Reversible phosphorylation of the signal transduction complex in Drosophila photoreceptors

In the Drosophila visual cascade, the transient receptor potential (TRP) calcium channel, phospholipase Cbeta (no-receptor-potential A), and an eye-specific isoform of protein kinase C (eye-PKC) comprise a multimolecular signaling complex via their interaction with the scaffold protein INAD. Previously, we showed that the interaction between INAD and eye-PKC is a prerequisite for deactivation of a light response, suggesting eye-PKC phosphorylates proteins in the complex. To identify substrates of eye-PKC, we immunoprecipitated the complex from head lysates using anti-INAD antibodies and performed in vitro kinase assays. Wild-type immunocomplexes incubated with [(32)P]ATP revealed phosphorylation of TRP and INAD. In contrast, immunocomplexes from inaC mutants missing eye-PKC, displayed no phosphorylation of TRP or INAD. We also investigated protein phosphatases that may be involved in the dephosphorylation of proteins in the complex. Dephosphorylation of TRP and INAD was partially suppressed by the protein phosphatase inhibitors okadaic acid, microcystin, and protein phosphatase inhibitor-2. These phosphatase activities were enriched in the cytosol of wild-type heads, but drastically reduced in extracts prepared from glass mutants, which lack photoreceptors. Our findings indicate that INAD functions as RACK (receptor for activated PKC), allowing eye-PKC to phosphorylate INAD and TRP. Furthermore, dephosphorylation of INAD and TRP is catalyzed by PP1/PP2A-like enzymes preferentially expressed in photoreceptor cells.     

An essential role for Grk2 in Hedgehog signalling downstream of Smoothened

The G‐protein‐coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock‐down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP‐dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase‐dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C‐terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho‐null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.     

Fused-dependent Hedgehog signal transduction is required for somatic cell differentiation during Drosophila egg chamber formation

The fused gene encodes a serine/threonine kinase involved in Hedgehog signal transduction during Drosophila embryo and larval imaginal disc development. Additionally, fused mutant females exhibit reduced fecundity that we report here to be associated with defects in three aspects of egg chamber formation: encapsulation of germline cysts by prefollicular cells in the germarium, interfollicular stalk morphogenesis and oocyte posterior positioning. Using clonal analysis we show that fused is required cell autonomously in prefollicular and pre-stalk cells to control their participation in these aspects of egg chamber formation. In contrast to what has been found for Hedgehog and other known components of Hedgehog signal transduction, we show that fused does not play a role in the regulation of somatic stem cell proliferation. However, genetic interaction studies, as well as the analysis of the effects of a partial reduction in Hedgehog signaling in the ovary, indicate that fused acts in the classical genetic pathway for Hedgehog signal transduction which is necessary for somatic cell differentiation during egg chamber formation. Therefore, we propose a model in which Hedgehog signals at least twice in germarial somatic cells: first, through a fused-independent pathway to control somatic stem cell proliferation; and second, through a classical fused-dependent pathway to regulate prefollicular cell differentiation.     

Hedgehog signal transduction: recent findings

The Hedgehog (Hh) family of signaling molecules are key agents in patterning numerous types of tissues. Mutations in Hh and its downstream signaling molecules are also associated with numerous oncogenic and disease states. Consequently, understanding the mechanisms by which Hh signals are transduced is important for understanding both development and disease. Recent studies have clarified several aspects of Hh signal transduction. Several new Sonic Hedgehog binding partners have been identified. Cholesterol and palmitic acid modifications of Hh and Sonic hedgehog have been examined in greater detail. Characterization of the trafficking patterns of the Patched and Smoothened proteins has demonstrated that these two proteins function very differently from the previously established models. The Fused kinase has been demonstrated to phosphorylate the kinesin-like protein Costal2 and the sites identified, while Cubitus interruptus has been shown to be phosphorylated in a hierarchical manner by three different kinases. Finally, the interactions, both genetic and physical, between Fused, Costal2, Cubitus interruptus, and Suppressor of Fused have been further elucidated.     

