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Gene name - puckered Synonyms - <> Cytological map position - 84E10--84E13 Function - phosphatase Keywords - dorsal closure, ectoderm, JNK pathway |
Symbol - puc FlyBase ID:FBgn0004210 Genetic map position - 3-[48] assification - dual specificity phosphatase KH-1 subfamily Cellular location - unspecified |
| Recent literature | Karkali, K. and Martin-Blanco, E. (2021).. Dissection of the Regulatory Elements of the Complex Expression Pattern of Puckered, a Dual-Specificity JNK Phosphatase. Int J Mol Sci 22(22). PubMed ID: 34830088
Summary: For developmental processes, most of the gene networks controlling specific cell responses. It still has to be determined how these networks cooperate and how signals become integrated. The JNK pathway is one of the key elements modulating cellular responses during development. Yet, still little is known about how the core components of the pathway interact with additional regulators or how this network modulates cellular responses in the whole organism in homeostasis or during tissue morphogenesis. A promoter analysis was performed, searching for potential regulatory sequences of puckered (puc) and identified different specific enhancers directing gene expression in different tissues and at different developmental times. Remarkably, some of these domains respond to the JNK activity, but not all. Altogether, these analyses show that puc expression regulation is very complex and that JNK activities participate in non-previously known processes during the development of Drosophila. |
Karkali, K., Vernon, S. W., Baines, R. A., Panayotou, G., Martin-Blanco, E. (2023). Puckered and JNK signaling in pioneer neurons coordinates the motor activity of the Drosophila embryo. Nat Commun, 14(1):8186 PubMed ID: 38081827
Summary: Central nervous system organogenesis is a complex process that obeys precise architectural rules. The impact that nervous system architecture may have on its functionality remains, however, relatively unexplored. To clarify this problem, the development of the Drosophila embryonic Ventral Nerve Cord (VNC) was studied. VNC morphogenesis requires the tight control of Jun kinase (JNK) signaling in a subset of pioneer neurons, exerted in part via a negative feedback loop mediated by the dual specificity phosphatase Puckered. The JNK pathway autonomously regulates neuronal electrophysiological properties without affecting synaptic vesicle transport. Manipulating JNK signaling activity in pioneer neurons during early embryogenesis directly influences their function as organizers of VNC architecture and, moreover, uncovers a role in the coordination of the embryonic motor circuitry that is required for hatching. Together, these data reveal critical links, mediated by the control of the JNK signaling cascade by Puckered, between the structural organization of the VNC and its functional optimization. |
Dorsal closure [Images] of the Drosophila embryo provides a good example of cell differentiation and how this is usually coupled to morphogenetic events and movements that shape the embryo in the late stages of development. Half way through embryogenesis, the dorsal surface of the embryo is covered by an extraembryonic membrane, the amnioserosa, which contacts the epidermis. After cell proliferation stops, the epidermis stretches dorsally, and as it does, it encroaches upon the amnioserosa and covers, closes the existing gap. The successful completion of this event may be divided into three phases: the dorsal-ward movement of the epidermal cells; the anteroposterior stretching of the embryo, and the seaming of the dorsal epidermis over the amnioserosa. The completion of this process takes several hours and is associated with specialized behavior of the dorsal-most epidermal cells. These cells display planar polarity reflected in the arrangement of the cytoskeleton, which is essential for the normal process of dorsal closure. Dorsal cuticle puckering and dorsal holes are indicative of a defect in dorsal closure, probably dependent on cell shape changes. During dorsal closure, the epidermal cells in the dorsal region of the embryo change shape dramatically. Beginning with cells immediately flanking the amnioserosa, there is an elongation along the dorsoventral axis; this change in cell shape gradually spreads ventrally through the epidermis, causing the opposing sides of the lateral epidermis to stretch until they meet along the dorsal midline. In puckered mutants, dorsal closure takes place, but an abnormal organization of the cells at the leading edge of the epidermis results in a defective process and puckering, which gives the mutant its descriptive name (Ring, 1993).
