Molecular and cell biology analysis of Rgnef, Trim71 and Gli2 in regulation of the Shh signalling pathway

Kayvan Hakim-Rad

Research output: ThesisDoctoral Thesis

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The Sonic hedgehog signalling pathway is of key importance during embryonic development as well as adult disease. While much is known regarding the main intracellular signalling components involved, precise details of the regulation of the pathway remain undiscovered. In this thesis, I aim to further our understanding of the regulation of the Shh signalling pathway by investigating aspects of the molecular mechanism.
We have previously shown that Tulp3 acts as a negative regulator of the Shh pathway. More recently, we have found that Tulp3 interacts with Rgnef and Trim71. In this thesis, I test the hypothesis that Rgnef and Trim71 act as regulators of the Shh pathway. My investigations centre on assessing pathway activity in relation to changes in Rgnef and Trim71 expression, using a cell biology assay. I show that reducing Rgnef expression leads to a reduction in Shh pathway activity, while increasing Rgnef expression stimulates pathway activity. I show also that increasing Rgnef expression significantly reduces the proportion of cells with a primary cilium, an organelle fundamental to Shh signalling. Within the embryo, I show that Rgnef is expressed in the developing neural tube. Shh pathway suppression was also induced by Trim71 siRNA, but through validation tests I show this to be a false positive result and investigate the possible causes.
In addition, I have used mass spectrometry to identify putative novel sites of Gli2 post translational modification. Gli2 is one of three transcription factors conserved in vertebrate Shh signalling, and which are the ultimate target effectors of pathway activity. Gli2 is predominantly involved in pathway activation however precise regulation of Gli2 activity remains poorly understood. By using mass spectrometry, I aimed to reveal novel sites of protein modification that would help to understand the role of Gli2. Two potential novel sites of Gli2 phosphorylation have been discovered.
Original languageEnglish
Awarding Institution
  • Royal Holloway, University of London
  • Murdoch, Jenny, Supervisor
  • Wilkinson, Christopher, Advisor
Thesis sponsors
Award date1 May 2015
Publication statusUnpublished - 2015


  • Shh signalling pathway
  • Rho-guanine nucleotide exchange factor
  • p190-rhoGEF
  • ARHGEF28
  • trim71
  • mlin41
  • Glioma associated oncogene
  • tulp3
  • Neural Tube Defects

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