Protein turnover of Patched, the Hedgehog receptor and key negative regulator of Hedgehog signaling, is controlled by the ubiquitin E3 ligase, Smurf, in a manner that depends on activation of signal transducer, Smoothened.
Hedgehog signaling plays conserved roles in controlling embryonic development; its dysregulation has been implicated in many human diseases including cancers. Hedgehog signaling has an unusual reception system consisting of two transmembrane proteins, Patched receptor and Smoothened signal transducer. Although activation of Smoothened and its downstream signal transduction have been intensively studied, less is known about how Patched receptor is regulated, and particularly how this regulation contributes to appropriate Hedgehog signal transduction. Here we identified a novel role of Smurf E3 ligase in regulating Hedgehog signaling by controlling Patched ubiquitination and turnover. Moreover, we showed that Smurf-mediated Patched ubiquitination depends on Smo activity in wing discs. Mechanistically, we found that Smo interacts with Smurf and promotes it to mediate Patched ubiquitination by targeting the K1261 site in Ptc. The further mathematic modeling analysis reveals that a bidirectional control of activation of Smo involving Smurf and Patched is important for signal-receiving cells to precisely interpret external signals, thereby maintaining Hedgehog signaling reliability. Finally, our data revealed an evolutionarily conserved role of Smurf proteins in controlling Hh signaling by targeting Ptc during development.
Hedgehog (Hh) signaling is a pathway renowned for its roles in controlling embryonic development and tumorigenesis. Signaling via this pathway proceeds when Hh ligands bind to the receptor Patched (Ptc), thereby preventing Ptc from inhibiting the signal transducer, Smoothened (Smo), and thus allowing Smo to accumulate on the cell surface where it becomes activated and promotes downstream signal transduction. In the absence of Hh ligands, Ptc inhibits Smo and is a key negative regulator of Hh signaling. In this study, we investigate how protein turnover of Ptc is controlled to ensure tight regulation of Hh signaling. Using Drosophila as a model system, we provide biochemical and genetic evidence to show that the E3 ligase, Smurf, directly controls Ptc protein turnover in developing wing discs. Moreover, we found that Smurf mediates Ptc degradation in a manner that depends on Smo signaling activity: activated Smo forms a complex with Smurf to preferentially promote degradation of the ligand-unbound Ptc receptor. Using mathematic modeling we reveal that the control of Smo activation by the opposing activities of Smurf and Ptc, is important for cells receiving the Hh signal to precisely interpret and relay external signals. We show that this control mechanism is also active in vertebrates with evidence that zebrafish Smurf proteins target Ptc1 protein for degradation to control late somitogenesis during zebrafish embryogenesis.