There are several mutations that disrupt the process of dorsal closure. In basket (bsk) and hemipterous (hep) mutants, dorsal closure fails and a hole remains in the embryo's dorsal cuticle. hep encodes a Drosophila homolog of MKK7, a kinase that regulates Jun N-terminal kinase (JNK); bsk encodes a Drosophila homolog of JNK. The involvement of the JNK pathway in dorsal closure is further supported by the observation that mutants for Djun, a target of DJNK signaling, fail to close dorsally, and that ectopic expression of a dominant-negative form of Drac1 (DN-Drac1), the Drosophila homolog of Rac1, also leads to the same dorsal closure defects (Martin-Blanco, 1998 and references).
In bsk, hep, and Djun mutants, cell shape changes are disrupted. It is interesting that the expression of dominant negative Drac1 generates phenotypes that are similar to those of hep and bsk mutants, suggesting that Drac1 might initiate signaling through this cascade. Drac1 seems to be involved in the control of the cell cytoskeleton. In wild-type embryos, the onset of dorsal closure coincides with a specific subplasmalemmal accumulation of nonmuscle myosin at the leading edge of the dorsal-most epidermal cells. It is likely that nonmuscle myosin contributes to the elongation of these cells by participating with actin in forming a dorsal constriction. In DN-Drac1 embryos, nonmuscle myosin and actin in epidermal cells are strongly reduced. These changes in the cytoskeleton are also evident in Djun mutant embryos (Martin-Blanco, 1998 and references) .
The effects of the absence of puc (pucE69) and its overexpression were compared o the levels and organization of the actin cytoskeleton and nonmuscle myosin. In pucE69 mutants, the expression of myosin and actin does not change dramatically in the periphery of the cells in lateral regions of the embryo, but these proteins fail to accumulate along the leading edge of the epidermis. Cell shape changes proceed almost normally. In contrast, epidermal cells of embryos overexpressing puc fail to change their shapes and accumulate low levels of spatially disorganized myosin at the leading edge. In these embryos, actin fails to be expressed in the amnioserosa and its levels are reduced in the epidermis. Actin and myosin tend to form clumps in these epidermal cells. Ths puc is an essential component in the control of the different steps of dorsal closure progression; it acts by modulating the apical accumulation of actin and myosin at the leading edge. These results correlate with those of the effects of overexpression of DN-Drac1 and Djun mutants, and further suggest a role for Puc in the control of JNK activity over the cytoskeleton (Martin-Blanco, 1998).
The expression of most members of the VH-1 family of PTPs is subject to tight transcriptional
regulation. The same is likely to be true for puckered because it displays dynamic patterns of expression in the embryo and the adult. During and after germ band shortening, puc is expressed in the dorsal-most epidermal cells that play a leading role in the process of dorsal closure. In embryos mutant for the JNKK encoded by hemipterous or for the JNK encoded by basket, there is no puc expression in these cells, and dorsal closure fails in a manner similar to that produced by the overexpression of puc (Glise, 1995 and Riesgo-Escovar, 1996). These results suggest a model in which signaling through Hep and Bsk leads to the expression of effectors of dorsal closure and a regulator encoded by puc. The function of the latter is to exert a negative feedback on the signaling cascade of hep and bsk. Interestingly, in mutants for Djun (a likely target of JNK activity), puc expression is absent at the leading edge of the epidermis (N. Perrimon, pers. comm. to Martin-Blanco, 1998), suggesting a transcriptional link between the activity of the JNK encoded by bsk and the expression of puc. Thus, the activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities during dorsal closure. In this system, Puckered seems to act in a feedback loop. Puckered expression is upregulated by DJun and in turn, Puckered inactivates MAPK, whose function is the activation of DJun downstream of Rac signaling (Martin-Blanco, 1998).
