The Hedgehog pathway controls development and tissue homeostasis. Ligands of the Hedgehog family bind to the receptor Patched, which alleviates repression of the transmembrane protein Smoothened (Smo), enabling downstream signaling. Smo activity is also regulated by localization and by posttranslational modifications, such as phosphorylation, which enhances Hedgehog signaling, and ubiquitylation, which limits Hedgehog signaling (see Qi et al.). Ma et al. identified proteins involved in the addition and removal of the protein SUMO in a screen for Hedgehog pathway regulators in Drosophila. Knockdown of the enzymes involved in SUMOylation (Ubc9 or dPIAS) further impaired Hedgehog pathway activity in fly wings and imaginal discs (the embryonic tissue that produces several adult organs, including the wings) of flies with a dominant-negative mutant Smo, whereas knocking down the enzyme that removes SUMO (Ulp1) restored Hedgehog signaling in these Smo-mutant flies. Based on changes in the intensity of antibody staining of Smo in imaginal discs, impairing SUMOylation by knocking down Ubc9 or dPIAS reduced the abundance of Smo, and transcript analysis showed that this was not through a change in mRNA abundance. The C-terminal tail of Smo contains a consensus SUMOylation site, and a mutation in this site (K851R) reduced the abundance of tagged Smo expressed in cultured cells and prevented any stabilizing effect of overexpression of SUMO-conjugating enzymes or an increase in Smo abundance in response to the addition of Hedgehog (in conditioned medium). Directly conjugating SUMO to the C terminus of either wild-type tagged Smo or the K851R mutant stabilized Smo expressed in cultured cells, and both forms rescued Hedgehog signaling in Smo-deficient imaginal discs, whereas without this C-terminal SUMO, the K851R mutant did not rescue. Coimmunoprecipitation experiments with tagged proteins expressed in cultured fly cells indicated that Hedgehog stimulation reduced the amount of the SUMO-removing enzyme Ulp1 that coimmunoprecipitated with Smo, and this reduction occurred with phosphorylation-deficient or phosphorylation-mimetic mutants of Smo, indicating that the SUMO-mediated regulation and phosphorylation-mediated regulation of Smo were independent. Ubiquitylation of Smo was increased in cells in which Ubc9 or dPIAS were knocked down, as well as in cells expressing the K851R mutant, suggesting that SUMOylation reduced Smo ubiquitylation, a modification that promotes internalization and degradation. Hedgehog reduces Smo ubiquitylation through a process dependent on the deubiquitylating enzyme UBPY, which contains three putative SUMO-interacting motifs (SIMs). Consistent with SUMO modification of Smo recruiting this enzyme, the K851R mutant appeared to coimmunoprecipitate less UBPY, whereas wild-type Smo with SUMO conjugated directly to the C terminus appeared to coimmunoprecipitate more UBPY, and the interaction was reduced in cells expressing UBPY with mutated SIMs. Analysis of Smo in mouse NIH3T3 cells indicated that Sonic hedgehog (Shh) stimulated the attachment of SUMO2 to Smo and that overexpression of the deSUMOylating enzyme SENP1, but not a catalytically inactive form, reduced both basal and Shh-stimulated SUMOylation of Smo. Overexpression of SENP1 reduced the activation of a Hedgehog pathway reporter, and expression of mouse Smo conjugated to SUMO on the C terminus enhanced basal and Shh-stimulated reporter activity. Furthermore, overexpressing a membrane-targeted SENP1 reduced the Shh-stimulated accumulation of Smo in the primary cilia, whereas the C-terminally SUMO-conjugated form of Smo accumulated in the primary cilium of more cells even under basal conditions, and Shh-mediated stimulation of its accumulation in the cilia was not affected by overexpression of SENP1. Thus, similar to phosphorylation, SUMOylation of Smo enhances Hedgehog signaling by stabilizing Smo. The mechanism of SUMO-mediated stabilization appears to be through the recruitment of a SIM-containing deubiquitylase.
G. Ma, S. Li, Y. Han, S. Li. T. Yue, B. Wang, J. Jiang, Regulation of Smoothened trafficking and Hedgehog signaling by the SUMO pathway. Dev. Cell 39, 438–451 (2016). [PubMed]
Y. Qi, H. Liu, X. Lin, Sumoylation stabilizes Smoothened to promote Hedgehog signaling. Dev. Cell 39, 385–387 (2016). [PubMed]