The centrosomal Deubiquitylase USP21 regulates Gli1 transcriptional activity and stability.

USP21 is a centrosome-associated deubiquitylase (DUB) that has been implicated in the formation of primary cilia, critical organelles for the regulation of the Hedgehog (Hh) signaling pathway in vertebrates. Here we identify KCTD6, a Cullin3 E3-ligase substrate adapter previously linked to Hh-signaling, as well as Gli1, the key transcription factor responsible for Hh signal amplification, as novel interacting partners of USP21. We identify a cryptic structured protein interaction domain in KCTD6, which is predicted to bear fold similarity 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 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 DUB and a kinase, our study highlights the centrosome as an important hub for signal coordination.