Gene name - punt
Cytological map position - 88D2-3
Function - receptor - serine/threonine kinase
Symbol - put
Genetic map position - chr3R:10,444,784-10,451,456
Classification - Type II TGF beta receptor homolog
Cellular location - surface
|Recent literature||Martins, T., Eusebio, N., Correia, A., Marinho, J., Casares, F. and Pereira, P. S. (2017). TGFβ/Activin signalling is required for ribosome biogenesis and cell growth in Drosophila salivary glands. Open Biol 7(1). PubMed ID: 28123053
Signalling by TGFβ superfamily factors plays an important role in tissue growth and cell proliferation. In Drosophila, the activity of the TGFβ/Activin signalling branch has been linked to the regulation of cell growth and proliferation, but the cellular and molecular basis for these functions are not fully understood. This study shows that both the RII receptor Punt (Put) and the R-Smad Smad2 are strongly required for cell and tissue growth. Knocking down the expression of Put or Smad2 in salivary glands causes alterations in nucleolar structure and functions. Cells with decreased TGFβ/Activin signalling accumulate intermediate pre-rRNA transcripts containing internal transcribed spacer 1 regions accompanied by the nucleolar retention of ribosomal proteins. Thus, these results show that TGFβ/Activin signalling is required for ribosomal biogenesis, a key aspect of cellular growth control. Importantly, overexpression of Put enhanced cell growth induced by Drosophila Myc, a well-characterized inducer of nucleolar hypertrophy and ribosome biogenesis.
|Eusebio, N., Tavares, L. and Pereira, P. S. (2018). CtBP represses Dpp-dependent Mad activation during Drosophila eye development. Dev Biol. PubMed ID: 30031756
Complex networks of signaling pathways maintain the correct balance between positive and negative growth signals, ensuring that tissues achieve proper sizes and differentiation pattern during development. In Drosophila, Dpp, a member of the TGFbeta family, plays two main roles during larval eye development. In the early eye primordium, Dpp promotes growth and cell survival, but later on, it switches its function to induce a developmentally-regulated cell cycle arrest in the G1 phase and neuronal photoreceptor differentiation. To advance in the identification and characterization of regulators and targets of Dpp signaling required for retinal development, an in vivo eye-targeted double-RNAi screen was carried out to identify punt (Type II TGFbeta receptor) interactors. Using a set of 251 genes associated with eye development, CtBP, Dad, Ago and Brk were identified as punt genetic interactors. This study shows that downregulation of Ago, or conditions causing increased tissue growth including overexpression of Myc or CyclinD-Cdk4 are sufficient to partially rescue punt-dependent growth and photoreceptor differentiation. Interestingly, a novel role is shown for the transcriptional co-repressor CtBP in inhibiting Dpp-dependent Mad activation by phosphorylation, downstream or in parallel to Dad, the inhibitory Smad. Furthermore, CtBP downregulation activates JNK signaling pathway, implying a complex regulation of signaling pathways by CtBP during eye development.
|Peterson, A. J., Murphy, S. J., Mundt, M. G., Shimell, M., Leof, E. B. and O'Connor, M. B. (2022). A juxtamembrane basolateral targeting motif regulates signaling through a TGF-beta pathway receptor in Drosophila. PLoS Biol 20(5): e3001660. PubMed ID: 35594316
In polarized epithelial cells, receptor-ligand interactions can be restricted by different spatial distributions of the 2 interacting components, giving rise to an underappreciated layer of regulatory complexity. This study explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue featuring the TGF-β family member Decapentaplegic (Dpp) as a morphogen controlling growth and patterning. Dpp protein has been observed in an extracellular gradient within the columnar cell layer of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of 2 distinctly localized ligand pools. The Dpp Type II receptor Punt was found to be enriched at the basolateral membrane and depleted at the junctions and apical surface. Wit, a second Type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified a short juxtamembrane sequence required for basolateral restriction of Punt in both wing discs and mammalian Madin-Darby canine kidney (MDCK) cells. This basolateral targeting (BLT) determinant can dominantly confer basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility, and significant lethality. This study found that basolateral presentation of Punt is required for optimal signaling. Finally, evidence is presented that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.
Punt, along with Saxophone and Thick veins, constitute the Drosophila receptors for decapentaplegic. These receptors mediate the transduction of DPP signals into the cell. Mutations in these genes have the same effect as mutation in dpp, since without DPP's receptors, its signals fail to be communicated to the proper cells. The results are developmental confusion on a grand scale.
