The mechanism of hedgehog signal transduction.

Hedgehog (Hh) proteins are one of a small number of families of secreted signalling proteins that are responsible for cell interactions during development in many animals. As such, Hh signals produce many different responses at different times and in different cells. As for other multifunctional ligands, this requires regulated patterns of expression, special mechanisms for ligand movement between cells and ligand destruction, and mechanisms for integrating a generic signalling state (on or off) with the status of responding cells in order to produce an appropriate cell-specific response. Here I discuss what is known about the biochemical mechanisms by which an Hh signal is transduced in order to change the patterns of gene transcription.

Hedgehog acts as a somatic stem cell factor in the Drosophila ovary.

Secreted signalling molecules of the Hedgehog (Hh) family have many essential patterning roles during development of diverse organisms including Drosophila and humans. Although Hedgehog proteins most commonly affect cell fate, they can also stimulate cell proliferation. In humans several distinctive cancers, including basal-cell carcinoma, result from mutations that aberrantly activate Hh signal transduction. In Drosophila, Hh directly stimulates proliferation of ovarian somatic cells. Here we show that Hh acts specifically on stem cells in the Drosophila ovary. These cells cannot proliferate as stem cells in the absence of Hh signalling, whereas excessive Hh signalling produces supernumerary stem cells. We deduce that Hh is a stem-cell factor and suggest that human cancers due to excessive Hh signalling might result from aberrant expansion of stem cell pools.

Proteolysis of cubitus interruptus in Drosophila requires phosphorylation by protein kinase A.

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.

Generation of a novel A kinase anchor protein and a myristoylated alanine-rich C kinase substrate-like analog from a single gene.

A unique Drosophila gene encodes two novel signaling proteins. Drosophila A kinase anchor protein 200 (DAKAP200) (753 amino acids) binds regulatory subunits of protein kinase AII (PKAII) isoforms in vitro and in intact cells. The acidic DAKAP200 polypeptide (pI approximately 3.8) contains an optimal N-terminal myristoylation site and a positively charged domain that resembles the multifunctional phosphorylation site domain of vertebrate myristoylated alanine-rich C kinase substrate proteins. The 15-kilobase pair DAKAP200 gene contains six exons and encodes a second protein, DeltaDAKAP200. DeltaDAKAP200 is derived from DAKAP200 transcripts by excision of exon 5 (381 codons), which encodes the PKAII binding region and a Pro-rich sequence. DeltaDAKAP200 appears to be a myristoylated alanine-rich C kinase substrate analog. DAKAP200 and DeltaDAKAP200 are evident in vivo at all stages of Drosophila development. Thus, both proteins may play important physiological roles throughout the life span of the organism. Nevertheless, DAKAP200 gene expression is regulated. Maximal levels of DAKAP200 are detected in the pupal phase of development; DeltaDAKAP200 content is elevated 7-fold in adult head (brain) relative to other body parts. Enhancement or suppression of exon 5 excision during DAKAP200 pre-mRNA processing provides potential mechanisms for regulating anchoring of PKAII and targeting of cAMP signals to effector sites in cytoskeleton and/or organelles.

Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator.

In Drosophila, signalling by the protein Hedgehog (Hh) alters the activity of the transcription factor Cubitus interruptus (Ci) by inhibiting the proteolysis of full-length Ci (Ci-155) to its shortened Ci-75 form. Ci-75 is found largely in the nucleus and is thought to be a transcriptional repressor, whereas there is evidence to indicate that Ci-155 may be a transcriptional activator. However, Ci-155 is detected only in the cytoplasm, where it is associated with the protein kinase Fused (Fu), with Suppressor of Fused (Su(fu)), and with the microtubule-binding protein Costal-2. It is not clear how Ci-155 might become a nuclear activator. We show here that mutations in Su(fu) cause an increase in the expression of Hh-target genes in a dose-dependent manner while simultaneously reducing Ci-155 concentration by some mechanism other than proteolysis to Ci-75. Conversely, eliminating Fu kinase activity reduces Hh-target gene expression while increasing Ci-155 concentration. We propose that Fu kinase activity is required for Hh to stimulate the maturation of Ci-155 into a short-lived nuclear transcriptional activator and that Su(fu) opposes this maturation step through a stoichiometric interaction with Ci-155.

