Targets of Activity

Mutants of punt lack Ultrabithorax and dpp expression in the visceral mesoderm and fail to induce labial in the adjacent endodermal cells. In addition, tinman expression in cardioblasts and even skipped expression in pericardial cells are strongly reduced in punt and tkv mutant stage 14 embryos (Ruberte, 1995).

Decapentaplegic, through its receptors Thickveins and Punt target optimotor blind and spalt transcription in the wing imaginal disc. The range of DPP action is wide, affecting spalt and omb expression on both sides of the anterior-posterior compartment boundary. The finding of an extended range of action for DPP is unexpected. DPP diffusion away from its site of expression may be limited by its tendency to be sequestered by components of the extracellular matrix. spalt and omb respond differently to the DPP concentration gradient, with omb showing a wider range of response due to its greater sensitivity to low DPP concentrations (Nellen, 1996).

During Drosophila embryogenesis the two halves of the lateral epidermis migrate dorsally over a surface of flattened cells, the amnioserosa, and meet at the dorsal midline in order to form the continuous sheet of the larval epidermis. During this process of epithelial migration, known as dorsal closure, signaling from a Jun-amino-terminal-kinase cascade causes the production of the secreted Tgf-beta-like ligand, Decapentaplegic. Binding of Decapentaplegic to the putative Tgf-beta-like receptors Thickveins and Punt activates a Tgf-beta-like pathway that is also required for dorsal closure. Mutations in genes involved in either the Jun-amino-terminal-kinase cascade or the Tgf-beta-like signaling pathway can disrupt dorsal closure. Although these pathways are linked they are not equivalent in function. Signaling by the Jun-amino-terminal-kinase cascade may be initiated by the small Ras-like GTPase Drac1 and acts to assemble the cytoskeleton and specify the identity of the first row of cells of the epidermis prior to the onset of dorsal closure. Signaling in the Tgf-beta-like pathway is mediated by Dcdc42, and acts during the closure process to control the mechanics of the migration process, most likely via its putative effector kinase DPAK (Ricos, 1999).

The Drosophila ovary is an attractive system to study how niches control stem cell self-renewal and differentiation. The niche for germline stem cells (GSCs) provides a Dpp/Bmp signal, which is essential for GSC maintenance. bam is both necessary and sufficient for the differentiation of immediate GSC daughters (cystoblasts). Bmp signals directly repress bam transcription in GSCs in the Drosophila ovary. Similar to dpp, gbb encodes another Bmp niche signal that is essential for maintaining GSCs. The expression of phosphorylated Mad (pMad), a Bmp signaling indicator, is restricted to GSCs and some cystoblasts, which have repressed bam expression. Both Dpp and Gbb signals contribute to pMad production. bam transcription is upregulated in GSCs mutant for dpp and gbb. In marked GSCs mutant for two essential Bmp signal transducers (Med and punt) bam transcription is also elevated. Finally, Med and Mad are shown to directly bind to the bam silencer in vitro. This study demonstrates that Bmp signals maintain the undifferentiated or self-renewal state of GSCs, and directly repress bam expression in GSCs by functioning as short-range signals. Thus, niche signals directly repress differentiation-promoting genes in stem cells in order to maintain stem cell self-renewal (Song, 2004).

Protein Interactions

Punt binds mammalian BMP2 in concert with TKV or SAX, forming complexes with these receptors (Ruberte, 1995 and Letsou, 1995).

The immunophilin FKBP12 binds to the cytoplasmic domain of TGFß type I receptors and is released upon a ligand-induced, type II receptor mediated phosphorylation of the type I receptor. Blocking FKBP12/type I receptor interaction with immunophilin FK506 nonfunctional derivatives enhances the ligand activity, indicating that FKBP12 binding is inhibitory to the signaling pathways of the TGFß family ligands. Overexpression of FKBP12 specifically inhibits pathways activated by TGFß, and point mutations in FKBP12 abolish its inhibitory activity. FKBP12 functions as an immunosuppressive in vertebrates by binding macrolides FK506 and rapamycin and recruiting and thereby inactivating calcineurin and the serine kinase FRAP, respectively, resulting in the blockage of the signaling pathways mediated by calcineurin or FRAP. Since calcineurin is a serine/threonine phosphatase while type I receptors are serine/threonine kinases, and phosphorylation of the type I receptor as well as its downstream substrates is essential for signaling via the type I receptor, one plausible mechanism is proposed for FKBP12 action whereby calcineurin could inhibit type I signaling activity by dephosphorylating type I receptor or its bound substrates. A novel cDNA that is 66% identical to mouse FKBP12 was isolated as the predominant interactor for Drosophila type I receptor Thick veins. FKBP12 interacts with Saxophone as well (Wang, 1996).