Decapentaplegic is responsible for induction of dorsal-ventral polarity in the fly. Loss of dpp through mutation yields catastrophic, indeed, topsy-turvy effects: the fly's back turns into its front. The back fails to develop normally and becomes instead a neurogenic ectoderm resembling tissue usually found in the ventral portion of the trunk.
Punt is homologous to a tumor growth factor (TGF-beta) receptor found in vertebrates. A digression to have a closer look at TGF-beta receptors is now in order. Much work has been carried out on TGF-beta receptors in vertebrates, providing detailed information on their nature and function. They have been classified into three types, non-descriptively named types I, II and III. Based on structural comparisons with the vertebrate TGF-beta receptors, Thick veins and Saxophone are homologous to type I and Punt to type II. Both receptor types are transmembrane proteins and their N-terminal domains are extracellular.
The TGF-beta receptor is really a heterodimer, consisting of both a type I and a type II receptor, each of which possess intracellular kinase domains. The kinase carries out the task of phosphorylation, a way of sending signals from one protein to another. A third subunit known as a type III receptor has a short cytoplasmic region lacking a kinase domain.
The type II receptor is essential for all known TGF-beta initiated signals. Homodimers of the type II and III receptors exist on the cell surface in the absence of TGF-beta. Type II and type III receptors interact in the presence of ligand. The type I receptors are also transmembrane serine-threonine kinases. Type II receptors require the corresponding type I receptor for signaling. Binding of TGF-beta or activin-A to type I receptors requires the presence of the corresponding type II receptor. Type I and type II receptors form heteromeric complexes after ligand binding (Lin, 1995 and references). The type II receptors act upstream of type I receptors, and so one may think ofthese components as primary receptors and transducers, respectively (Massagué, 1996).
Back to Punt: how then does Punt carry out its function? DPP binds to the extracellular domain of the heterodimer or heterotrimer DPP receptor. The DPP signal is transduced across the membrane activating the kinase function in the intracellular domain. The targets of the kinase function of Punt are not yet known. Phosphorylation of the type I receptor follows the DPP signal, in a transphosphorylation event carried out by the type II receptor. Phosphorylation activates the kinase activity of the type I receptor allowing it to transduce signals to unknown substrates (Wrana, 1994).
Punt is require for both SAX and TKV signaling. sax appears to be expressed more ubiquitously than thickveins, but paradoxically is required less ubiquitously than thick veins. SAX requires the function of both Thick veins and Punt to mediate signal transduction (Wharton, 1995, and Ruberte, 1995). Since sax is only necessary for the specification of the dorsalmost cell fate (amnioserosa) which requires the highest levels of DPP activity, SAX might serve as either part of the receptor complex or function independently to interpret peak levels of DPP.
Punt and Thickveins are both required for the establishment of dorsoventral polarity in the early embryo, the closure of the dorsal epidermis, and the correct formation of the visceral mesoderm and the tracheal system. All of these processes depend on DPP signaling (Ruperte, 1995 and Letsou, 1995).
Proper nerve connections form when growing axons terminate at the correct postsynaptic target. Transforming growth factor β (TGFβ) signals regulate axon growth. In most contexts, TGFβ signals are tightly linked to Smad transcriptional activity. Although known to exist, how Smad-independent pathways mediate TGFβ responses in vivo is unclear. In Drosophila mushroom body (MB) neurons, loss of the TGFβ receptor Baboon (Babo) results in axon overextension. Conversely, misexpression of constitutively active Babo results in premature axon termination. Smad activity is not required for these phenotypes. This study shows that Babo signals require the Rho GTPases Rho1 and Rac, and LIM kinase1 (LIMK1), which regulate the actin cytoskeleton. Contrary to the well-established receptor activation model, in which type 1 receptors act downstream of type 2 receptors, this study shows that the type 2 receptors Wishful thinking (Wit) and Punt act downstream of the Babo type 1 receptor. Wit and Punt regulate axon growth independently, and interchangeably, through LIMK1-dependent and -independent mechanisms. Thus, novel TGFβ receptor interactions control non-Smad signals and regulate multiple aspects of axonal development in vivo (Ng, 2008).
