Sequential Phosphorylation of Smoothened Transduces Graded Hedgehog Signaling

Abstract

The correct interpretation of a gradient of the morphogen Hedgehog (Hh) during development requires phosphorylation of the Hh signaling activator Smoothened (Smo); however, the molecular mechanism by which Smo transduces graded Hh signaling is not well understood. We show that regulation of the phosphorylation status of Smo by distinct phosphatases at specific phosphorylated residues creates differential thresholds of Hh signaling. Phosphorylation of Smo was initiated by adenosine 3′,5′-monophosphate (cAMP)–dependent protein kinase (PKA) and further enhanced by casein kinase I (CKI). We found that protein phosphatase 1 (PP1) directly dephosphorylated PKA-phosphorylated Smo to reduce signaling mediated by intermediate concentrations of Hh, whereas PP2A specifically dephosphorylated PKA-primed, CKI-phosphorylated Smo to restrict signaling by high concentrations of Hh. We also established a functional link between sequentially phosphorylated Smo species and graded Hh activity. Thus, we propose a sequential phosphorylation model in which precise interpretation of morphogen concentration can be achieved upon versatile phosphatase-mediated regulation of the phosphorylation status of an essential activator in developmental signaling.

Hedgehog signalling

“…When activated, Smo accumulates at the plasma membrane, where it becomes hyperphosphorylated through the coordinated activities of Protein kinase A (PKA), Casein kinase 1 (CK1), Glycogen synthase kinase 3 (GSK3; Shaggy -FlyBase) and G-protein coupled receptor kinase 2 (Gprk2) (Su et al, 2011). Phosphorylation occurs on multiple sites in the Smo intracellular C-terminal tail, leading to a conformational change that recruits the kinesin family protein Costal-2 (Cos2; Costa -FlyBase) and promotes its dimerisation (Shi et al, 2011).…”

Genome-wide identification of phospho-regulators of Wnt signaling inDrosophila

“…The following Drosophila strains were used: w 1118 (wild type), dpp-Gal4/ TM6B, C5-Gal4, MS1096-Gal4, omb-Gal4, 71B-Gal4, UAS-flp, UAS-dicer-2, UAS-p35, dpp-lacZ/CyO, Cdc37 eD4 FRT79/TM6B, FRT82,GFP/TM6B, MARCM79, hs-flp;;FRT82,GFP/TM6B and gish EY15206 (Bloomington Drosophila Stock Center); hh-Gal4/TM6B and eyFlp;ey-Gal4,GMR-Gal4; sev>y+>wg (Port et al, 2011); UAS-hhN (Su et al, 2011); and FRT82 gish e01759 /TM6B (Gault et al, 2012). The transgenic RNAi strains used for the screens were obtained from the Vienna Drosophila RNAi Center (Dietzl et al, 2007), National Institute of Genetics and Harvard Transgenic RNAi Project (supplementary material Fig.…”

Ubpy controls the stability of the ESCRT-0 subunit Hrs in development

“…ap-Gal4 (Du et al, 2011), MS1096-Gal4 (Zhang et al, 2012), ptc-lacZ, dpplacZ (Su et al, 2011), dor 8 (gift of Helmut Krämer) (Sevrioukov et al, 1999), hrs D28 (gift of Hugo Bellen) (Lloyd et al, 2002), Ubpy KO (gift of Satoshi Goto) (Mukai et al, 2010), vps2 PP6 and vps22 ZZ13 (gift of David Bilder) (Vaccari et al, 2009) alleles were described previously. Transgenic RNAi flies targeting different regions of Ubpy gene were obtained from Dr Satoshi Goto (Mitsubishi-Kagaku Institute of Life Sciences, Machida, Japan) (5798R-1 and 5798R-2) and the Vienna Drosophila RNAi Center (VDRC #107623).…”

“…A DNA template targeting Ubpy (encoding amino acids 333-435) was generated by PCR and used for dsRNA synthesis. dsRNA targeting yeast gal80 coding sequence was used as a negative control (Su et al, 2011). For RNAi knockdown, S2 cells were cultured in full medium containing 40 nM indicated dsRNA for 4 days.…”