Critical periods are temporally-restricted, early-life windows when sensory experience remodels synaptic connectivity to optimize environmental input. In the Drosophila juvenile brain, critical period experience drives synapse elimination, which is transiently reversible. Within olfactory sensory neuron (OSN) classes synapsing onto single projection neurons extending to brain learning/memory centers, glia were found to mediate experience-dependent pruning of OSN synaptic glomeruli downstream of critical period odorant exposure. Glial projections were found that infiltrate brain neuropil in response to critical period experience, and use Draper (MEGF10) engulfment receptors to prune synaptic glomeruli. Downstream, antagonistic Basket (JNK) and Puckered (DUSP) signaling was found to be required for the experience-dependent translocation of activated Basket into glial nuclei. Dependent on this signaling, critical period experience was found to drives expression of the F-actin linking signaling scaffold Cheerio (FLNA), which is absolutely essential for the synaptic glomeruli pruning. Cheerio was found to mediate experience-dependent regulation of the glial F-actin cytoskeleton for critical period remodeling. These results define a sequential pathway for experience-dependent brain synaptic glomeruli pruning in a strictly-defined critical period; input experience drives neuropil infiltration of glial projections, Draper/MEGF10 receptors activate a Basket/JNK signaling cascade for transcriptional activation, and Cheerio/FLNA induction regulates the glial actin cytoskeleton to mediate targeted synapse phagocytosis (Nelson, 2024).
An experience-dependent glial pruning mechanism in a critical period of the powerful Drosophila genetic system. Glia were found to be recruited to synaptic glomeruli in response to critical period sensory experience to mediate dose-dependent pruning. Using a combination of mutants, transgenic RNAi and glial-targeted expression studies, this study dissected core mechanisms of critical period pruning. The glial Draper engulfment receptor (MEGF10/Jedi) drives experience-dependent pruning. Downstream signaling antagonism between positive Basket (JNK) and negative Puckered (DUSP) functions controls critical period glial pruning. draper RNAi with a draper null, and basket RNAi results with puckered phosphatase overexpression. Early-life sensory odorant experience induces activated Basket translocation into remodeling glia nuclei, driving cheerio gene transcription to strongly upregulate Cheerio (FLNA) expression in the glia infiltrating synaptic glomeruli. This F-actin linking signaling scaffold is absolutely essential for targeted critical period glial pruning, and consequently sensory experience drives the remodeling of the F-actin cytoskeleton in glia infiltrating synaptic glomeruli. Together, these results reveal a glial pruning mechanism that is experience-dependent and temporally-restricted, connecting Draper receptor activation, nuclear translocation signaling, and F-actin cytoskeleton regulation (Nelson, 2024).
Different classes of olfactory sensory neurons can either expand or retract synaptic arbors based on critical period experience. The Or42a OSNs exhibit striking synapse elimination. Glia infiltrate synaptic glomeruli in response to critical period experience to mediate dose-dependent pruning. Only the EB-responsive VM7 glomerulus has been tested so far, and studies are needed for other odorant-selective glomeruli to determine the generalization of this mechanism. Glia subclasses differentially refine OSN synaptic architecture in a Draper-dependent mechanism. Three glial classes function as phagocytes and can act cooperatively for neuronal phagocytosis in Drosophila juvenile brains. In the critical period, only ensheathing glia employ Draper for experience-dependent synapse pruning. Draper receptors activate Basket/JNK signaling to induce neuronal phagocytosis in early development (larval-pupal transition) and following injury. However, there was no link to experience or circuit remodeling. This study discovered glial Draper→Basket signaling is essential for experience-dependent and temporally-restricted glial pruning. Draper also activates Src42a/Shark signaling, which has not been implicated in this study. The Puckered phosphatase inhibits glial pruning. Glial-targeted RNAi of the other pathway components (e.g.hep, jra, kay), could provide additional insights for determining the signaling mechanisms controlling critical period experience-dependent glial pruning (Nelson, 2024).
Draper triggers phosphorylated Basket nuclear translocation for transcriptional activation in glia. The glial translocation signaling mechanism is imaged using a glial-targeted basket::GFP transgenic reporter with the glial nucleus co-labeled using a Repo antibody. Critical period sensory experience was found to drive very striking Basket translocation into remodeling glial nuclei. The glial nuclei remain outside of the synaptic glomeruli, and extend infiltrating membrane projections into the neuropil to mediate experience-dependent pruning. Circuit-localized signaling was found around EB-responsive VM7 glomeruli. Nuclear Basket activates Jun-related antigen (Jra; Jun homolog)/Kayak (Fos homolog) heterodimers, which regulate the Activator Protein 1 (AP-1) transcription at target sites, with Jra and Kayak acting in concert. Given homodimers do not replicate heterodimer activity, it would be predicted that glial-targeted RNAi against either one would reveal a role in critical period glial pruning. Basket signaling acts in both neurons and glia, but the results indicate a selective glial requirement in experience-dependent pruning. Whether Basket signaling has a neuronal function in the critical period mechanism could also be determine. Thiw iw the first w5uey to discover a Basket signaling requirement in the glial pruning of normally-developing brain circuits in Drosophila, or conserved signaling in any other model system (Nelson, 2024).
