ABSTRACT
Smoothened (SMO) is a G-protein-coupled receptor-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in Drosophila, we identified new SMO phosphosites that enhance the effects of the PKA/CKI kinases on SMO accumulation, its localization at the plasma membrane and its activity. Surprisingly, phosphorylation at these sites is induced by the kinase Fused (FU), a known downstream effector of SMO. In turn, activation of SMO induces FU to act on its downstream targets. Overall, our data provide evidence for a SMO/FU positive regulatory loop nested within a multikinase phosphorylation cascade. We propose that this complex interplay amplifies signaling above a threshold that allows high HH signaling.
Subject(s)
Casein Kinase I/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster , Hedgehog Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Smoothened Receptor/metabolism , Animals , Animals, Genetically Modified , Casein Kinase I/genetics , Cell Membrane/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Hedgehog Proteins/genetics , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Recombinant Fusion Proteins/genetics , Signal Transduction , Smoothened Receptor/genetics , Wings, Animal/embryology , Wings, Animal/metabolismABSTRACT
Microtubules (MTs) are essential for cell division, shape, intracellular transport, and polarity. MT stability is regulated by many factors, including MT-associated proteins and proteins controlling the amount of free tubulin heterodimers available for polymerization. Tubulin-binding cofactors are potential key regulators of free tubulin concentration, since they are required for α-ß-tubulin dimerization in vitro. In this paper, we show that mutation of the Drosophila tubulin-binding cofactor B (dTBCB) affects the levels of both α- and ß-tubulins and dramatically destabilizes the MT network in different fly tissues. However, we find that dTBCB is dispensable for the early MT-dependent steps of oogenesis, including cell division, and that dTBCB is not required for mitosis in several tissues. In striking contrast, the absence of dTBCB during later stages of oogenesis causes major defects in cell polarity. We show that dTBCB is required for the polarized localization of the axis-determining mRNAs within the oocyte and for the apico-basal polarity of the surrounding follicle cells. These results establish a developmental function for the dTBCB gene that is essential for viability and MT-dependent cell polarity, but not cell division.
