title: Proteolysis of cubitus interruptus in Drosophila requires phosphorylation by protein kinase A.
abstract: The Hedgehog signal transduction pathway is involved in diverse patterning events in many organisms. In Drosophila, Hedgehog signaling regulates transcription of target genes by modifying the activity of the DNA-binding protein Cubitus interruptus (Ci). Hedgehog signaling inhibits proteolytic cleavage of full-length Ci (Ci-155) to Ci-75, a form that represses some target genes, and also converts the full-length form to a potent transcriptional activator. Reduction of protein kinase A (PKA) activity also leads to accumulation of full-length Ci and to ectopic expression of Hedgehog target genes, prompting the hypothesis that PKA might normally promote cleavage to Ci-75 by directly phosphorylating Ci-155. Here we show that a mutant form of Ci lacking five potential PKA phosphorylation sites (Ci5m) is not detectably cleaved to Ci-75 in Drosophila embryos. Moreover, changes in PKA activity dramatically altered levels of full-length wild-type Ci in embryos and imaginal discs, but did not significantly alter full-length Ci5m levels. We corroborate these results by showing that Ci5m is more active than wild-type Ci at inducing ectopic transcription of the Hh target gene wingless in embryos and that inhibition of PKA enhances induction of wingless by wild-type Ci but not by Ci5m. We therefore propose that PKA phosphorylation of Ci is required for the proteolysis of Ci-155 to Ci-75 in vivo. We also show that the activity of Ci5m remains Hedgehog responsive if expressed at low levels, providing further evidence that the full-length form of Ci undergoes a Hedgehog-dependent activation step.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Price
authors.forename: D
authors.initials: D
authors.lastname: Kalderon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila / Q000196:embryology / Q000378:metabolism*; D029721:Drosophila Proteins; D004625:Embryo, Nonmammalian / Q000378:metabolism; D053823:Hedgehog Proteins; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014158:Transcription, Genetic; D014921:Wings, Animal / Q000196:embryology
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D053823:Hedgehog Proteins; D019476:Insect Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C092937:ci protein, Drosophila; C077245:hh protein, Drosophila; D017868:Cyclic AMP-Dependent Protein Kinases