SCFβTrCP, discovering new sides of the E3 ligase by studying its substrates

TY - THES

T1 - SCFβTrCP, discovering new sides of the E3 ligase by studying its substrates

AU - Kruiswijk, F.

N1 - Reporting year: 2013

PY - 2013

Y1 - 2013

N2 - The ubiquitin-proteasome system controls molecular networks that underlie fundamental cellular functions such as DNA replication, DNA repair, transcription, protein synthesis, cell differentiation and apoptosis. Aberrant functions of components of the ubiquitin-proteasome system (particularly of ubiquitin ligases that provide substrate specificity) have been implicated in the pathogenesis of many human cancers. One of these ubiquitin ligases is SCFβTrCP, a multi-subunit complex that targets proteins involved in DNA damage signaling and cell cycle progression, as well as known tumour suppressors, for proteasomal degradation. These findings have raised interest in the use of SCFβTrCP as a therapeutic target. The vast array of SCFβTrCP substrates and their diverse functions, however, are a complicating factor in the development of any therapy that targets SCFβTrCP, as it might have unexpected off-target effects. Thorough knowledge of all processes regulated by SCFβTrCPis therefore required to aid the development of the most effective therapy, with limited unwanted effects. In an attempt to fill these gaps in our knowledge, we have developed an unbiased, mass spectrometry based, screen aimed at identifying novel SCFβTrCPsubstrates. In this thesis, two of these novel substrates are described. First, a link between DNA damage signaling and protein synthesis regulation is established through the elucidation of the coupled activation and degradation of eukaryotic elongation factor 2 kinase (eEF2K) during DNA damage signaling. In response to genotoxic stress, eEF2K is activated by AMPK-mediated phosphorylation. Activated eEF2K phosphorylates eEF2 and induces a temporary ribosomal slowdown at the stage of elongation. Subsequently, during checkpoint silencing, eEF2K is degraded by the ubiquitin-proteasome system via the SCFβTrCP ubiquitin ligase to allow rapid resumption of translation elongation. This event requires eEF2K autophosphorylation on a canonical βTrCP-binding domain; inability to degrade eEF2K during checkpoint silencing causes delayed resumption of translation elongation. Second, a Golgi-associated guanine-nucleotide exchange factor (GEF) is shown to be a bona fide substrate of SCFβTrCP, by biochemical characterization of the binding, as well as the identification of a βTrCP-specific binding site in this GEF and assessment of the capability of SCFβTrCP to ubiquitylate it in vitro. To provide insight in the organism-wide effects of inhibiting βTrCP-mediated degradation of a single βTrCP substrate, an in vivo model is required.

AB - The ubiquitin-proteasome system controls molecular networks that underlie fundamental cellular functions such as DNA replication, DNA repair, transcription, protein synthesis, cell differentiation and apoptosis. Aberrant functions of components of the ubiquitin-proteasome system (particularly of ubiquitin ligases that provide substrate specificity) have been implicated in the pathogenesis of many human cancers. One of these ubiquitin ligases is SCFβTrCP, a multi-subunit complex that targets proteins involved in DNA damage signaling and cell cycle progression, as well as known tumour suppressors, for proteasomal degradation. These findings have raised interest in the use of SCFβTrCP as a therapeutic target. The vast array of SCFβTrCP substrates and their diverse functions, however, are a complicating factor in the development of any therapy that targets SCFβTrCP, as it might have unexpected off-target effects. Thorough knowledge of all processes regulated by SCFβTrCPis therefore required to aid the development of the most effective therapy, with limited unwanted effects. In an attempt to fill these gaps in our knowledge, we have developed an unbiased, mass spectrometry based, screen aimed at identifying novel SCFβTrCPsubstrates. In this thesis, two of these novel substrates are described. First, a link between DNA damage signaling and protein synthesis regulation is established through the elucidation of the coupled activation and degradation of eukaryotic elongation factor 2 kinase (eEF2K) during DNA damage signaling. In response to genotoxic stress, eEF2K is activated by AMPK-mediated phosphorylation. Activated eEF2K phosphorylates eEF2 and induces a temporary ribosomal slowdown at the stage of elongation. Subsequently, during checkpoint silencing, eEF2K is degraded by the ubiquitin-proteasome system via the SCFβTrCP ubiquitin ligase to allow rapid resumption of translation elongation. This event requires eEF2K autophosphorylation on a canonical βTrCP-binding domain; inability to degrade eEF2K during checkpoint silencing causes delayed resumption of translation elongation. Second, a Golgi-associated guanine-nucleotide exchange factor (GEF) is shown to be a bona fide substrate of SCFβTrCP, by biochemical characterization of the binding, as well as the identification of a βTrCP-specific binding site in this GEF and assessment of the capability of SCFβTrCP to ubiquitylate it in vitro. To provide insight in the organism-wide effects of inhibiting βTrCP-mediated degradation of a single βTrCP substrate, an in vivo model is required.

M3 - PhD thesis (Proefschrift)

SN - 978-90-393-6011-8

ER -