The AP-1 complex binds to four separate promoter sites to regulate transcription of the cheerio locus, encoding the Filamin A (FLNA) homolog. Mutant basketDN and kayak nulls have also been shown to regulate Cheerio/FLNA transcription in a Drosophila epithelial tumor disease model. Consistently, this study discovered both glial-targeted draper and basket RNAi dramatically reduce glial Cheerio expression within synaptic glomeruli during the critical period. It is assumed that the changes in Cheerio levels shown closely reflect AP-1 transcriptional regulation, but direct fluctuations of AP-1 activity could potentially also be tested with TPA-responsive element (TRE) GFP reporters, which might show whether reductions in glial Cheerio expression are caused by reduced AP-1 activity. Note that simultaneous manipulations in separate cell types (neurons and glia) requires dual transgenic systems with separable drivers/responders, which has not yet been achievable in critical period studies. This study discovered that Cheerio expression is experience-dependent and upregulated specifically in the EB-responsive VM7 synaptic glomeruli dependent on Draper-Basket nuclear signaling. In addition to the cheerio gene, AP-1 also regulates the transcriptional activity at other genetic loci. One pertinent example is AP-1 transcriptional regulation of secreted matrix metalloproteinase 1 (MMP1), which has a proposed role in glial phagocytosis. Future work could test Draper→Basket regulation of MMP1, or even Cheerio and MMP1 both working together, in orchestrating glial pruning functions in the juvenile brain critical period (Nelson, 2024).
Cheerio/FLNA supports microfilaments in orthogonal arrays in dynamic membrane movements, cross-linking F-actin filaments, and functioning as a vital intracellular signaling scaffold to control force-generating cytoskeletal motor activities. Regulation of the F-actin cytoskeleton is thus central to cell motility and the complex processes of membrane engulfment and phagocytosis. Consistently, glial-targeted cheerio RNAi utterly blocks experience-dependent glial pruning during the critical period. Relatively little is known about F-actin regulation in glia and almost nothing is known about glial actin cytoskeleton regulation within early-life critical periods. However, visualizing the glial-targeted F-actin marker LifeAct::GFP74, sensory experience-dependent rearrangement of the glial actin cytoskeleton circuit-localized to the EB-responsive VM7 synaptic glomeruli was clearly observed. Given the absolute requirement of Cheerio for the glial pruning of these connections, glial-targeted cheerio RNAi prevents the F-actin cytoskeleton rearrangements in response to experience during the early-life critical period. Similarly, glial-targeted draper RNAi also blocks the experience-dependent regulation of the glial F-actin cytoskeleton. Future work may also reveal that additional actin regulatory proteins, such as the Rho GTPase Rac1, also facilitate this targeted glial pruning mechanism (Nelson, 2024).
Glial phagocytosis of supernumerary synaptic connections during multiple stages of brain development is a central mechanism in the refinement and remodeling of neural circuits. EB exposure outside the critical period does not result in significant pruning of the Or42a OSN innervation, showing this glial phagocytosis mechanism is temporally restricted. A large body of work has revealed that mammalian glia utilize a variety of mechanisms to phagocytose and eliminate differentially-active synapses, including the MEGF10 receptor for the activity-dependent pruning of retinogeniculate synapses. This study showed that Drosophila glia employ the conserved Draper engulfment receptor to prune central brain olfactory synaptic glomeruli in an early-life, experience-dependent critical period mechanism, highlighting the ever-growing similarity between mammalian and Drosophila glial functions. However, very few studies have explored glial phagocytic pruning functions to grossly remodel synaptic connections during critical periods, and none have done so in the normally-developing Drosophila brain. Thus, this study presents an invaluable new model to explore glial pruning mechanisms during a temporally-restricted critical period of heightened brain circuit plasticity, providing a novel forward genetic system to complement the ongoing mammalian model glial studies. Future work will continue to build upon this new Drosophila genetic model to elucidate the conserved molecular mechanisms directing glia to infiltrate specific brain neuropils to mediate experience-dependent phagocytosis of targeted synapses. Overall, this work reveals an essential role for glial pruning in the juvenile brain olfactory circuitry during the temporally-transient and experience-dependent critical period (Nelson, 2024).