Baboon has been shown to function through the newly identified dSmad2 (Smad on X), a Drosophila homolog of mammalian Smad2 and Smad3, which function in Activin signaling. Activated Babo induces phosphorylation on the last two serines of dSmad2. These data suggest that dSmad2 is a downstream target of Babo and is phosphorylated on the last two serine residues in the carboxyl terminus. Babo-dependent phosphorylation of dSmad2 also induces association with Medea, a homolog of mammalian Smad4. Phosphorylation of dSmad2 on the last two serines is necessary for receptor-dependent induction of heteromeric complexes of dSmad2 and Medea. Mammalian Activin receptors require dimerization with a type II receptor for their function. Punt, the Drosophila type II receptor was shown to function to activate Babo. dSmad2 interacts transiently and specifically with Punt-Babo receptor complexes. Taken together, these functional and biochemical analyses strongly suggest that dSmad2 is a Drosophila homolog of Smad2/Smad3 and functions as a downstream signaling component that directly interacts with Babo (Brummel, 1999).

Interactions between the various components of the putative activin pathway of Drosophila were characterized. In vertebrates, R-Smads have been shown to associate with, and be phosphorylated by, specific type I receptors. Baboon, along with thick veins (tkv) and saxophone (sax) have been cloned in searches for Drosophila TGFbeta-like receptors. Since Sax and Tkv participate in the Dpp pathway, and biochemical studies have shown that Tkv activates Mad, it was possible that Baboon could be the receptor responsible for activating Smox. In addition, a previous study (Wrana, 1994) had shown that Baboon could bind human Activin, supporting the view that Smox, along with Baboon, may comprise part of a Drosophila activin pathway (Das, 1999).

The activation of Smox was studied by two methods. First, the ability of Baboon to phosphorylate Smox was examined. An antiphosphoserine antibody was used that has been shown to recognize the ligand-dependent phosphorylation of R-Smads, including Smads 1, 2, 3 and 5 and Drosophila Mad. N-terminally FLAG-tagged Smox (FLAG-Smox) was transfected into COS cells with baboon and the Drosophila type II receptor punt, and cells were treated with Activin A. Cells were then lysed and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting using antiphosphoserine antibody. Co-expression of baboon and punt induces a dramatic increase in the phosphorylation of Smox. The phosphorylation of Smox in the absence of Activin A may have been induced by the spontaneous association of type I and type II receptors in transfected cells. The expression of punt alone induces a weak phosphorylation of Smox, which may be due to the interaction of Punt with endogenous type I receptors in COS cells. Mutations of Thr-204 in the TGFbeta type I receptor and corresponding threonine or glutamine residues in other type I receptors to acidic amino acids leads to the constitutive activation of these type I receptors. The constitutively active form of Punt (CA-Punt) induces a weak phosphorylation of Smox. As expected, however, a kinase-inactive (dn) form of Punt (Punt-KR) does not phosphorylate Smox (Das, 1999).

Since Mad has been shown to be phosphorylated by Tkv, another Drosophila type I receptor, the specificity of activation of the Drosophila R-Smads, Smox and Mad, by type I receptors, was examined. Punt was used as the type II receptor in these experiments, since it has been shown to bind both Activin and BMP-like ligands, together with Punt and Tkv, respectively. In the presence of Punt, Baboon induces the phosphorylation of Smox but not of Mad, while MAD is phosphorylated by the constitutively active form of Tkv (CA-Tkv), but Smox is not. Thus, Smox acts as a downstream component of Baboon, whereas MAD acts downstream of Tkv (Das, 1999).

R-Smads translocate into the nucleus following phosphorylation and oligomerization. The nuclear translocation of Smox was examined using transfected COS cells. COS cells were transfected with expression plasmids for Smox, Baboon and punt, and the subcellular localization of Smox was determined by immunofluorescent staining. In unstimulated cells, Smox is distributed throughout the cell, but after activation by Baboon and Punt, Smox accumulates in the nucleus (Das, 1999).

The interaction between PP1 and Punt requires the type I receptor Thick-veins

Two proteins have been identified that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. Modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants (Bennett, 2002; Parker, 2002). These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGFß superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, this study shows that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1 (Bennett, 2003).

