Regulation of Gli ciliary localization and Hedgehog signaling by the PY-NLS/karyopherin-β2 nuclear import system

Hedgehog (Hh) signaling in vertebrates depends on primary cilia. Upon stimulation, Hh pathway components, including Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting remains largely unknown. Here, we show that the PY-type nuclear localization signal (PY-NLS)/karyopherin beta 2 (Kap beta 2) nuclear import system regulates Gli ciliary localization and Hh pathway activation. Mutating the PY-NLS in Gli or knockdown of Kap beta 2 diminished Gli ciliary localization. Kap beta 2 is required for the formation of Gli activator (Gli A) in wild-type but not in Sufu mutant cells. Knockdown of Kap beta 2 affected Hh signaling in zebrafish embryos, as well as in vitro cultured cerebellum granule neuron progenitors (CGNPs) and SmoM2-driven medulloblastoma cells. Furthermore, Kap beta 2 depletion impaired the growth of cultured medulloblastoma cells, which was rescued by Gli overexpression. Interestingly, Kap beta 2 is a transcriptional target of the Hh pathway, thus forming a positive feedback loop for Gli activation. Our study unravels the molecular mechanism and cellular machinery regulating Gli ciliary localization and identifies Kap beta 2 as a critical regulator of the Hh pathway and a potential drug target for Hh-driven cancers.