< PUC mRNA accumulates preferentially in the CMFSs and cells of the elongating dorsal appendages. Proper levels of Puc activity in the follicle cells are critical for the production of a normal egg: either reduced or
increased Puc activity results in incomplete nurse cell
dumping and aberrant dorsal appendages. Phenotypes
associated with puc mutant follicle cells include altered DE-cadherin (Shotgun) expression in the follicle cells and a failure of
nurse cell dumping to coordinate with dorsal appendage
elongation, leading to the formation of cup-shaped egg
chambers. The JNK pathway target A251-lacZ shows cell-type-specific differences in its regulation by puc and by the
small GTPase Rac1. puc mutant cells display region-specific
ectopic expression of the A251-lacZ enhancer trap
whereas overexpression of a transgene encoding Puc is
sufficient to suppress lacZ expression in a cell autonomous
fashion. Strikingly, decreased or increased puc function
leads to a corresponding increase or decrease, respectively,
of Fos and Jun protein levels. Taken together, these data
indicate that puc modulates gene expression responses by
antagonizing a Rho GTPase signal transduction pathway that stabilizes the AP-1 transcription factor. Consistent with this, overexpression of a dominant negative Rac1 results in lower levels of Fos/Jun (Dobens, 2001).
Production of puc mutant clones in the ovary leads to the formation of small eggs with anterior eggshell defects. The junction between the operculum
and ventral collar is aberrant, so that the eggshells are open
in the anterior, and the dorsal appendages are short and broad.
The presence of opercula and micropyles indicate that
centripetal migration occurs, but the specific defects
suggest that it does not proceed normally. Late-staged egg
chambers from these females retain significant nurse cell
material at the anterior after the eggshell is deposited,
indicating that nurse cell dumping is not coordinated with
follicle cell morphogenesis. Germline clonal analysis indicates
that these puc mutant phenotypes derive solely from a
requirement for puc in the soma. Overexpression of Puc in the
FC also leads to production of small eggs with dorsal
appendage defects. Because either increased or decreased
puc function disrupts the coordination of follicle cell
morphogenesis and nurse cell dumping, it is concluded that Puc
activity must be poised at the proper level to coordinate anterior
morphogenesis. These data parallel the subtly different defects in dorsal closure that result from either reduced or increased Puc activity, and point to a general role for Puc in coordinating epithelial sheet movements (Dobens, 2001).
Loss of puc in the follicle cells
leads to aberrant cell shapes. Puc mutant dorsal appendage FCs
accumulate elevated levels of Shotgun and fail to extend
properly. Late in oogenesis the mainbody FCs are irregular in
shape when mutant for puc. It is concluded that the level of Puc
activity finely modulates the morphogenesis of the follicular
epithelium. This requirement is most evident in the dorsal
appendage FCs, but is also important for morphogenesis of the
operculum boundary (Dobens, 2001).
Enhancer traps A251-lacZ and E69-lacZ are inserted into the
same region of the puc second intron. Both exhibit leading
edge cell expression during embryonic dorsal closure and nurse
cell FC-specific expression during nurse cell dumping. In
contrast, PUC mRNA expression is more widespread in both
tissues. In the egg chamber, it accumulates at low levels
throughout the FC and at high levels in the centripetally
migrating FCs, dorsal appendage FCs and posterior pole FCs.