I-2Dm and NIPP1Dm are potent inhibitors of PP1 in vitro. Ectopic I-2Dm and ectopic NIPP1Dm also inhibit PP1c in vivo, resulting in titration of PP1c from its other functions. First, PP1 activity is reduced in extracts from flies ectopically expressing I-2Dm or NIPP1Dm. NIPP1Dm has a larger effect on PP1 activity than I-2Dm does, consistent with the potent inhibition of PP1c by NIPP1Dm in vitro. NIPP1Dm degradation in tissue extracts releases NIPP1 from PP1c, thereby restoring PP1 activity. Consequently, the reduction in PP1c activity in ectopic NIPP1Dm flies is probably higher than that measured in extracts. This is consistent with the phenotypic effect of NIPP1Dm overexpression. Second, ectopic expression of I-2Dm or NIPP1Dm results in phenotypes resembling those of PP1c loss-of-function mutations. In conjunction with arm-GAL4, ectopic NIPP1Dm flies phenotypically resemble PP1alpha87B-/- and PP1ß9C-/- mutants. In conjunction with vg-GAL4, which expresses only weakly in the wing, ectopic NIPP1Dm resembles strong PP1alpha87B-/+ mutants in combination with type II TGFß superfamily receptor Punt. In contrast, overexpression of I-2Dm in the wing resembles the effect of weak PP1alpha87B-/+ mutants in combination with Punt, indicating that the effects of I-2Dm overexpression are similar to, but weaker than, the effects of ectopic NIPP1Dm. Lastly, PP1 activity is reduced in flies modestly overexpressing I-2Dm, but can be restored by reactivation of I-2Dm:PP1c with GSK3ß + MgATP. This implies that I-2Dm and NIPP1Dm sequester PP1c away from other functions and suggests that there is no compensation for titration of endogenous PP1c by production of additional active PP1c. This might be because PP1 is normally in excess, which would also explain why PP1c overexpression in a wild-type background has no phenotypic effect. However, the effects of ectopic I-2Dm and NIPP1Dm on their own, in combination with each other, or in a sensitized background, can be suppressed by coexpression of PP1c, indicating that exogenous PP1c can restore PP1c levels by titrating additional inhibitor (Bennett, 2003).

Co-overexpression of either I-2Dm or NIPP1Dm with PP1 has no phenotypic effect. This implies that neither inhibitor:PP1c complex (I-2Dm:PP1c or NIPP1Dm:PP1c) has any significant function when in excess, and may simply be inactive, as expected if the binding proteins are simply inhibitors of PP1. The ability of I-2 to convert recombinant PP1 to more native-like activity upon phosphorylation of I-2 has led to the suggestion that I-2 may be a molecular chaperone of PP1 as well as an inhibitor of PP1. This biochemical property is also conserved in I-2Dm (Bennett, 1999). However, the net effect of I-2Dm overexpression in flies is to reduce PP1 activity. This indicates that phosphorylation of I-2Dm and reactivation of PP1 is rate limiting, at least when I-2Dm is in excess. Alternatively, reactivation of PP1 in vivo might serve another purpose, namely to allow a pool of inactive (I-2Dm-bound) PP1 to be recruited by targeting subunits to specific subcellular loci. Indeed there is some precedent for cycling of PP1c from inhibitors to targeting subunits: for instance phosphorylation and inactivation of Inhibitor-1 (I-1) by PKA releases I-1-bound PP1 and might be coordinated with phosphorylation and activation of glycogen-binding subunit of PP1 to recruit PP1 to glycogen particles. The isolation of loss-of-function mutations in I-2Dm and NIPP1Dm will help to resolve the question of whether these proteins act solely as inhibitors of PP1c (Bennett, 2003).

To explore the utility of PP1c inhibitors in vivo the effect of I-2Dm and NIPP1Dm on TGFß signaling was examined. Ectopic expression of I-2Dm or NIPP1Dm in the cells expressing ectopic Punt had the same effect as PP1alpha87B mutants, which reduce PP1 levels across the whole wing disc. Since genetic analysis indicates that both Babo signaling and Tkv/Sax signaling have a role in regulating growth of the wing, attempts were made to identify the relevant type I receptor responsible for these effects. In a sensitized genetic background in which Punt is not limiting (vg-GAL4, UAS-punt), the wing phenotype is sensitive to the level of Tkv but not the other type I receptors. The overgrowth phenotype is enhanced by extra Tkv (UAS-tkv), and this closely resembles the phenotype of extra Punt in a background of reduced PP1 (vg-GAL4, UAS-punt PP1-/+). Furthermore, the wing phenotype of elevated punt in a background of reduced PP1 (vg-GAL4, UAS-punt PP1-/+) is suppressed by reducing the level of Tkv (tkv-/+) and enhanced by elevating the level of Tkv (UAS-tkv) but is not affected by similar manipulation of the levels of Sax or Babo. Therefore the overgrowth wing phenotypes are entirely regulated by Tkv (Bennett, 2003).

Displacement of PP1 from Sara has been shown to induced TGFß superfamily signaling (Bennett, 2002), implying that the role of Sara-bound PP1 may be to prevent inappropriate ligand-independent signaling by the receptors. This study shows that the interaction between PP1 and Punt is not sensitive to the levels of the ligand Dpp and that reduction of PP1 activity results in ectopic expression of Bi, a target of Tkv/Dpp signaling, beyond the region in which Dpp is known to elicit signaling (reported to be up to 25 cell diameters from its source). Since PP1 loss of function has the same effect on TGFß/Dpp signaling as a PP1c-nonbinding mutant of Sara, the possibility can be ruled out that free PP1 goes off and does something else, ultimately leading to the increased Dpp signaling by some other mechanism. Taken together, this suggests that reduction of PP1 activity can activate the downstream Dpp signaling pathway in cells that receive low levels or no Dpp and is consistent with a role for PP1 in preventing inappropriate activation of the signaling pathway where the ligand is low or absent (Bennett, 2003).

