Hedgehog signaling plays important roles in embryonic patterning of multicellular organisms. This pathway is ultimately transmitted by the zinc-finger transcriptional factor Gli, of which activity is suppressed by Sufu, a negative regulator of this signaling. To clarify this regulation to more detail, we screened for Sufu-binding proteins. We identified GSK3 beta as a specific binding partner of Sufu by mass spectrometric analysis. GSK3 beta bound to Sufu both in vitro and in vivo. Down-regulation of GSK3 beta expression by RNAi in Hedgehog-responsive cells attenuated Hedgehog signaling, suggesting that GSK3 beta functions as a positive regulator of Hedgehog signaling, In addition, an in vitro kinase assay showed that GSK3 beta phosphorylates Sufu and phosphorylation-mimicking mutant of Sufu showed significantly decreased ability to bind Gli1 and could not suppress the Gli-mediated expression of a reporter gene efficiently. These results strongly suggest that GSK3 beta phosphorylates Sufu to positively regulate Hedgehog signaling in mammalian cells. (c) 2006 Elsevier Inc. All rights reserved.