Suppressor of fused (Sufu) is an essential negative regulator of the sonic hedgehog (Shh) pathway, but little is known about how Sufu itself is normally regulated. Here, we report that Sufu is phosphorylated at Ser-342 and Ser-346 by GSK3β and cAMP-dependent protein kinase A (PKA), respectively, and phosphorylation at this dual site stabilizes Sufu against Shh signaling-induced degradation. We further show that localization of Sufu in the primary cilium is induced by Shh signaling and is required for the turnover of both phosphorylated and total Sufu. Perturbing Sufu phosphorylation with PKA inhibitors or replacing Ser-346 with alanine reduced the stay and replacing Ser-342 and Ser-346 with aspartic acid prolonged the stay of Sufu in the cilia. Finally, ciliary localization of Gli2/3 also required Smo and was similarly influenced by perturbations of PKA activity or mutations at the dual Sufu phosphorylation site. Thus, Shh likely induced trafficking of phospho-Sufu into the primary cilium in a complex with Gli2/3, and dephosphorylation triggered a retrograde export, allowing Sufu to be degraded by the ubiquitin-proteasome system.

Cell surface reception of Sonic hedgehog (Shh) must ensure that the graded morphogenic signal is interpreted accordingly in neighboring cells to specify tissue patterns during development. Here, we report endocytic sorting signals for the receptor Patched1 (Ptch1), comprising two ‘PPXY’ motifs, that direct it to degradation in lysosomes. These signals are recognized by two HECT-domain ubiquitin E3 ligases, Smurf1 and Smurf2, which are induced by Shh and become enriched in Caveolin-1 lipid rafts in association with Ptch1. Smurf-mediated endocytic turnover of Ptch1 is essential for its clearance from the primary cilium and pathway activation. Removal of both Smurfs completely abolishes the ability of Shh to sustain the proliferation of postnatal granule cell precursors in the cerebellum. These findings reveal a novel step in the Shh pathway activation as part of the Ptch1 negative feedback loop that precisely controls the signaling output in response to Shh gradient signal.

Vertebrate muscle differentiation is coordinated by an intricate network of transcription factors requiring proliferating myogenic precursors to withdraw irreversibly from the cell cycle. Recent studies have implicated a large number of microRNAs exerting another layer of control in many aspects of muscle differentiation. By annealing to short recognition sequences in the 3'-untranslated region, microRNAs attenuate target gene expression through translation repression or mRNA degradation. Here, we show that miR-214 promotes myogenic differentiation in mouse C2C12 myoblasts at a step preceding the induction of p21 and myogenin. Blocking miR-214 function with a 2'-O-methylated double-stranded inhibitor maintained C2C12 cells in the active cell cycle, thereby inhibiting the myogenic differentiation. By global gene expression profiling, we identified the proto-oncogene N-ras as one of miR-214 targets. Furthermore, manipulating the N-Ras level with small interfering RNA or adenovirus-mediated forced expression either augmented or attenuated the effect of miR-214, respectively. Thus, our data uncovered a novel microRNA-mediated mechanism that controls myogenic differentiation.

Gastric cancer is one of the leading causes of cancer death world-wide and carries a high rate of metastatic risk. In addition to other protein-coding oncogenes and tumor suppressor genes, microRNAs play an important role in gastric cancer tumorigenic progression. Here, we show that miR-206 is expressed at markedly low levels in a cohort of gastric tumors compared to their matching normal tissues, and in a number of gastric cancer cell lines. Down-regulation of miR-206 was particularly significant in tumors with lymphatic metastasis, local invasion, and advanced TNM staging. We find that forced expression of miR-206 suppressed the proliferation, colony-formation, and xenograft tumorigenesis of SCG-7901 cells, a line of gastric cancer cells. Forced expression of miR-206 also suppressed SCG-7901 cell migration and invasion, as well as metastasis in cell culture or tail-vein injected mouse models, respectively. The anti-metastatic effect of miR-206 is likely mediated by targeting metastasis regulatory genes STC2, HDAC4, KLF4, IGF1R, FRS2, SFRP1, BCL2, BDNF, and K-ras, which were drastically down-regulated by stable expression of exogenous miR-206 in SCG-7901 cells. Taken together, our results indicate that miR-206 is a tumor suppressor of gastric cancer acting at steps that regulate metastasis.

Cellular responses to the graded Sonic hedgehog (Shh) morphogenic signal are orchestrated by three Gli genes that give rise to both transcription activators and repressors. An essential downstream regulator of the pathway, encoded by the tumor suppressor gene Suppressor of fused (Sufu), plays critical roles in the production, trafficking and function of Gli proteins, but the mechanism remains controversial. Here, we show that Sufu is up-regulated in active Shh responding tissues and accompanies Gli activators translocating into and Gli repressors out of the nucleus. Trafficking of Sufu to the primary cilium, potentiated by Gli activators but not repressors, was found to be coupled to its nuclear import. We have identified a NES motif of Sufu in juxtaposition to the PKA and GSK3 dual phosphorylation site and show that Sufu binds the chromatin with both Gli1 and Gli3. Close comparison of neural tube development among individual Ptch1^-/-, Sufu^-/- mutant and Ptch1^-/-;Sufu^-/- double mutant embryos indicates that Sufu is critical for the maximal activation of Shh signaling essential to the specification of ventral most neurons. These data define Sufu as a novel class of molecular chaperone required for every aspect of Gli regulation and function.