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Hedgehog-Induced Phosphorylation by CK1 Sustains the Activity of Ci/Gli

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Hedgehog-Induced Phosphorylation by CK1 Sustains the Activity of Ci/Gli Hedgehog-induced phosphorylation by CK1 sustains PNAS PLUS the activity of Ci/Gli activator Qing Shia, Shuang Lia, Shuangxi Lia,b, Alice Jianga, Yongbin Chenc,d, and Jin Jianga,e,1 Departments of aDevelopmental Biology and ePharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235; bInstitute of Biomedical Sciences, East China Normal University, Shanghai 200062, China; and cKey Laboratory of Animal Models and Human Disease Mechanisms and dYunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China Edited by Norbert Perrimon, Harvard Medical School, Howard Hughes Medical Institute, Boston, MA, and approved November 11, 2014 (received for review August 28, 2014) Hedgehog (Hh) signaling governs many developmental processes CiA/GliA by dissociating Ci/Gli from Cos2/Kif7 and Sufu (8, 21– by regulating the balance between the repressor (CiR/GliR) and 27). The Drosophila Ser/Thr kinase Fused (Fu) is required to activator (CiA/GliA) forms of Cubitus interruptus (Ci)/glioma- antagonize Cos2- and Sufu-mediated inhibition of Ci (28–30), associated oncogene homolog (Gli) transcription factors. Although but its mammalian counterpart remains to be identified. much is known about how CiR/GliR is controlled, the regulation of CiA is unstable and is degraded by the ubiquitin/proteasome A A Ci /Gli remains poorly understood. Here we demonstrate that pathway mediated by the MATH- and BTB-domain containing Casein kinase 1 (CK1) sustains Hh signaling downstream of Costal2 protein HIB (also called “Rdx”) (8, 31, 32). Interestingly, HIB is A and Suppressor of fused (Sufu) by protecting Ci from premature up-regulated in response to Hh in both embryos and imaginal degradation. We show that Hh stimulates Ci phosphorylation by discs (31, 32), and HIB also down-regulates Sufu through Crn CK1 at multiple Ser/Thr-rich degrons to inhibit its recognition by (33), thus forming feedback loops to fine-tune CiA activity. the Hh-induced MATH and BTB domain containing protein (HIB), However, it is not clear how HIB-mediated degradation of CiA is a substrate receptor for the Cullin 3 family of E3 ubiquitin ligases. kept in check to prevent premature loss of Hh signaling activity. In Hh-receiving cells, reduction of CK1 activity accelerated HIB-me- CK1 plays a dual role in both Drosophila and vertebrate Hh diated degradation of CiA, leading to premature loss of pathway signaling (11). In the absence of Hh, CK1 phosphorylates Ci/Gli activity. We also provide evidence that GliA is regulated by CK1 in after PKA-primed phosphorylation, which is essential for the a similar fashion and that CK1 acts downstream of Sufu to pro- R R – mote Sonic hedgehog signaling. Taken together, our study not production of Ci /Gli (34 38); however, in the presence of Hh, only reveals an unanticipated and conserved mechanism by which CK1 phosphorylates Smo and likely Fu, to activate the Hh – phosphorylation of Ci/Gli positively regulates Hh signaling but pathway (15, 30, 39 41). Here we uncover an unanticipated A also provides the first evidence, to our knowledge, that substrate positive role of CK1 in the regulation of Ci downstream of Smo recognition by the Cullin 3 family of E3 ubiquitin ligases is nega- and Fu. We show that reduction in CK1 activity leads to de- A tively regulated by a kinase. stabilization of Ci and diminished Hh pathway activity. Mecha- nistically, we provide biochemical evidence that CK1 phosphorylates Hedgehog | CK1 | Ci | Gli | SPOP multiple Ser/Thr-rich degrons in Ci to attenuate HIB recognition and thus reduce the rate of HIB-mediated CiA degradation. he evolutionarily conserved Hedgehog (Hh) signaling path- Blockage of the HIB-mediated degradation either by inactivating Tway governs embryogenesis and adult tissue homeostasis by HIB or by mutating the HIB degrons bypasses the requirement of A A tightly controlling the balance between the repressor (CiR/GliR) CK1 in the stabilization of Ci . Importantly, we show that Gli is and activator (CiA/GliA) forms of Cubitus interruptus (Ci)/Gli regulated by CK1 in a conserved manner and that CK1 positively transcription factors (1–5). In Drosophila wing discs, Hh secreted regulates Gli activity in Sufu mutant cells. from posterior (P) compartment cells moves into the anterior (A) compartment to form a local activity gradient near the A/P Significance boundary. Low, intermediate, and peak levels of Hh differen- R A tially regulate the Ci /Ci ratio to activate decapentaplegic (dpp), Hedgehog (Hh) signaling controls development and tissue ho- patched (ptc), and engrailed (en), respectively (6–8). In humans, R A meostasis through the Cubitus interruptus (Ci)/glioma-associated imbalance between Gli and Gli causes various birth defects oncogene homolog (Gli) transcription factors, and abnormal Gli and