puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila - PubMed (original) (raw)

Comparative Study

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

E Martín-Blanco et al. Genes Dev. 1998.

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.

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Figures

Figure 1

Figure 1

Characterization of the puc gene and predicted protein. (A) Genomic organization of the puc locus. Structure of cDNA exons (shaded boxes) and introns (broken lines) is shown below the genomic map. Exon/intron boundaries are approximate to within the restriction fragment indicated. The P-element integration site of pucB48 is located in the first intron of cDNA12, pucE69, pucA251.1, and puc320 are located within the second intron of cDNA12. (E) _Eco_RI; (H) _Hin_dIII; (B) _Bam_HI; (S) _Sal_I; (X) _Xho_I. (B) DNA sequence of puc cDNA12 and predicted amino acid sequence. Identified motifs are the signature sequence for PTPases (boxed) and potential MAPK phosphorylation sites (P/L-X-S/T-P; circled letters).

Figure 1

Figure 1

Characterization of the puc gene and predicted protein. (A) Genomic organization of the puc locus. Structure of cDNA exons (shaded boxes) and introns (broken lines) is shown below the genomic map. Exon/intron boundaries are approximate to within the restriction fragment indicated. The P-element integration site of pucB48 is located in the first intron of cDNA12, pucE69, pucA251.1, and puc320 are located within the second intron of cDNA12. (E) _Eco_RI; (H) _Hin_dIII; (B) _Bam_HI; (S) _Sal_I; (X) _Xho_I. (B) DNA sequence of puc cDNA12 and predicted amino acid sequence. Identified motifs are the signature sequence for PTPases (boxed) and potential MAPK phosphorylation sites (P/L-X-S/T-P; circled letters).

Figure 2

Figure 2

Homology of Puc to VH-1 family phosphatases. (A) Sequence alignment (ClustalV) of Puc and other VH-1 family phosphatases [Drosophila Puc; human CL100 (Keyse and Emslie 1992); human Pac1 (Rohan et al. 1993); human HVH-2 (Guan and Butch 1995); human HVH-3 (Ishibashi et al. 1994; Kwak and Dixon 1995); human HVH-5 (Martell et al. 1995); human Pyst1 (Groom et al. 1996); human Pyst2 (Groom et al. 1996); human MKP-4 (Muda et al. 1997); C. elegans CEL-F08B1 (Wilson et al. 1994) and Saccharomyces cerevisiae MSG5 (Doi et al. 1994)]. Identical residues are in black, conservative changes in blue. CEL-F08B1, Pyst1 and homologs, Pyst2, and HVH-5 gave the highest homology scores to Puc in BLAST/BEAUTY searches (BCM Launcher). The other enzymes complete the whole series of distinct human dual phosphatases isolated so far. Yeast MSG-5, which share some characteristics with Puc, is also included. Phylogenetic trees (DNAstar program) point to the C. elegans CEL-F08B1 as the closest relative of Puc in the databases. CEL-F08B1 has been identified recently in the C. elegans Genome Project, but its function is unknown. Residues in the alignment highlighted in red represent putative MAPK phosphorylation sites. Interestingly, they seem to cluster for almost every protein in a low homology region at the carboxy-terminal end of the catalytic domain (double underlined), which suggest a possible functional homology. (B) Matrix alignment of Puc with itself shows the existence of an internal repeat in the protein. These domains correspond to the putative MAPK phosphorylated region and a further sequence close to the carboxy-terminal end of the molecule. Again, in the second repeat, several tentative phosphorylation sites can be identified.

Figure 3

Figure 3

puc encodes a JNK phosphatase. (A) In vitro phosphatase activity of a GST–Puc fusion protein. Results of PNPP assay in which cleavage of PNPP was measured by change in absorbance at 410 nm as a function of added protein. (•) Purified GST–Puc protein; (○) control points from extracts obtained from untransformed bacteria following similar protocols. (Bottom) Schematic representation of the fusion protein. The phosphatase catalytic domain is highlighted in black (residues 214–222). (B) Endogenous JNK and ERK activity of wild-type (wt), pucE69/pucE69 (puc) and hep1/hep1 (hep) embryos. (Top) JNK assays were performed with normalized amounts of embryo extracts (1 embryo/μl) prepared in the presence of phosphatase inhibitors (as indicated in Materials and Methods). Kinase activity is measured in arbitrary units from imaging analysis. (Solid circles) Wild-type (wt) extracts; (shaded triangles) puc embryo extracts; (shaded squares) hep embryos. JNK activity increases twofold in puc mutants and reduced threefold in hep. (Bottom) ERK assays were performed by in-gel kinase assay with a normalized amount of extract, in the linear range for JNK, equivalent to five embryos. Histograms represent quantitation of kinase activity (arbitrary units). Wild-type, puc, and hep extracts have equivalent levels of ERK activity. (C) Puc phosphatase activity on heterologous JNK and ERK. (Top) JNK activity induced in HeLa cells was measured in the absence of any extract to deduce the basal level of activity (100% JNK activity—broken line). Equivalent amounts were incubated with normalized embryo extracts (1 embryo/μl) prepared in the absence of phosphatase inhibitors. The results are expressed in percentage of JNK activity. (Solid circles) Wild-type extracts; (shaded triangles) puc embryo extracts; (shaded squares) hep embryos. Wild-type embryos have high levels of JNK phosphatase activity (HeLa JNK activity is reduced fivefold). Puc extracts do not show JNK phosphatase activity, indeed HeLa JNK activity gets increased because of the high levels of JNK activity of puc extracts (it can be brought back to basal levels by previous heat inactivation; see also Discussion). In hep extracts, JNK phosphatase activity is reduced to 50% of that of wild-type embryos. (Bottom) ERK activity of preactivated human ERK (hERK*) was assayed as indicated in Materials and Methods. Extracts (5 embryos) from wild-type, puc, and hep embryos display the same level of ERK phosphatase activity, reducing hERK* activity by 40%. Histograms represent quantitation of kinase activity (arbitrary units). Positive controls were performed with purified CL100 phosphatase (50 μg/ml) (data not shown).