Tripartite mushroom body architecture revealed by antigenic markers.

We have explored the organization of the axonal lobes in Drosophila mushroom bodies by using a panel of immunohistochemical markers. These markers consist of antibodies to eight proteins expressed preferentially in the mushroom bodies: DAMB, DCO, DRK, FASII, LEO, OAMB, PKA RII, and RUT. Previous to this work, four axonal lobes, two projecting dorsally (alpha and alpha') and two medially (beta and gamma), had been described in Drosophila mushroom bodies. However, our analysis of immunohistochemically stained frontal and sagittal sections of the brain revealed three medially projecting lobes. The newly distinguished lobe, which we term beta', lies along the dorsal surface of beta, just posterior to gamma. In addition to resolving a fifth lobe, our studies revealed that there are specific lobe sets defined by equivalent marker expression levels. These sets are (1) the alpha and beta lobes, (2) the alpha' and beta' lobes, and (3) the gamma lobe and heel (a lateral projection formed by a hairpin turn of some of the peduncle fibers). All of the markers we have examined are consistent with these three sets. Previous Golgi studies demonstrate that each mushroom body cell projects one axon that branches into a dorsal lobe and a medial lobe, or one unbranched axon that projects medially. Taken together with the lobe sets listed above, we propose that there are three major projection configurations of mushroom body cell axons: (1) one branch in the alpha and one in the beta lobe, (2) one branch in the alpha' and one in the beta' lobe, and (3) one unbranched axon projecting to the heel and the gamma lobe. The fact that these neuron types exhibit differential expression levels of a number of mushroom body genes suggests that they may have corresponding functional differences. These functions may be conserved in the larvae, as several of these genes were expressed in larval and embryonic mushroom bodies as well. The basic mushroom body structure, including the denritic calyx, peduncle, and lobes, was already visible by the late stages of embryogenesis. With new insights into mushroom body organization, and the characterization of markers for developing mushroom bodies, we are beginning to understand how these structures form and function.

Drosophila melanogaster deficient in protein kinase A manifests behavior-specific arrhythmia but normal clock function.

Drosophila melanogaster bearing mutations in the DCO gene, which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), displays arrhythmic locomotor activity strongly suggesting a role for PKA in the circadian timing system. This arrhythmicity might result from a requirement for PKA activity in photic resetting pathways, the timekeeping mechanism itself, or downstream effector pathways controlling overt behavioral rhythms. To address these possibilities, we examined the protein and mRNA products from the clock gene period (per) in PKA-deficient flies. The per protein (PER) and mRNA products undergo daily cycles in the heads and bodies of DCO mutants that are indistinguishable from those observed in control wild-type flies. These results indicate that PKA deficiencies affect the proper functioning of elements downstream of the Drosophila timekeeping mechanism. The requirement for PKA in the manifestation of rhythmic activity was preferentially greater in the absence of environmental cycles. However, PKA does not appear to play a universal role in output functions because the clock-controlled eclosion rhythm is normal in DCO mutants. Our results suggest that PKA plays a critical role in the flow of temporal information from circadian pacemaker cells to selective behaviors.

Dual pathways for induction of wingless expression by protein kinase A and Hedgehog in Drosophila embryos.