Hedgehog secretion and signal transduction in vertebrates

Signaling by the Hedgehog (Hh) family of secreted proteins is essential for proper embryonic patterning and development. Dysregulation of Hh signaling is associated with a variety of human diseases ranging from developmental disorders such as holoprosencephaly to certain forms of cancer, including medulloblastoma and basal cell carcinoma. Genetic studies in flies and mice have shaped our understanding of Hh signaling and revealed that nearly all core components of the pathway are highly conserved. Although many aspects of the Drosophila Hh pathway are conserved in vertebrates, mechanistic differences between the two species have begun to emerge. Perhaps the most striking divergence in vertebrate Hh signaling is its dependence on the primary cilium, a vestigial organelle that is largely absent in flies. This minireview will provide an overview of Hh signaling and present recent insights into vertebrate Hh secretion, receptor binding, and signal transduction.     

Hrs Promotes Ubiquitination and Mediates Endosomal Trafficking of Smoothened in Drosophila Hedgehog Signaling

In Hedgehog (Hh) signaling, the seven-transmembrane protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation, ubiquitination, and cell surface accumulation. However, it is not clear how Smo cell surface accumulation and intracellular trafficking are regulated. Here, we demonstrate that inactivation of Hrs by deletion or RNAi accumulates Smo in the late endosome that is marked by late endosome markers. Inactivation of Hrs enhances the wing defects caused by dominant-negative Smo. We show that Hrs promotes Smo ubiquitination, deleting the ubiquitin-interacting-motif (UIM) in Hrs abolishes the ability of Hrs to regulate Smo ubiquitination. However, the UIM domain neither recognizes the ubiquitinated Smo nor directly interacts with Smo. Hrs lacking UIM domain still downregulates Smo activity even though to a less extent. We have characterized that the N-terminus of Hrs directly interacts with the PKA/CK1 phosphorylation clusters to prevent Smo phosphorylation and activation, indicating an ubiquitin-independent regulation of Smo by Hrs. Finally, we found that knockdown of Tsg101 accumulates Smo that is co-localized with Hrs and other late endosome markers. Taken together, our data indicate that Hrs mediates Smo trafficking in the late endosome by not only promoting Smo ubiquitination but also blocking Smo phosphorylation.     

Scube activity is necessary for Hedgehog signal transduction in vivo

The Hedgehog (HH) signaling pathway is a central regulator of embryonic development, controlling the pattern and proliferation of a wide variety of organs. Previous studies have implicated the secreted protein, Scube2, in HH signal transduction in the zebrafish embryo (Hollway et al., 2006; Kawakami et al., 2005; Woods and Talbot, 2005) although the nature of the molecular function of Scube2 in this process has remained undefined. This analysis has been compounded by the fact that removal of Scube2 activity in the zebrafish embryo leads to only subtle defects in HH signal transduction in vivo (Barresi et al., 2000; Hollway et al., 2006; Ochi and Westerfield, 2007; van Eeden et al., 1996; Wolff et al., 2003). Here we present the discovery of two additional scube genes in zebrafish, scube1 and scube3, and demonstrate their roles in facilitating HH signal transduction. Knocking down the function of all three scube genes simultaneously phenocopies a complete loss of HH signal transduction in the embryo, revealing that Scube signaling is essential for HH signal transduction in vivo. We further define the molecular role of scube2 in HH signaling.     

Hedgehog signal transduction: from flies to vertebrates

The patterning and morphogenesis of multicellular organisms require a complex interplay of inductive signals which control proliferation, growth arrest, and differentiation of different cell types. A number of such signaling molecules have been identified in vertebrates and invertebrates. The molecular dissection of these pathways demonstrated that in vertebrates, mutations or abnormals function of these signaling pathways were often associated with developmental disorders and cancer formation. The Hedgehog (Hh) family of secreted proteins provides a perfect example of such signaling proteins. In the following review, we will not discuss in detail the role of Hh as a morphogen, but rather focus on its signal transduction pathway and its role in various human disorders.