puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila

Abstract

The activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities. The latter is mediated by a subset of phosphatases with dual specificity (VH-1 family). Here, we describe a new member of this family encoded by the puckered gene of Drosophila. Mutations in this gene lead to cytoskeletal defects that result in a failure in dorsal closure related to those associated with mutations in basket, the Drosophila JNK homolog. We show that puckered mutations result in the hyperactivation of DJNK, and that overexpression of puc mimics basket mutant phenotypes. We also show that puckered expression is itself a consequence of the activity of the JNK pathway and that during dorsal closure, JNK signaling has a dual role: to activate an effector, encoded by decapentaplegic, and an element of negative feedback regulation encoded by puckered. In many cases, cell differentiation represents a transition between two states of cellular activity-one in which cells proliferate and acquire information about their fates and identities, and another in which cells stop dividing and manifest the information gathered earlier.Many of the signaling pathways leading to cell differentiation depend on phosphorylation cascades. Mounting evidence points to signaling through MAP kinase (MAPK) pathways as a key component in this transition. Three distinct types of MAPK pathways have been identified: p42-p44 ERKs (extracellular signal-related kinases), p38 kinases, and p46-p54 JNKs (Jun N (amino)-terminal kinases). These major subfamilies transduce signals from different stimuli. The ERKs respond predominantly to growth factors and hormones and are activated in a Ras-dependent manner. The p38 and JNKs respond to different environmental stresses and are activated preferentially downstream of Rac1 and Cdc42 small G proteins (for review, see Canman and Kastan 1996). In most cases, MAPK activation is a transient event, even in the continuing presence of the stimulus that leads to its activation. MAPK activity is controlled by the balance of MAPK kinase and MAPK phosphatase activities.The dorsal closure of the Drosophila embryo provides an example of cell differentiation and how this is usually coupled to morphogenetic events and movements that shape late stages in development. Half way through embryogenesis, the dorsal surface of the embryo is covered by an extraembryonic membrane, the amnioserosa, which contacts the epidermis. After proliferation stops, the epidermis stretches dorsally and, as it encroaches the amnioserosa, closes the existing gap. Three phases lead to the successful completion of this event. The dorsalward movement of the epidermal cells, an anteroposterior stretching of the embryo and the seaming of the dorsal epidermis (Martinez-Arias 1993). 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...

Stress signaling in Drosophila

“…In contrast, multiple lines of evidence suggest that the essential function of Rac is the ultimate activation of Jun/Fos AP-1 and transcriptional regulation mediated through the JNK pathway. For example, mutations in hep can suppress activated Rac, and Puc phosphatase overexpression mimics the e ects on the LE cytoskeleton of both JNK loss-offunction and expression of DN-Rac (Glise and Noselli, 1997;Martin-Blanco et al, 1998). These data call into question the role of each GTPase in DC and the extent of branching signals upstream of the JNK pathway.…”

“…In fact puc loss-of-function mutant embryo lysates have increased JNK activity in kinase assays, and enhanced dpp expression in embryos (Martin-Blanco et al, 1998). In contrast, ubiquitous expression in embryos of wildtype Puc leads to dorsal cuticle holes and a loss of dpp expression indicating a substantial downregulation of JNK pathway activity (Martin-Blanco et al, 1998). Therefore, Puc serves a key regulatory role in JNK signaling as a transcriptional target of the pathway that builds up and feeds back negatively on the activity of Bsk to moderate signaling through the pathway.…”

“…Puc functions to moderate signaling through the JNK pathway by antagonizing the kinase activity of Bsk. In fact puc loss-of-function mutant embryo lysates have increased JNK activity in kinase assays, and enhanced dpp expression in embryos (Martin-Blanco et al, 1998). In contrast, ubiquitous expression in embryos of wildtype Puc leads to dorsal cuticle holes and a loss of dpp expression indicating a substantial downregulation of JNK pathway activity (Martin-Blanco et al, 1998).…”

“…puc encodes a dual-speci®city protein phosphatase of the VH-1 family of MAP kinase phosphatases (MKPs), that is localized specifically in LE cells (Martin-Blanco et al, 1998;Ring and Martinez-Arias, 1993). Puc functions to moderate signaling through the JNK pathway by antagonizing the kinase activity of Bsk.…”

Puckered‐GAL4 driving in JNK‐active cells

“…The GAL4/ UAS system (Brand and Perrimon, 1993), which functions in JNK-active cells, is a good tool to study in this respect. Here I engineer a GAL4 enhancer trap of the puckered (puc) gene, which encodes a MAPK phosphatase family member to inactivate JNK (Martín-Blanco et al, 1998). The expression of puc is known to be induced by the JNK signal, thereby creating a negative regulatory circuit of JNK signaling.…”