This pattern is recapitulated by the B48-lacZ enhancer trap
insertion located in puc intron 1. Consistent with this, the puc
gene is required in more FCs than the limited expression of
A251-lacZ would predict. Given the widespread expression of
PUC RNA in the embryo, A251-lacZ reveals only a subset of
the complete puc expression pattern in this tissue as well (Dobens, 2001).
These lacZ reporters have been useful to dissect the
regulation of epithelial cell morphogenesis during dorsal
closure in embryos. Reporter expression is positively regulated
by components of the JNK pathway, and negatively regulated
by Puc itself. It has not been reported whether PUC RNA levels
are similarly regulated. A251-lacZ expression is
also negatively regulated by Puc in the FC. (1) When PUC
mRNA levels are low, before stage 10, there is a requirement
for Puc activity to prevent A251-lacZ expression throughout
the FC. (2) After stage 10, when clear FC domains are
established, striking regional differences are observed in the
requirement for Puc to regulate A251-lacZ expression.
In the nurse cell FC, A251-lacZ expression is increased by
reduced Puc function and repressed by overexpression of UAS-Puc.
Because PUC mRNA levels are low in these cells at this
stage, it is inferred that Puc activity is at an intermediate level so
that Jun kinase-like signaling is only partially repressed.
In the centripetally migrating FCs, low A251-lacZ levels are
Puc dependent because reduction of Puc function results in
ectopic A251-lacZ expression. It is concluded that Jun kinase-like
signaling is active in these cells, but strongly suppressed
by the high levels of Puc activity normally there. Consistent
with this, high levels of PUC mRNA are seen in these cells. High
PUC mRNA levels correlate with strong repression of A251-lacZ at stage 10 in the posterior pole FCs, as well. The strong
requirement for Puc activity in anterior FCs correlates well with
the puc mutant defects seen in anterior FC morphogenesis (Dobens, 2001).
Low levels of A251-lacZ in the main body FC are not
dependent on puc: mutant clones show only a slight
derepression of A251-lacZ at very late stages.
Consistent with this, these cells express only low levels of PUC
mRNA, and overexpression of Puc has no visible effect on the
morphology of these cells. However, Puc is active in the main
body FCs, for these cells accumulate Fos and Jun when mutant (Dobens, 2001).
The Rho family of small GTPases are important modulators of
cell shape and epithelial cell reorganization. Specific Rho
proteins can manifest specialized cellular functions. The cell-type
specific effects extend to their abilities to stimulate the
JNK pathway. The differing requirements for Puc in different regions of the
follicular epithelium suggest that these regions show
distinct sensitivities to the actions of Rho family GTPases.
Because of evidence that Rac1 and Cdc42 regulate
expression of A251-lacZ during embryogenesis, the ability of these small GTPases to regulate
A251-lacZ expression in the FC was tested. Two opposing
effects were found: activated Rac1 and dominant negative Cdc42
activate A251-lacZ expression in posterior cells; dominant
negative Rac1 and activated Cdc42 repress A251-lacZ
expression in anterior cells. Clones for activated Rac1 were
not recovered in the anterior nurse cell FCs, suggesting that
these cells are highly sensitive to increased Rac activity. These
data suggest that a balance between these two small GTPases
may define the level of A251-lacZ expression in the nurse cell
FCs and the posterior pole FCs. This may reflect the shared
origins of these distinct cell groups as terminal follicle cells (Dobens, 2001).
Neither Rac1 nor Cdc42 appeared to regulate A251-lacZ
expression in the main body FCs or the centripetally migrating
FCs. This latter observation is surprising, given the strong
requirement for Puc activity in these cells. Perhaps a distinct
Rho family GTPase is active in these cells, such as Rho1.
Consistent with this, mutants in PAK kinase, which acts in
parallel to JNK signaling during dorsal closure, have a variable
defect in centripetal migration (Dobens, 2001).
Activated GTPase expression in the follicle cells results in
additional phenotypes. Activated Rac1 has non-autonomous
phenotypes in the posterior pole FCs. Similarly,
activated Rac1 has non-autonomous effects on gene
expression in the dorsal ectoderm of the embryo. This suggests that in both tissues, a short-range
signal dependent on Rac1 signaling can upregulate
expression of these intron II enhancer traps in adjacent cells.