Once growing axons reach the correct postsynaptic target, axon outgrowth terminates and synaptogenesis begins. These studies suggest that TGFβ signals play a role. When Babo is inactivated, MB axon growth does not terminate properly and overextends across the midline. Consistent with this, CA Babo expression results in precocious termination, forming axon truncations. How Babo is spatially and temporally regulated remains to be determined. Analogous to the Drosophila NMJ, MB axon growth might be terminated through retrograde signalling. Target-derived TGFβ ligands could signal to Babo (on MB axon growth cones) and stop axons growing further. In an alternative scenario, TGFβ ligands might act as a positional cue that prevents MB axons from crossing the midline. Recent data have shown that Babo acting through Smad2 restricts individual R7 photoreceptor axons to single termini. Loss of Babo, Smad2, or the nuclear import regulator Importin α3 (Karyopherin α3 - FlyBase), results in R7 mutant axons invading neighbouring R7 terminal zones. With the phenotype described in this study, Babo could similarly be restricting MB axons to appropriate termination zones, its loss resulting in inappropriate terminations on the contralateral side (Ng, 2008).
In contrast to MB neurons, Babo inactivation in AL and OL neurons resulted in axon extension and targeting defects. This might reflect cell-intrinsic differences in the response in different neurons to a common Babo signalling program. This may be the case for MB axon pruning and DC axon extension, which require Babo/Smad2 signals. Whether these differences derive from cell-intrinsic properties, or from Babo signal transduction, they underline the importance of Smad-independent signals in many aspects of axonal development (Ng, 2008).
The results suggest that Smad-independent signals involve Rho GTPases. One caveat in genetic interaction experiments is that the loss of any given gene might not be dosage-sensitive with a particular assay. Nevertheless, all the manipulations together suggest that Babo-regulated axon growth requires Rho1, Rac and LIMK1. How Babo signals involve Rho GTPases remains to be fully determined. In addition to LIMK1, which binds to Wit, one possibility, as demonstrated for many axon guidance receptors, is that the RhoGEFs, RhoGAPs and Rho proteins might be linked to the Babo receptor complex. Thus, ligand-mediated changes in receptor properties would lead to spatiotemporal changes in Rho GTPase and LIMK1 activities (Ng, 2008).
The data suggest that a RhoGEF2/Rho1/Rok/LIMK1 pathway mediates Babo responses. Whether Rac activators are required is unclear, as tested RacGEFs do not genetically interact with babo. In this respect, rather than through GEFs, Babo might regulate Rac through GAPs, by inhibiting Tumbleweed (Tum) activity (Ng, 2008).
Do mutations in Rho1 and Rac components phenocopy babo phenotypes? β lobe overextensions are observed in Rok, Rho1 and Rac mutant neurons. In MB neurons, Rac GTPases also control axon outgrowth, guidance and branching. Rho1 also has additional roles in MB neurons. Although Rho1 mutant neuroblasts have cell proliferation defects, single-cell αβ clones do show β lobe extensions. RhoGEF2 strong loss-of-function clones do not exhibit axon overextension. As there are 23 RhoGEFs in the Drosophila genome, there might well be redundancy in the way Rho1 is activated. LIMK1 inactivation in MB neurons was reported previously. Axon overextensions were not observed as LIMK1 loss results in axon outgrowth and misguidance phenotypes. This suggests that LIMK1 mediates multiple axon guidance signals, of which TGFβ is a subset in MB morphogenesis (Ng, 2008).
Although their phenotypes are similar, several lines of evidence indicate that CA Babo does not simply reflect LIMK1 misregulation in MB neurons. First, whereas LIMK1 genetically interacts with most Rho family members and many Rho regulators, CA babo is dosage-sensitive only to Rho1 and Rac and specific Rho regulators, suggesting that Babo regulates LIMK1 only through a subset of Rho signals (Ng, 2008).
Second, the LIMK1 misexpression phenotype is suppressed by expression of wild-type cofilin (Twinstar Tsr), S3A Tsr, or the cofilin phosphatase Slingshot (Ssh). By contrast, only wild-type Tsr, but not S3A Tsr or Ssh, suppresses CA Babo. The suppression by wild-type Tsr might reflect a restoration of the endogenous balance or spatial distribution of cofilin-on (unphosphorylated) and -off (phosphorylated) states within neurons. Indeed, optimal axon outgrowth requires cofilin to undergo cycles of phosphorylation and dephosphorylation. Since S3A forms of cofilin cannot be inactivated and recycled from actin-bound complexes, wild-type cofilin is more potent in actin cytoskeletal regulation (Ng, 2008).
CA Babo might not simply misregulate LIMK1 but also additional cofilin regulators. Recent data suggest that extracellular cues (including mammalian BMPs) can regulate cofilin through Ssh phosphatase and phospholipase Cγ activities. In different cell types, cofilin phosphorylation and phospholipid binding (which also inhibits cofilin activity) states vary and potently affect cell motility and cytoskeletal regulation. Whether a combination of LIMK1, Ssh and phospholipid regulation affects cofilin-dependent axon growth remains to be determined (Ng, 2008).
