Abstract
In Hedgehog (Hh) signaling, the seven-transmembrane protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation, ubiquitination, and cell surface accumulation. However, it is not clear how Smo cell surface accumulation and intracellular trafficking are regulated. Here, we demonstrate that inactivation of Hrs by deletion or RNAi accumulates Smo in the late endosome that is marked by late endosome markers. Inactivation of Hrs enhances the wing defects caused by dominant-negative Smo. We show that Hrs promotes Smo ubiquitination, deleting the ubiquitin-interacting-motif (UIM) in Hrs abolishes the ability of Hrs to regulate Smo ubiquitination. However, the UIM domain neither recognizes the ubiquitinated Smo nor directly interacts with Smo. Hrs lacking UIM domain still downregulates Smo activity even though to a less extent. We have characterized that the N-terminus of Hrs directly interacts with the PKA/CK1 phosphorylation clusters to prevent Smo phosphorylation and activation, indicating an ubiquitin-independent regulation of Smo by Hrs. Finally, we found that knockdown of Tsg101 accumulates Smo that is co-localized with Hrs and other late endosome markers. Taken together, our data indicate that Hrs mediates Smo trafficking in the late endosome by not only promoting Smo ubiquitination but also blocking Smo phosphorylation.
Highlights
The Hedgehog (Hh) morphogen controls such development processes as cell proliferation, embryonic patterning, and cell growth [1,2,3]
We recently showed that ubiquitination promotes Smo intracellular trafficking that is mediated by endosomes [13]
We found that the physical interaction between Smo and HGF-regulated tyrosine kinase substrate (Hrs) was inhibited by Hh-treatment (Fig. 3B, 2nd panel), indicating that the downregulation of Smo ubiquitination correlates with the disassociation of Hrs
Summary
The Hedgehog (Hh) morphogen controls such development processes as cell proliferation, embryonic patterning, and cell growth [1,2,3]. In Drosophila, the Hh signal is transduced through a reception system at the plasma membrane, which includes the receptor complexes Ptc-Ihog and the signal transducer Smo [7,8,9]. Using an antibody uptake assay, we found that Hh treatment inhibited Smo endocytosis and reduced the ratio of early endosome-localized Smo [13]. It is clearly important to understand how Hh regulates Smo trafficking and how ubiquitination promotes Smo endocytosis. Among the proteins that regulate receptor intracellular trafficking, the components from the Endosomal Sorting Complex Required for Transport (ESCRT) are critical. In Drosophila, the endosomal sorting of Smo is likely regulated by the Drosophila homolog of HGF-regulated tyrosine kinase substrate (Hrs), as Smo accumulation has been observed in cells mutating hrs [14,16]. The mechanisms by which the ciliary localization of vertebrate Smo is controlled remain unclear
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