The secreted Drosophila Hedgehog (Hh) protein induces transcription of specific genes by an unknown mechanism that requires the serpentine transmembrane protein Smoothened (Smo) and the transcription factor Cubitus interruptus (Ci). Protein kinase A (PKA) has been implicated in the mechanism of Hh signal transduction because it acts to repress Hh target genes in imaginal disc cells that express Ci. Changes in Ci protein levels, detected by an antibody that recognizes an epitope in the carboxy-terminal half of Ci, have been suggested to mediate the positive effects of Hh and the negative effects of PKA on Hh target gene expression in imaginal discs. Here we show that PKA inhibition, like Hh, leads to increased "carboxy-terminal" Ci staining and Hh target gene expression in embryos. In addition, we find that Hh and Smo can stimulate target gene expression at constant Ci levels and that increased PKA activity can induce ectopic Hh target gene expression in a manner that requires Smo and Ci activities but does not involve changes in Ci protein concentration. This suggests a branching pathway of Hh signal transduction downstream of Smo and that PKA exerts opposite effects on the two branches. Finally we show that Hh signaling in embryos does not depend on cAMP-dependent regulation of PKA activity.

Hedgehog signalling: Ci complex cuts and clasps.

Hedgehog (Hh) signalling in Drosophila inhibits partial proteolysis of the transcription factor Cubitus interruptus (Ci), and the ability of a complex containing Ci and the kinesin-related protein Costal-2 to bind microtubules. These changes are proposed to turn Ci from a repressor to an activator of Hh-target genes.

Activity, expression and function of a second Drosophila protein kinase A catalytic subunit gene.

The DC2 gene was isolated previously on the basis of sequence similarity to DC0, the major Drosophila protein kinase A (PKA) catalytic subunit gene. We show here that the 67-kD Drosophila DC2 protein behaves as a PKA catalytic subunit in vitro. DC2 is transcribed in mesodermal anlagen of early embryos. This expression depends on dorsal but on neither twist nor snail activity. DC2 transcriptional fusions mimic this embryonic expression and are also expressed in subsets of cells in the optic lamina, wing disc and leg discs of third instar larvae. A saturation screen of a small deficiency interval containing DC2 for recessive lethal mutations yielded no DC2 alleles. We therefore isolated new deficiencies to generate deficiency trans-heterozygotes that lacked DC2 activity. These animals were viable and fertile. The absence of DC2 did not affect the viability or phenotype of imaginal disc cells lacking DC0 activity or embryonic hatching of animals with reduced DC0 activity. Furthermore, transgenes expressing DC2 from a DC0 promoter did not efficiently rescue a variety of DC0 mutant phenotypes. These observations indicate that DC2 is not an essential gene and is unlikely to be functionally redundant with DC0, which has multiple unique functions during development.

Morphogenetic signalling. Responses to hedgehog.

Protein kinase A activity is required for signalling by the extracellular molecule Hedgehog in developing Drosophila imaginal discs, but does the kinase actually respond to the Hedgehog signal?

Localization and functions of protein kinase A during Drosophila oogenesis.

We have characterized the requirements for the Protein Kinase A (PKA) catalytic subunit, DC0, in Drosophila oogenesis. Intercellular bridges in egg chambers from PKA deficient females are unstable, leading to the formation of multinucleate nurse cells by fusions of adjacent cells. Germline clones of cells homozygous for null mutations of DC0 indicate that PKA acts autonomously in the germline. Highest levels of PKA catalytic subunit protein are associated with germ cell membranes, suggesting that targets of PKA are associated with the membrane or membrane skeleton and contribute to the stabilization of intercellular bridges. The migration of a subset of follicle cells, the border cells, is also disrupted by germline PKA mutations, implying that nurse cell junctions provide an essential path for border cell migrations.

Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development.

Reduced protein kinase A (PKA) activity in anterior imaginal disc cells leads to cell-autonomous induction of decapentaplegic (dpp), wingless (wg), and patched (ptc) transcription that is independent of hedgehog (hh) gene activity. The resulting nonautonomous adult wing and leg pattern duplications are largely due to induced dpp and wg expression and resemble phenotypes elicited by ectopic hh expression. Inhibition of PKA in anterior cells close to the posterior compartment can substitute for hh activity to promote growth of imaginal discs, whereas overexpression of PKA can counteract transcriptional induction of ptc by hh in these cells. PKA therefore appears to be an integral component of the mechanism by which hh regulates the expression of key patterning molecules in imaginal discs.

