquitin-proteasome system, describing how substrate proteins are targeted for proteasomal degradation by a well-defined enzymatic cascade catalyzed by E1, E2 and E3 enzymes. E3 ubiquitin ligases confer specificity to this process by binding target proteins and mediating the transfer of ubiquitin. By regulating the proteolysis of proteins involved in important cellular functions, E3s play crucial roles in the control of many biological processes. In Chapter 2, we report that, by employing modified versions of tandem affinity immunopurification coupled to mass spectrometry analysis, we have recovered most previously reported substrates of the SCFβTrCP ubiquitin ligase, thus demonstrating the validity and reliability of our approach. Moreover, we describe the identification of a number of putative new SCFβTrCP substrates, which are now undergoing validation. In Chapter 3, we investigate the molecular mechanisms controlling the βTrCP-mediated degradation of PDZGEF1, an activator of Rap1. PDZGEF1 was found in our screen as an interactor of βTrCP and validated as a novel substrate of SCFβTrCP. We show that in response to factors that stimulate cell motility, IKKβ stimulates the CK1α-dependent phosphorylation of PDZGEF1 and consequent PDZGEF1 binding to βTrCP. PDZGEF1 is then ubiquitylated and degraded by the proteasome, resulting in Rap1 inactivation. We also report that defective degradation of PDZGEF1 inhibits migration and invasiveness of epithelial cells in response to metastatic factors. In Chapter 4, we study the degradation of Tiam1, a second novel SCFβTrCP substrate identified in our proteomic screen. We show that Tiam1, a specific guanine nucleotide exchange factor of the small GTPase Rac, interacts and is ubiquitylated by SCFβTrCP. The interaction depends on a conserved phosphodegron motif and is regulated by CK1. Furthermore, we demonstrate that Tiam1 is targeted for proteasomal degradation by βTrCP in response to mitogenic stimulation. In Chapter 5, we describe the role of affinity purification-mass spectrometry in characterizing E3 ligase-substrate complexes and new strategies developed to screen for binding partners of both deubiquitylating enzymes (DUBs) and ubiquitin binding domain (UBDs). We also review the possibility of applying mass spectrometry to study different poly-ubiquitin linkages. In Chapter 6, we discuss strengths and limitations of our affinity purification methods for the identification of substrates of E3 ubiquitin ligases and illustrate the role of E3 ubiquitin ligases as potential targets in drug discovery.