Phosphorylation of the PTCH1 C-terminal domain: profiling and significance

The Hedgehog (HH) signalling pathway is a well-established signalling cascade, and aberrant activity of the pathway is associated with the development of many cancer types. Much of the pathway hyperactivity stems from mutations within the primary HH receptor protein, PTCH1, a transmembrane protein with a long cytoplasmic C-terminal domain (CTD). Within the CTD are several proline-rich motifs, and within the first one is a TPSP motif which is a hotspot site for mutation within various cancers. The TPSP motif is the primary focus of this thesis, in Chapter 1, I show the changing phosphorylation states of the TPSP motif between wild-type (WT) and disease-associated mutants by mass spectrometry.

In Chapter 4, I investigate the structure of the proximal region of the PTCH1 CTD by nuclear magnetic resonance, confirming its intrinsically disordered nature and single phosphorylation of T1195 in the TPSP motif by in vitro phosphorylation assays with a S/T-P directed kinase, ERK2. In Chapter 5, I investigate the functional role of the TPSP motif, showing that mutations within the TPSP motif increase the levels of phosphorylated ERK1/2 and increased cell proliferation compared to PTCH1, indicating that the TPSP motif regulates Erk1/2 phosphorylation, which could explain the role of TPSP mutations in cancer.

In Chapter 6, I describe a novel interaction between PTCH proteins and the peptidyl-prolyl isomerase Pin1. The findings point towards a complex interaction which requires multiple binding sites within each PTCH protein but does not confirm the role of the TPSP motif in the interaction. In Chapter 7, I describe my contribution to a side project that demonstrated the ability of PTCH1 and PTCH2 to form heterodimers. In this project, I demonstrated that heterodimers are equally responsive to rSHH than homodimers of PTCH1 or PTCH2 and that the difference in activity observed between PTCH1 and PTCH2 is not due to the lack of conservation of the CTD.

In summary, this thesis improves the understanding of the structure and functionality of the first proline-rich motif of the PTCH1 CTD and uncovers a novel interaction between PTCH proteins and Pin1, adding of the complex nature of PTCH1.