cancers (1, 9, 10). R R activity causes congenital diseases and cancers. Here we show BIOLOGY Generation of Ci /Gli occurs in the absence of Hh. The that Ci/Gli phosphorylation by Casein kinase 1 positively regu- DEVELOPMENTAL kinesin-like proteins Costal2 (Cos2)/Kinesin superfamily member lates Hh pathway activity, providing insights into the regulation 7 (Kif7) and the tumor suppressor Suppressor of fused (Sufu) form F F of Ci/Gli activity. By showing that phosphorylation protects the protein complexes with full-length Ci/Gli (Ci /Gli ) to prevent its Ci/Gli activator from premature degradation, our study not only nuclear localization and promote its phosphorylation by multiple sheds lights on how the production and degradation of Ci/Gli kinases, including Protein kinase A (PKA), Casein kinase 1 (CK1), activator are delicately balanced to achieve optimal pathway and Glycogen synthase kinase 3 (GSK3), which targets it for Su- activity but also provides the first evidence (to our knowledge) pernumerary limbs (Slimb)/β-Transducin repeat containing E3 β that protein degradation by the Cullin 3 family of E3 ubiquitin ubiquitin protein ligase ( TRCP)-mediated processing to generate ligases is negatively regulated by phosphorylation. truncated repressor forms (11). The production of CiA/GliA requires the binding of Hh ligand to the transmembrane receptor Author contributions: Q.S. and J.J. designed research; Q.S., Shuang Li, Shuangxi Li, A.J., Ptc, which alleviates the inhibition of the transmembrane signal and Y.C. performed research; Q.S., Shuang Li, Shuangxi Li, Y.C., and J.J. analyzed data; transducer Smoothened (Smo) by Ptc (1–3, 12, 13). Smo and Q.S. and J.J. wrote the paper. undergoes phosphorylation by multiple kinases that promote The authors declare no conflict of interest. its active conformation and cell surface (Drosophila)/primary This article is a PNAS Direct Submission. cilium (vertebrates) accumulation (11, 14–20). Smo-mediated 1To whom correspondence may be addressed. Email: [email protected]. intracellular signal transduction abrogates Ci/Gli processing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. R R into Ci /Gli and converts accumulated full-length Ci/Gli into 1073/pnas.1416652111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1416652111 PNAS | Published online December 15, 2014 | E5651–E5660 Downloaded by guest on October 1, 2021 Results regulating both Ci processing and Smo activation (34, 39, 42). CK1 and PKA Differentially Regulate Ci Levels in Hh-Receiving Cells. In Of note, CRL also affected Hh-independent en expression wild-type wing discs of late third-instar larvae, CiF is accumulated in posterior (P) compartment cells as observed previously in A-compartment cells near the A/P boundary because of the (Fig. 1D′) (39). inhibition of Ci processing by Hh secreted from P-compartment The down-regulation of Hh target genes at the A/P boundary cells (arrows in Fig. 1 A and B) (7); however, in A-compartment of MS > CRL wing discs could be attributed to compromised A cells immediately adjacent to the A/P boundary that receive high Smo/Fu activation and consequent failure of Ci production. levels of Hh, Ci staining is diminished because of the conversion of However, we noticed that Ci levels in A-compartment cells near CiF into labile CiA (arrowheads in Fig. 1 A and B)(8),asevi- the A/P boundary were much lower than in A-compartment cells denced by the expression of the high-threshold Hh target gene en away from the boundary (arrowheads in Fig. 1 C and D), a result in these cells (arrowhead in Fig. 1B′). that would not be expected if MS > CRL blocked both Ci pro- Inactivation of CK1 using a wing-specific Gal4 driver MS1096 cessing and CiF-to-CiA conversion. This finding is in sharp con- to express CRL (MS > CRL), a UAS-CK1-RNAi transgene that trast to the nearly uniform accumulation of Ci in A-compartment knocks down both CK1α and CK1e (35), ectopically stabilized cells of MS > R* wing discs (Fig. S1), a phenotype expected with CiF in A-compartment cells distant from the A/P boundary the blockage of both Ci processing and CiA production. Thus, the (arrows in Fig. 1 C and D), consistent with previous findings that mechanism(s) by which CRL diminishes Hh signaling activity phosphorylation of CiF by CK1 is required for its proteolytic may differ from the mechanism(s) by which PKA is inactivated. processing (35). MS > CRL also reduced ptc expression at the Of note, coexpressing either CK1α or CK1e blocked CRL-medi- A/P boundary and blocked Hh-dependent en expression in A- ated down-regulation of Ci in A-compartment cells near the A/P compartment cells (Fig. 1 C′ and D′), as is consistent with CK1 boundary and rescued ptc and en expression (Fig. S1), confirming having a positive role in Smo/Fu activation (30, 39–41). Similar that the effect of CRL on Ci level and activity results from the loss results were obtained when PKA was inactivated by expressing of CK1α/e activity.
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