RNA localization along the anteroposterior axis of the Drosophila oocyte requires PKA-mediated signal transduction to direct normal microtubule organization.

Microtubule polarity has been implicated as the basis for polarized localization of morphogenetic determinants that specify the anteroposterior axis in Drosophila oocytes. We describe mutation affecting Protein Kinase A (PKA) that act in the germ line to disrupt both microtubule distribution and RNA localization along this axis. In normal oocytes, the site of microtubule nucleation shifts from posterior to anterior immediately prior to polarized localization of bicoid and oskar RNAs. In PKA-deficient oocytes, posterior microtubules are present during this transition, oskar RNA fails to accumulate at the posterior, and bicoid RNA accumulates at both ends of the oocyte. Similar RNA mislocalization patterns previously reported for Notch and Delta mutants suggest that PKA transduces a signal for microtubule reorganization that is sent by posteriorly located follicle cells.

Altered circadian pacemaker functions and cyclic AMP rhythms in the Drosophila learning mutant dunce.

Neural circadian pacemakers can be reset by light, and the resetting mechanism may involve cyclic nucleotide second messengers. We have examined pacemaker resetting and free-running activity rhythms in Drosophila dunce (dnc) and DC0 mutants, which identify a cAMP specific phosphodiesterase and the catalytic subunit of cAMP-dependent protein kinase, respectively. dnc mutants exhibit augmented light-induced phase delays and shortened circadian periods, which indicate altered pacemaker function. Interestingly, however, light-induced phase advances are normal in dnc, suggesting a selective effect on one component of the pacemaker resetting response. Furthermore, we demonstrate the presence of circadian rhythms in cAMP content in head tissues and show that dnc mutations increase the amplitude of daily cAMP peaks. These results show that cAMP levels are not chronically elevated in the dnc mutant. A role for cAMP signaling in circadian processes is also suggested by an analysis of DC0 mutants, which have severe kinase deficits and display arrhythmic locomotor activity.

Genetic investigation of cAMP-dependent protein kinase function in Drosophila development.

The cAMP-dependent protein kinase (PKA) has been shown to mediate the vast majority of cellular responses to the intracellular second messenger, cAMP, in eukaryotes. To study the role of cAMP signal transduction in Drosophila development, we have isolated and molecularly characterized mutations of varying severity in the Drosophila PKA gene, DC0. Biochemical measurements indicate that DC0 is either the sole or the major PKA catalytic subunit gene in Drosophila. Adult females heterozygous for a strong and a weak DC0 allele fail to lay eggs and show a striking and novel defect in oogenesis that includes the formation of egg chambers containing multinucleate nurse cells. Females heterozygous for two weak DC0 alleles are fertile but produce offspring showing a variety of defects in embryogenesis, including preblastoderm arrest and alterations in cuticular patterning. Animals zygotically null for DC0 die as morphologically normal first-instar larvae, implying that maternally encoded protein, which perdures for at least 12 hr, suffices for embryogenesis. Animals hemizygous for weak DC0 alleles survive for several days as larvae but grow slowly. Mitotic recombination experiments in the adult eye indicate that the DC0 gene is not required autonomously either for cell viability or normal growth rates. These results argue that cAMP-mediated signal transduction is essential at a variety of stages during the development of a metazoan.

Biochemical characterization of phosphorylation site mutants of simian virus 40 large T antigen: evidence for interaction between amino- and carboxy-terminal domains.

The simian virus 40 large T antigen is phosphorylated at eight or more sites that are clustered in an amino-terminal region and a carboxy-terminal region of the protein. Mutants carrying exchanges at these phosphorylation sites have been generated in vitro by bisulfite or oligonucleotide-directed mutagenesis and analyzed for their phosphorylation patterns. Two-dimensional phosphopeptide analyses of the mutant large T antigens confirmed most of the previously identified phosphorylation sites, namely, serine residues 106, 112, 123, 639, 677, and 679 and threonine residues 124 and 701. In addition, serine residue 120 was identified as a new site, whereas serines residues 111 and 676 were excluded. Interestingly, several of the mutants exhibited secondary effects in that a mutation in the amino-terminal region affected phosphorylation at distant and even carboxy-terminal sites and vice versa. Thus, the amino- and carboxy-terminal domains appear to be in close proximity in the three-dimensional structure of large T antigen. The possible consequences of the above findings and the role of phosphorylation are discussed.

