Suppressor of fused impedes Ci/Gli nuclear import by opposing Trn/Kapβ2 in Hedgehog signaling

The Hedgehog (Hh) family of secreted proteins governs many key developmental processes by regulating Ci/Gli transcription factors at multiple levels including nuclear-cytoplasmic shuttling. Here, we investigate the mechanism underlying the regulation of Ci/Gli subcellular localization by identifying and characterizing a novel nuclear localization sequence (NLS) in the N-terminal conserved domain of Ci/Gli that matches the PY-NLS consensus. We demonstrate that the PY-NLS functions in parallel with a previously identified bipartite NLS to promote nuclear localization and activity of full-length Ci. We find that Transportin (Trn), the Drosophila homolog of Kap beta 2, is responsible for PY-NLS-mediated nuclear localization of Ci. Furthermore, we show that the tumor suppressor and conserved Hh pathway component Suppressor of fused (Sufu) opposes Trn-mediated Ci nuclear import by masking its PY-NLS. Finally, we provide evidence that Gli proteins also contain a functional PY-NLS and that mammal Sufu uses a similar mechanism to regulate nuclear translocation of Gli. Our study not only provides a mechanistic insight into how Sufu regulates Hh signaling and the subcellular localization of Ci/Gli, but also reveals a role for Trn/Kap beta 2 in developmental regulation.