In summary, a method has been developed of inhibiting PP1 activity in a cell- and tissue-specific manner. I-2Dm and NIPP1Dm do not discriminate between different PP1c isoforms; therefore the role of PP1 in a given pathway can be easily tested without having to test the separate isoforms individually. The role of PP1c has been examined in wing development using ectopic inhibitors of PP1c; that reduction of PP1c activity enhances the effect of the TGFß superfamily type II receptor Punt, giving rise to overgrowth of the wing. The basis of this interaction was examined using I-2Dm; this interaction is shown to require the type I receptor Tkv and is accompanied by induction of a downstream target Bi, suggesting that PP1c negatively regulates Dpp/Tkv signaling during wing morphogenesis. It is anticipated that ectopic inhibitors of PP1c can be used in a wide variety of contexts to test for the effect of reducing PP1 activity on specific developmental processes. While these inhibitors are very useful for identifying a role for PP1 in a particular developmental process, they are not able to dissect the role of a specific PP1c species. The demonstration of the usefulness of ectopic expression of PP1c inhibitors in Drosophila, together with the highly conserved nature of both PP1 and the inhibitors of PP1, suggests that this approach is also applicable to other, less genetically tractable systems (Bennett, 2003).

The divergent TGF-β ligand Dawdle utilizes an activin pathway to influence axon guidance in Drosophila

Axon guidance is regulated by intrinsic factors and extrinsic cues provided by other neurons, glia and target muscles. Dawdle (Daw), a divergent TGF-β superfamily ligand expressed in glia and mesoderm, is required for embryonic motoneuron pathfinding in Drosophila. In daw mutants, ISNb and SNa axons fail to extend completely and are unable to innervate their targets. Daw initiates an activin signaling pathway via the receptors Punt and Baboon (Babo) and the signal-transducer Smad2. Mutations in these signaling components display similar axon guidance defects. Cell-autonomous disruption of receptor signaling suggests that Babo is required in motoneurons rather than in muscles or glia. Ectopic ligand expression can rescue the daw phenotype, but has no deleterious effects. These results indicate that Daw functions in a permissive manner to modulate or enable the growth cone response to other restricted guidance cues, and support a novel role for activin signaling in axon guidance (Parker, 2006).

Cell signaling assays and phenotypic analyses indicate that Daw affects motoneuron pathfinding by acting through Put, Babo and Smad2. Supporting this idea, the incidence of ISNb pathfinding defects increases when animals with a single copy of the receptors Put and Babo are further depleted of Daw ligand. Mutations in Daw and its receptors result in a similar range and penetrance of phenotypes, arguing that Daw is the primary contributor to activin signaling in motoneuron pathfinding and that the canonical pathway can fully account for the ability of Daw to influence axon guidance. The slightly higher penetrance of ISNb defects in babo as compared with daw maternal/zygotic nulls (59% versus 50%), raises the possibility that an additional ligand could contribute to embryonic motor axon guidance. Both Activin and Myoglianin can bind Babo, and are expressed in neural or muscle cells compatible with such a role. Intriguingly, overexpression of Activin (and to a lesser extent Myg) can partially rescue daw- pathfinding defects. However, an assessment of their roles in axon pathfinding must await the recovery of mutations in these genes. Furthermore, daw may have other functions in addition to embryonic pathfinding. A majority of daw mutants die during pupal stages despite the fact that pathfinding defects are largely corrected by the third larval instar (Parker, 2006).

Daw could act as a paracrine signal from the muscle or glia to influence motoneurons. Alternatively, it could provide an autocrine signal that supports glial or muscle growth/function and affects axon outgrowth indirectly. The data show that cell-autonomous disruption of activin signaling in muscles or glia does not disrupt motoneuron pathfinding, ruling out an autocrine mechanism. By contrast, expression of BaboΔI and PutΔI receptors in motoneurons effectively phenocopies daw-, suggesting that axon guidance defects could arise from the inability of motoneurons to respond to a paracrine Daw signal. Interestingly, the retrograde Gbb/BMP signal transduced by Wit/Tkv and Mad that regulates synapse morphology and function in larval motoneurons, shows minimal crosstalk despite acting in the same tissue. Disruption of BMP signaling, by expression of TkvΔI in motoneurons or mutations in wit, does not affect axon guidance although it affects neuromuscular junction (NMJ) function (Parker, 2006).

punt: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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