Dissociation of Rb-binding and anchorage-independent growth from immortalization and tumorigenicity using SV40 mutants producing N-terminally truncated large T antigens.

The large T antigen of SV40 is both necessary and sufficient for conversion of primary mouse cells to cells with fully transformed phenotype. In this investigation, the influence of the N-terminal portion of T antigen on individual transformed cell characteristics was probed by using mutants bearing deletions in the 5'T antigen coding sequence. Specifically, DNA constructs expected to produce T antigens missing the first 109, 127, 150, or 176 amino acids or internal amino acid segments between 117 and 250 were tested for the ability to immortalize C57Bl/6 mouse embryo fibroblasts. The transformed cell properties displayed by clonally derived cell lines were then examined. The results indicated that neither the first 127 amino acids nor amino acids 127-250 of T antigen were necessary for efficient immortalization of primary cells or for their tumorigenicity. Functions mapped within these regions, including binding of the retinoblastoma susceptibility gene product (Rb) and transactivation of heterologous promoters, therefore, were not required to confer either of these growth properties. In addition the results showed that anchorage-independent growth was separable genetically from tumorigenicity and that removal of amino acids within the first 250 residues of T antigen compromised other transformed cell growth properties.

cGMP-dependent protein kinase genes in Drosophila.

Two Drosophila genes encoding products related to cGMP-dependent protein kinase have been isolated by cross-hybridization to a Drosophila cAMP-dependent protein kinase catalytic subunit gene. Both genes encode products with putative cGMP binding and kinase domains on the same polypeptide chain, as found for the prototypical bovine lung cGMP-dependent protein kinase. The deduced product of one gene (DG1; cytological position, 21D) is 14% larger than the bovine enzyme and differs substantially in sequence at the amino terminus, the region responsible in the bovine enzyme for dimerization. The second gene (DG2; cytological position, 24A) is transcribed into three major RNA species of different size. The largest (DG2; T1) and smallest (DG2;T3) RNAs encode overlapping polypeptides of similar sequence to the whole length of bovine lung cGMP-dependent protein kinase. The translation product of the third major RNA (DG2;T2) lacks sequences similar to those that constitute the dimerization and kinase inhibitory domains of the bovine enzyme. The percentage amino acid identity between DG1 or DG2 and bovine lung cGMP-dependent protein kinase is 55 and 64%, respectively. A common progenitor of the two cGMP-dependent protein kinase genes, DG1 and DG2, is strongly suggested by the conserved positions of introns in these genes.

Isolation and characterization of Drosophila cAMP-dependent protein kinase genes.

We have used mammalian probes to clone genes encoding the catalytic (C) and type I regulatory (RI) components of the cAMP-dependent protein kinase in Drosophila. Both Drosophila gene products are very similar in amino acid sequence (RI, 71%; C, 82%) to their respective mammalian counterparts, implying homologous activity. A single Drosophila type I regulatory subunit gene is the source of at least three distinct transcripts originating from different promoters and spliced to a common body that would encode a full-length analog and two amino-terminally truncated variants of the mammalian RI protein. The RI locus also includes two intronic genes of unknown function. A single highly conserved catalytic subunit gene (DC0) was found that codes for a single polypeptide. It was used to isolate 11 further more distantly related apparent protein kinase genes. Two of these genes (DC1 and DC2) are sufficiently similar to DC0 in sequence (45% and 49% amino acid identity, respectively) that they could conceivably encode products of overlapping function. Two further genes are very similar in sequence to bovine cGMP-dependent protein kinase. The remaining putative gene products include amino acid sequence motifs characteristic of serine-threonine protein kinases but cannot, from the available data, be defined as homologous to specific protein kinases of other organisms.