Figure 4

Figure 4

puc expression: Its modulation by puc activity. (A) Northern analysis of puc RNA expression in embryos at various times during development. (A) 0–4 hr; (B) 4–8 hr; (C) 8–12 hr; (D) 12–16 hr; and (E) 16–20 hr. The 2.9-kb puc transcript is apparent. (B) puc RNA detected in stage 13 by whole mount in situ hybridization. The expression in the dorsal-most epidermal cells is indicated. (C) stage 13 pucE69 heterozygous embryos stained with an antibody against β-galactosidase. The arrowhead points to the cells of the leading edge of the epidermis expressing β-gal. Notice that these are the same cells as in B. At early stages, evident puc expression is present in amnioserosa cells. (D) β-galactosidase expression of puc320 heterozygous embryos (stage 14). (F) A considerably higher number of cells, and at higher levels, express β-galactosidase in puc320/puc320 stage 15 embryos (arrowhead).

Figure 5

Figure 5

Cuticle phenotypes of puc mutants and those generated by ectopic expression of puc and dpp. Rescue of hep phenotype by puc in heterozygous condition. (A) Dorsal view of pucEh/pucEh embryo. (B) Dorsolateral view of pucE69/pucE69 mutant embryos. (C) Dorsolateral view of ArmGal4/UASdpp embryo. Arrowheads point to defects, puckering, and naked cuticle, along the dorsal midline. (D–E) Embryos from a cross of flies carrying the UASpuc with flies carrying a hsGal4 insert that were exposed to a 30 min heat shock at various times during development. Cuticle preparations revealed three classes of phenotype depending on the age of the embryo at the time of heat shock. (D) puc loss of function-like; (E) dorsal hole; (F) dorsal open. Arrowheads indicate dorsal cuticle defects. Dorsal open embryos are observed more frequently after early heat shocks. Dorsal hole phenotypes appear at intermediate times and puc loss-of-function-like embryos are present mainly after late heat shocks (see columns at the right). (G) hep1 hemizygous embryo, note the dorsal open phenotype. (H) hep1 hemizygous, pucE69 heterozygous embryo. A partial rescue of the dorsal open phenotype leads to small dorsal holes (arrowhead).

Figure 6

Figure 6

puc controls the accumulation of actin and myosin in the leading edge of the epidermis during dorsal closure. Confocal fluorescent micrographs of the boundary between the amnioserosa and the epidermis in stage 13 embryos. The distribution of nonmuscle myosin (A,B,D,E,G,H) and filamentous actin (C,F,I) are shown in wild-type embryos (A–C), pucE69 embryos (D–F) and ArmGal4/UASpuc embryos (G–I). Embryos were stained for filamentous actin with phalloidin or for nonmuscle myosin with antibodies. Whereas actin and NMM are accumulated along the leading edge in wild-type embryos (arrowheads in B and C), in puc mutants their level decreases and it is possible to observe gaps (arrowheads in E and F) between the amnioserosa and the epidermis. After Puc overexpression, NMM is maintained in the amnioserosa (arrowhead in H) but the level of expression in the epidermis is severely reduced. Actin ceases to be expressed in the amnioserosa and it appears on patchy spots in the epidermis (arrowhead in I).

Figure 7

Figure 7

Effects of wild-type, mutant pucE69 and Puc expressed under the control of the Armadillo promoter on dpp transcription. (A,D,G) dpp RNA expression in ArmGal4/UASpuc embryos. (B,E,H) dpp RNA expression in wild-type embryos. (C,F,I) dpp RNA expression in pucE69 embryos. (A–C) lateral views of stage 11 embryos. Anterior is to the left; dorsal is up. At this early stage, it is possible to observe a reduction in dpp expression after ectopic expression of Puc and new cells expressing dpp along the epidermal border in puc mutants (arrowheads). (D–F) lateral views of stage 13 embryos. (G–I) dorsal views of stage 13 embryos. At this stage, dpp disappears from the dorsal-most epidermal cells after ectopic Puc expression and it is present in at least two rows of cells at the leading edge of each lateral hemisegment in puc mutants (arrowheads). The expression of dpp in the visceral mesoderm is unaffected.

Figure 8

Figure 8

A model for the role of puc in JNK signaling during dorsal closure. Halfway during embryogenesis, in the cells at the leading edge of the epidermis, the hep/bsk pathway becomes activated, probably by Drac. As a consequence, DJun is itself activated and gets involved in the maintenance of dpp and puc expression. Puc will drive its own down regulation through inactivation of bsk, and it will control the level of expression of dorsal closure effectors as dpp. dpp might have two different roles: to induce the cellular events required for dorsal closure in the lateral cells and to participate in the specializations in the dorsal-most cells required for the last steps of closure. In puc mutants, JNK signaling becomes hyperactivated in the leading edge, the dorsal-ward stretching of the lateral cells proceeds normally, but the excess of dpp interferes with proper cell differentiation and affects midline alignment. When puc is ectopically expressed early throughout the epidermis, it blocks signaling through the bsk pathway leading to the disappearance of dpp from the dorsal-most cells and to a failure in dorsal closure. Late Puc overexpression it affects only the cellular differentiation of the leading edge cells.

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