Atypical Protein Kinase C (aPKC) isoforms are essential regulators of polarised cell behaviour and in migrating NRK cells translocate to the leading edge in a complex with the exocyst and KIBRA. Engineered delivery of upstream signals to the plasma membrane places leading edge ERK activation downstream of aPKC and demonstrates partial sufficiency in regulating cell migration and adhesion. This model system provides the opportunity to probe the leading edge to better understand events downstream of aPKC. Multiple screening approaches have identified cytoskeletal and translation processes as putative targets of this pathway. Based on in silico candidate screening it is shown that multi-site phosphorylation of Parvin alpha is important for focal adhesion maturation. These phosphorylation events are triggered following acute focal adhesion turnover, which can be blocked by aPKC and MEK inhibition. Based upon proteomic approaches, a novel role for the putative aPKC/ERK substrate Cdc42 effector protein 1 (Cdc42ep1) has been identified. siRNA knockdown of Cdc42ep1 phenocopies aPKC loss; focal adhesions enlarge and turnover less efficiently. This impacts on polarized cell motility as knockdown prevents cell orientation and efficient wound closure. Finally, a novel role for aPKC is reported in relation to leading edge translation. Active translation at the leading edge is reduced following aPKC and MEK inhibition and compartmentalised distribution of translation factors is modulated following pathway intervention. This includes the eukaryotic translation initiation factor 3A (eIF3A), one hit identified by proteomic screening. eIF3A interacts with the exocyst and localises to the leading edge in an aPKC-dependent fashion. In addition, eIF3A is shown to regulate polarised migration and adhesion maturation. The data presented in this thesis illustrate combined screening and validation to delineate compartmentalised signalling events. Localised aPKC/exocyst/ERK activity is necessary for cytoskeletal controls and the polarized delivery and activation of translation machinery at the leading edge.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:626783 |
| Date | January 2014 |
| Creators | Peel, N. R. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1419046/ |
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