Mechanisms of crosstalk between phosphorylation and ubiquitylation in the regulation of cell proliferation

Sara D'Annibale

Research output: PhD ThesisPhD thesis

Abstract

Cell cycle progression is tightly controlled by the ubiquitin-proteasome system. Cullin-RING ubiquitin ligases are the major players responsible for the periodic proteolysis of many regulators of the cell cycle. Deregulated protein degradation mediated by cullin-RING ubiquitin ligases may result in uncontrolled proliferation, genomic instability and cancer. The results described in this thesis provide important insights on novel functions of the UPS in the regulation of cell proliferation. Identifying substrates of ubiquitin ligases and characterizing the molecular mechanisms regulating their degradation is important as it could lead to the development of new strategies for anticancer therapies. In this thesis, we have studied how specific and timely cooperation between phosphorylation and ubiquitylation controls cell growth and proliferation by targeting specific substrate proteins for proteasomal degradation. The starting point of this study was an affinity purification coupled to a mass spectrometry analysis aimed at the identification of new substrates of the SCFβTrCP ubiquitin ligase. We decided to focus on the phosphorylation-dependent degradation of three previously unknown interactors of βTrCP, namely, TFAP4, DEC1 and AS160, as earlier literature had suggested a role of these proteins in the control of cell cycle progression and cellular growth. TFAP4 and DEC1 are both bHLH transcription factors, AS160 is a Rab-GTPase activating protein. TFAP4 is targeted by SCFβTrCP for ubiquitylation and proteasome-dependent degradation during the G2 phase of the cell cycle. We also show that failure to degrade TFAP4 results in a number of mitotic defects, including chromosome missegregation and multipolar spindles, which eventually cause the activation of the DNA damage checkpoint. Taken together these findings reveal that βTrCP-mediated proteolysis of TFAP4 is required for the fidelity of mitotic division. Moreover, I characterize the phosphodegron and phosphorylation events controlling the degradation of DEC1 by SCFβTrCP during recovery from G2 DNA damage checkpoint and, in particular, the role of CDK1-dependent phosphorylation in regulating DEC1 proteolysis. These findings contribute to uncover how a crosstalk between phosphorylation and ubiquitylation acts to control DEC1 proteolysis and the DNA damage response Finally, I describe for the first time the interaction between SCFβTrCP ubiquitin ligase and AS160, a substrate of AKT implicated in the insulin signaling pathway. The interaction depends on a specific phosphodegron motif and SCFβTrCP is directly responsible for AS160 ubiquitylation. We discuss a possible new regulatory mechanism by which the degradation of AS160 could exert an effect on the translocation of the glucose transporter GLUT4.
Original languageEnglish
Awarding Institution
  • Utrecht University
Supervisors/Advisors
  • de Laat, Wouter, Promotor
  • Guardavaccaro, Daniele, Co-promotor
Award date31 Oct 2014
Place of Publicationutrecht
Publisher
Publication statusPublished - 31 Oct 2014
Externally publishedYes

Keywords

  • cell growth and proliferation, ubiquitin-proteasome system, phosphorylation, SCFβTrCP, cancer

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