Third, by phalloidin staining, LIMK1, but not CA Babo, misexpression results in a dramatic increase in F-actin in MB neurons. Thus, CA Babo does not in itself lead to actin misregulation. Fourth, Babo also regulates axon growth independently of LIMK1 (Ng, 2008).
This study differs significantly from the canonical model of Smad signalling, in which type 1 receptors function downstream of the ligand-type 2 receptor complex. In this study, the gain- and loss-of-function results suggest that type 2 receptors act downstream of type 1 signals. Since ectopic only Wit and Put suppress the babo axon overextension phenotype, this implies that Smad-dependent and -independent signals have distinct type 1/type 2 receptor interactions. How these interactions propagate Smad-independent signals remains to be fully determined. Babo could act as a ligand-binding co-receptor with Wit and Put. In addition, Babo kinase activity could regulate type 2 receptor or Rho functions. The results suggest, however, that provided that Wit or Put signals are sufficiently high, Babo is not required. Whatever the mechanism(s), it is likely that Babo requires the Wit C-terminus-LIMK1 interaction to relay cofilin phosphoregulatory signals. How Put functions is unclear. Since the put135 allele (used in this study) carries a missense mutation within the kinase domain, this suggests that kinase activity is essential. put does not genetically interact with LIMK1. Since Put lacks the C-terminal extension of Wit that is necessary for LIMK1 binding, this suggests that Put acts independently of LIMK1. One potential effector is Rac, which, in the context of Babo signalling, also appears to be Pak1- and thus LIMK1-independent (Ng, 2008).
In MB neurons, Wit and Put can function interchangeably. In other in vivo paradigms, type 2 receptors are not interchangeable. However, since the Wit C-terminal tail is required to substitute for Put, this suggests that Wit axon growth signals are independent of its kinase activity. Together, this suggests that Smad-independent signals involve LIMK1-dependent and -independent mechanisms (Ng, 2008).
This study shows that Babo mediates two distinct responses in related MB populations. How do MB neurons choose between axon pruning and axon growth? The babo rescue studies suggest that whereas Baboa or Babob elicits Smad-independent responses, only Baboa mediates Smad-dependent responses. Since Babo isoforms differ only in the extracellular domain, differences in ligand binding could determine Smad2 or Rho GTPase activation. However, it is worth noting that in DC neurons, either isoform mediates axon extension through Smad2 and Medea. In addition, although expressed in all MB neurons, CA babo misexpression (which confers ligand-independent signals) perturbs only αβ axons. Thus, cell-intrinsic properties might also be essential in determining Babo responses (Ng, 2008).
Many TGFβ ligands signal through Babo. For example, Dawdle, an Activin-related ligand, patterns Drosophila motor axons, whereas Activin (Activin-β, FlyBase) is required for MB axon pruning. Whether these ligands regulate Babo MB, AL and OL axonal morphogenesis is unclear. Taken together, the evidence suggests that Babo signalling is varied in vivo and is involved in many aspects of neuronal development (Ng, 2008).
TGFβ signals are responsible for many aspects of development and disease and, throughout different models, Smad pathways are closely involved. Although Smad-independent pathways are known, their mechanisms and roles in vivo are unclear. TGFβ signals often drive cell shape changes in vivo. During epithelial-to-mesenchymal transition (EMT), cells lose their epithelial structure and adopt a fibroblast-like structure that is essential for cell migration during development and tumour invasion. TGFβ-mediated changes in the actin cytoskeleton and adherens junctions are necessary for EMT. Although Smads are crucial, TGFβ signals also involve the Cdc42-Par6 complex, resulting in cell de-adhesion and F-actin breakdown through Rho1 degradation. In other studies, however, TGFβ-mediated EMT has been shown to require Rho1, which can be regulated by Smad activity (Ng, 2008).
Many TGFβ-driven events in Drosophila are Smad-dependent. Whether Smad-independent roles exist beyond those identified in this study remains to be tested. This study therefore provides a framework to understand how non-Smad signals regulate cell morphogenesis during development (Ng, 2008).
The cysteine-rich extracellular domain contains three potential N-linked glycosylation sites. Most of the cytoplasmic region consists of a protein kinase domain. Two distinctive activin type II receptors and one TGF-beta type II receptor have been cloned from vertebrates, and each has a cytoplasmic protein-serine/threonine kinase domain, homologous to that of Punt. Another member of this receptor family is encoded by the C. elegans daf-1 gene, which controls larva development in response to an unknown ligand. There is also a Xenopus type II receptor homolog of Punt (Childs, 1993).
date revised: 22 November 2022
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