Delineating the Molecular Mechanisms of KIF7 in Vertebrate Hedgehog Signal Transduction

The Hedgehog (Hh) signalling pathway plays important roles in embryonic development and for adult tissue homeostasis and regeneration of multi-cellular organisms. Mutations in components of this pathway often result in human developmental disorders and cancer. The primary cilium, a microtubule based extension from the cell surface is required for Hh signalling in vertebrates. Hh ligand binding to the Patched (Ptch1) receptor leads to the ciliary accumulation and activation of the seven transmembrane protein Smoothened (Smo) and changes the localization of several pathway components within primary cilia subdomains. How Smo activation affects the localization of effector proteins in cilia and how this relates to the activation of target genes is unclear. Genetic and biochemical analysis revealed that the kinesin family protein 7 (Kif7) trafficks to the primary cilia upon pathway activation and fulfills dual positive and negative roles during mouse Hh signalling. Through proteomic and biochemical analysis, we aim to better understand the molecular mechanisms underlying the functions of Kif7 in vertebrate Hedgehog signalling. Using a mass spectrometry-based proteomic approach, we identified Liprin-1 (PPFIA1) and the PP2A phosphatase as Kif7-interacting proteins, and showed that these proteins regulate Kif7 phosphorylation and its activity. We further identified DYRK2 as a protein kinase that phosphorylates Kif7. Overall, our study identified phosphorylation as a post-translational modification that dictates Kif7 localization within primary cilia and its control of Gli transcriptional activity. Lastly, through biochemical assays we examined the relationship between SuFu and Kif7, two key regulators of the Hh pathway. Collectively, these studies highlight the importance of Kif7 in vertebrate Hh signalling.Ph.D