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Ubiquitylation is a reversible modification with a variety of roles ranging from governing protein homeostasis and trafficking to DNA damage and the immune response. A ubiquitin molecule is added on a substrate protein by an orchestrated cascade of reactions performed by the E1, E2 and E3 enzymes, while it is removed by the Deubiquitylases (DUBs). This work reports for the first time on a hitherto uncharacterised DUB enzyme, USP31. I identified USP31 in a screen for regulation of the MYCN oncogene in neuroblastoma. However, my initial characterisation showed that USP31 localises on microtubules and the centrosome. I proceeded to map the microtubule localising sequence of USP31 to the unstructured C-terminal of the protein. From that point, I developed a variety of cellular models in order to study its effect on microtubule network properties and the downstream processes they govern.

USP21 is a centrosome-associated deubiquitylase (DUB) that has been implicated in the formation of primary cilia – crucial organelles for the regulation of the Hedgehog (Hh) signaling pathway in vertebrates. Here, we identify KCTD6 – a cullin-3 E3-ligase substrate adapter that has been previously linked to Hh signaling – as well as Gli1, the key transcription factor responsible for Hh signal amplification, as new interacting partners of USP21. We identify a cryptic structured protein interaction domain in KCTD6, which is predicted to have a similar fold to Smr domains. Importantly, we show that both depletion and overexpression of catalytically active USP21 suppress Gli1-dependent transcription. Gli proteins are negatively regulated through protein kinase A (PKA)-dependent phosphorylation. We provide evidence that USP21 recruits and stabilises Gli1 at the centrosome where it promotes its phosphorylation by PKA. By revealing an intriguing functional pairing between a spatially restricted deubiquitylase and a kinase, our study highlights the centrosome as an important hub for signal coordination., Summary: We identify a Hedgehog-pathway-associated ubiquitin ligase adapter as a direct interaction partner of the deubiquitylase USP21 and discover a close interplay between USP21 and protein kinase A in regulating Gli1.