Dpp is a local signal to the dorsal ectoderm during dorsal
closure; however, the data suggest that Dpp is not the key
signal to induce A251-lacZ in the posterior FC. It is noted that loss of puc in marked clones never results in upregulation of A251-lacZ
expression in adjacent cells, indicating that whatever its
identity, the short range signal is not regulated by puc (Dobens, 2001).
Expression of activated Rac1 in the posterior pole FCs
causes domain-specific disorganization of the epithelium.
Dominant negative Cdc42 also causes this phenotype, similar
to genetic loss of Cdc42 activity. Similar phenotype are seen for activated RhoL. Thus the balance of Rho GTPases in the posterior follicular epithelium may be
important to maintain epithelial structure, similar to roles
ascribed to Rac in metastasis (Dobens, 2001).
Based on data presented here, it is proposed that puc functions
as a rheostat to modulate gene expression responses to Rho
family GTPases. This modulation is critical to
coordinate morphogenesis in distinct FC domains. Because Puc can act as a Jun kinase
phosphatase in the embryo, it has been speculated that Puc modulates
the activity of similar kinases that act downstream of the small
GTPases at the FC termini. It is likely that puc modulates Jun
kinase signal in the anterior FCs, since the Jun kinase kinase
encoded by hemipterous is required for anterior A251-lacZ expression. However, puc
is also required at the posterior, whereas late A251-lacZ
expression is not dependent on hep. Components of the
posterior pathway remain to be identified (Dobens, 2001).
The insensitivity of main body FCs to stimulation by Rac or
Cdc42 might suggest that these cells are incompetent to
respond to small GTPase activation during the late stages of
oogenesis. However, responses are suppressed by an independent mechanism. The dynamic
pattern of Jun and Fos and puc itself, suggests
that FCs may vary greatly, both in time and in space, in their
competence to respond to the JNK pathway. To complicate this
picture, the data indicates that puc regulates levels of Fos and
Jun, even in the main body FCs. Loss of puc leads to elevation
of Fos/Jun protein levels. Conversely overexpression of either
Puc or DN-Rac1 lowers Fos/Jun levels. Currently the direct mechanism for puc regulation of Fos/Jun levels is unknown, but these results recall observations that stability of Fos and Jun depends on phosphorylation. In cell culture, site-specific
mutations in Fos or Jun that block phosphorylation confer
instability; conversely, phosphate-mimetic mutations confer
greater stability. It is concluded that proper levels of Puc
phosphatase modulates Rho family GTPase signal output to
coordinate follicle cell morphogenesis. These results are
similar to the effect of both loss- and gain-of-function Puc in
blocking dorsal closure and a role for puc in disc epithelial morphogenesis. The precise regulation of Puc activity levels is critical to coordinate epithelial cell sheet spreading in at least three tissues (Dobens, 2001 and references therein).
Bases in 5' UTR - 405
Exons - 3
Bases in 3' UTR - 485
The Puc ORF encodes a protein tyrosine phosphatase, with a catalytic domain between amino acids 214 and 226 that includes the invariant cysteine known to be required for phosphatase activity. The protein contains eight putative sites for phosphorylation by MAPK, distributed throughout the carboxy-terminal part of the protein. The predicted protein contains no clear hydrophobic sequences indicative of either signal sequence or transmembrane domain, which suggests that the Puc protein is neither a secreted nor an integral membrane protein. The phosphatase encoded by puc has high similarity to nonreceptor dual specificity phosphatases of the VH-1 subfamily. Phylogenetic analysis, indicates that its closest relative is the protein encoded by Caenorhabditis elegans(identified in the C. elegans genome project) that has 38% identical residues over a 158 amino acid overlap. When conservative residues are taken into account, the comparison yields 59.5% similarity between the two sequences. Very high similarities with other proteins of this family highlight the conservation of their catalytic sites, which are identical at 9-11 of 13 amino acids. A single copy of an internally repeated domain of unknown function (amino acids 238-312; amino acids 386-460) is present in all VH-1 family phosphatases (Martin-Blanco, 1998 and references).
date revised: 1 September 2025
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