Smoothened Phosphorylation Promotes Hedgehog Signaling

Two research groups, Jia et al. and Zhang et al. , have implicated phosphorylation by protein kinase A (PKA) and casein kinase 1 (CK1) of the transmembrane protein Smoothened (which transduces developmental signals from the secreted Hedgehog proteins) as key to its regulation and to Hedgehog pathway activation. In Drosophila , Hedgehog binding to the Patched receptor relieves the inhibition by Patched of Smoothened, which leads to accumulation of activated Smoothened at the cell membrane and initiates a signaling cascade. Although PKA has an inhibitory effect on Hedgehog signaling downstream of Smoothened, Jia et al. found that moderate expression of constitutively activated PKA in Drosophila wing disc enhanced Smoothened accumulation and Hedgehog signaling, whereas blockade of PKA signaling inhibited them. Mutational analysis indicated that PKA phosphorylates Smoothened at several sites in its C terminus in vitro, which enables phosphorylation by CK1. Hedgehog stimulated Smoothened phosphorylation by PKA and CK1, and mutational analysis indicated that phosphorylation by these two kinases was necessary for Smoothened accumulation at the cell surface and Hedgehog signaling. Indeed, the degree of Smoothened phosphorylation correlated with its accumulation and activity. Zhang et al. immunoprecipitated Smoothened from Drosophila S2 cells treated with Hedgehog and used mass spectrometry to identify 26 C-terminally phosphorylated serines or threonines, including residues within PKA and CK1 consensus sequences. Analysis of Smoothened mutants expressed in cells in which endogenous Smoothened was inhibited with RNA interference indicated that collective and sequential phosphorylation of multiple residues contributed to Smoothened activity. Expression of mutant forms of PKA and CK1 substantiated their role in Smoothened phosphorylation. Zhang et al. found that Smoothened phosphorylation enhanced its stability and its activity but, unlike Jia et al. , did not find that it clearly influenced the fraction of total Smoothened on the cell surface. Nonetheless, both groups concluded that multiple phosphorylation of Smoothened by PKA and CK1 was critical to Hedgehog signaling. J. Jia, C. Tong, B. Wang, L. Luo, J. Jiang, Hedgehog signalling activity of Smoothened requires phosphorylation by protein kinase A and casein kinase I. Nature 432 , 1045-1050 (2004). [PubMed] C. Zhang, E. H. Williams, Y. Guo, L. Lum, P. A. Beachy, Extensive phosphorylation of Smoothened in Hedgehog pathway activation. Proc. Natl. Acad. Sci. U.S.A. 101 , 17900-17907 (2004). [Abstract] [Full Text]