AbstractPAWS1/FAM83G, a member of the poorly characterised FAM83 family of proteins that share the conserved domain of unknown function DUF1669, was identified as an interactor of the SMAD1 transcription factor. Because BMP signalling plays a fundamental role during embryogenesis, collaboration with Jim Smith (The Francis Crick Institute, London) led to the discovery that ectopic expression of PAWS1 mRNA in Xenopus embryos leads to a complete duplication of body axis. In similar assays, such a phenotype is associated with either the inhibition of canonical (SMAD4-dependent) BMP signalling or the activation of canonical Wnt/β-catenin signalling. PAWS1 has been reported to modulate only non-canonical BMP signalling and not influence canonical BMP signalling, findings which were also validated in Xenopus embryos and PAWS1-knockout U2OS cells in this thesis. Therefore, focus turned to investigating potential roles of PAWS1 in the regulation of the canonical Wnt/β-catenin signalling pathway.
The canonical Wnt/β-catenin signalling pathway plays critical roles during embryogenesis, stem cell self-renewal and in adult tissue homeostasis and is often misregulated in developmental defects, including skin and hair abnormalities, and cancer. In the absence of Wnt signals, sequential phosphorylation of the transcriptional co-factor β-catenin by CK1 and GSK3 results in the ubiquitylation of β-catenin, priming it for degradation via the proteasome. Upon Wnt-activation, β-catenin is stabilised and translocates to the nucleus, where it associates with TCF and LEF and regulates the expression of Wnt-target genes. Although the fundamental steps in Wnt signalling are established, many gaps remain in our understanding of the precise regulation of the pathway. It is demonstrated in this thesis that PAWS1 activates Wnt signalling in both Xenopus embryos and human cells. Furthermore, in PAWS1-knockout U2OS cells Wnt signalling is attenuated. Collectively, these data uncover a role for PAWS1 as a novel regulator of canonical Wnt/β-catenin signalling.
In search of molecular mechanisms through which PAWS1 regulates Wnt/β-catenin signalling, a proteomic approach on endogenous PAWS1 revealed the Ser/Thr protein kinase CK1α as a robust PAWS1 interactor. PAWS1 interacts and colocalises with endogenous CK1α. CK1 isoforms are key regulators of Wnt signalling and they phosphorylate many components of the pathway, however their precise regulation in cells, despite being critically important, is poorly understood. After mapping CK1-interaction sites to key residues within the conserved DUF1669 domain of PAWS1, it was possible to demonstrate that the interaction between PAWS1 and CK1α is critical for PAWS1 to activate Wnt signalling in both Xenopus embryos and U2OS cells. Although the phosphorylation of β-catenin on Ser45, which is reported to be phosphorylated by CK1 isoforms, appears to be unaltered by PAWS1-deficiency, the Wnt3A-induced nuclear translocation of β-catenin is slightly inhibited in PAWS1 knockout U2OS cells. It is likely that PAWS1 controls Wnt signalling by directing CK1α to key subcellular locations and substrates upon Wnt stimulation to regulate the nuclear translocation of β-catenin. Consistent with this hypothesis, a global phosphoproteomics analysis of wild type and PAWS1-/- U2OS cells has revealed differential phosphorylation of proteins that may be regulated by the PAWS1:CK1α interaction.
Interestingly, PAWS1 appears to control levels of endogenous CK1α protein and vice versa, although the mechanisms by which each achieves this are still unclear. The findings that the DUF1669 domain of PAWS1 interacts with CK1α led to the discovery that all FAM83 members bind to different CK1 isoforms through an identical mechanism. This has led to the hypothesis that FAM83 members serve as anchoring proteins for CK1 isoforms, and in doing so, they regulate CK1 subcellular localisation and substrate accessibility in cells.
Regulation of PAWS1 by post-translational modifications remains poorly defined. A proteomic approach identified calcium and calmodulin-dependent kinase isoforms D and G (CaMK2D and CaMK2G) as two novel interactors of PAWS1. CaMK2 enzymes are activated in response to calcium signals to control cytoskeletal rearrangements and cell movement. PAWS1 has been implicated in actin cytoskeletal dynamics and cell migration, through its dynamic interaction with the adapter protein CD2AP at the cell periphery. Here, PAWS1 has been shown to be phosphorylated at Ser356 by CaMK2D in cells and this phosphorylation event is demonstrated to be important for PAWS1-dependent cell migration.
Lastly, several PAWS1 mutations have been recently linked with the pathogenesis of skin diseases in dogs and humans. However, how these mutations relate to PAWS1 function in cells and potentially cause the disease phenotypes remain elusive. In this thesis, initial steps have been taken to address the potential impact of these pathogenic mutants on PAWS1 function.
|Date of Award||2018|
|Supervisor||Gopal Sapkota (Supervisor)|