Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1β (PP1β). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1 genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. Here, we show genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw¹, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. We show that puc expression is upregulated in wing imaginal discs mutant for flw¹ and puc^A251 and that this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw¹ enhanced by puc. Together, we show that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs.

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PROTEIN phosphatase type 1 (PP1), a major class of serine/threonine protein phosphatases, is one of the most conserved proteins in the animal kingdom and has been found in all eukaryotes examined to date (LIN 1999). PP1 is involved in the regulation of many cellular functions, including glycogen mechanism, muscle contraction, and mitosis (reviewed in BOLLEN 2001; COHEN 2002). In vitro, the catalytic subunit of PP1 (PP1c) dephosphorylates a wide variety of substrates, while in vivo PP1c is bound to a number of different regulators that modify its substrate activity and specificity and target it to specific locations.

Drosophila melanogaster has four genes encoding PP1c. Three code for the PP1 type (Pp1-13C, Pp1-87B, and PP1-96A), while one gene, flapwing (flw, PP1β9C), codes for the PP1β type. Of these genes, Pp1-87B is the major PP1 form and contributes 80% of the total PP1 activity in third instar larvae (DOMBRÁDI et al. 1990). Although the amino acid sequences of PP1 and PP1β are extremely similar, they show structural differences that are conserved in vertebrates; i.e., Drosophila PP1 is homologous to mammalian PP1 and PP1, and PP1β is homologous to mammalian PP1β/. This suggests a conserved functional difference between the two subtypes.

We previously showed that flw has a single essential function as a nonmuscle myosin phosphatase that is nonredundant with PP1. Flw, but not PP1, binds specifically to the myosin phosphatase-targeting subunit MYPT-75D, the homologue of mammalian MYPT3 (VERESHCHAGINA 2004). Recently, phosphorylation of human MYPT3 was shown to activate MYPT3-associated PP1 toward cytoplasmic myosin regulatory light chain (YONG 2006). In Drosophila, MYPT-75D targets Flw to nonmuscle myosin, where it presumably dephosphorylates the nonmuscle myosin regulatory light chain, Spaghetti squash (Sqh), thereby deactivating nonmuscle myosin. In clones mutant for the strong allele flw⁶, the level of phosphorylated Sqh (P-Sqh) is elevated and the nonmuscle myosin heavy chain Zipper (Zip) is hyperactivated. Reducing the amount of active Zip suppresses the semilethality of flw⁶, indicating that regulation of nonmuscle myosin activity is the only essential function of flw. Conversely, expressing a constitutively active form of Sqh enhances the weak viable allele flw¹ toward complete lethality (VERESHCHAGINA 2004).

Another myosin phosphatase-targeting subunit in Drosophila is the myosin-binding subunit (Mbs), which is the homologue of mammalian MYPT1/2; it binds both Flw and Pp1-87B in vitro (VERESHCHAGINA 2004). The level of P-Sqh is elevated in Mbs mutant clones (LEE and TREISMAN 2004). This suggests that Mbs targets PP1c to its substrate Sqh, and therefore flw might not be the only Sqh phosphatase. However, it is likely that the two presumptive nonmuscle myosin phosphatases Mbs/PP1c and MYPT-75D/Flw are localized differentially in cells because MYPT-75D has a prenylation motif while Mbs does not (VERESHCHAGINA 2004). This, together with the specificity of MYPT-75D for Flw, may account for the evident lack of redundancy between Flw and other potential Sqh phosphatases.

Drosophila Jun N-terminal kinase (DJNK, basket, bsk) is a mitogen-activated protein kinase (MAPK) that regulates several essential developmental processes in Drosophila (dorsal closure, thorax closure, dorsal appendage formation) as well as wound healing (MARTIN-BLANCO et al. 1998; ZEITLINGER 1999; SUZANNE 2001; RAMET et al. 2002). JNK is phosphorylated and activated by the MAPK kinase hemipterous (Hep) and phosphorylates Jun and Fos, which together form the active transcription factor AP-1. AP-1 induces the transcription of a number of genes, among them puckered (puc), which encodes a JNK phosphatase and thus provides a negative feedback loop (reviewed in KOCKEL 2001).

Some target genes of JNK-activated AP-1 in dorsal closure are components of the actomyosin network, for example, Drosophila profilin (chickadee), Tropomyosin1 (Tm1), Tropomyosin2 (Tm2), and myosin light chain 2 (mlc-2) (JASPER 2001). Nonmuscle myosin also has an essential role in dorsal closure (shown for zip, sqh, and Mbs) (YOUNG 1993; JORDAN 1995; MIZUNO 2002).

Even though regulation of nonmuscle myosin and JNK is crucial for such processes as dorsal closure or wound healing, no genetic or molecular interaction between the two has been found so far. In this study, we show that the nonmuscle myosin phosphatase flw genetically interacts with members of the JNK pathway. Mutants in JNK (bsk) suppress the strong semilethal allele flw⁶, while expression of hep^CA, a hyperactive form of the JNK kinase, enhances the weak viable allele flw¹. Hypomorphic mutants in the JNK phosphatase puc also enhance flw¹, leading to severe wing defects. This genetic interaction is further enhanced by a mutant in the myosin-binding subunit Mbs. We show that flw negatively regulates puc expression in wing imaginal discs and that this regulatory action is modulated by JNK and the nonmuscle myosin heavy chain Zip. Finally, the level of diphosphorylated (active) JNK is elevated in protein extracts from flw¹/Y ; puc^A251/+ wing imaginal discs. Ectopic activation of JNK in wing imaginal discs is known to induce apoptosis (ADACHI-YAMADA et al. 1999a), suggesting that the wing phenotype exhibited by flw¹/Y ; puc^A251/+ flies is due to increased cell death. Together, we show that flw can activate JNK through a function in the regulation of nonmuscle myosin.

Fly genetics and wing preparations:

D. melanogaster stocks and crosses were reared at 25°, or at 18° where indicated, on yeast/glucose/wheat flour medium under standard conditions. Most stocks were obtained from the Bloomington Drosophila Stock Center (Indiana University). The stocks w ; P{sqh^AA} and w ; P{sqh^EE} were kindly provided by Roger Karess. The allele puc^JK0 was generated by imprecise excision of l(3)j4E1 with P{2-3} transposase (LASKI 1986). The full genotype for flw¹ is y, cho, flw¹. Wings from adult flies were dissected in 70% ethanol and mounted in mounting medium (85% glycerol, 2.5% n-propylgallate).

X-gal staining of wing imaginal discs:

Wing imaginal discs from third instar wandering larvae were dissected in PBS (137 mM NaCl, 3 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄), fixed 15 min in 4% paraformaldehyde in PBS, and washed with X-gal buffer (7.2 mM Na₂HPO₄, 2.8 mM NaH₂HPO₄, 1 mM MgCl₂, 15 mM NaCl) before being incubated at 37° in X-gal staining solution [X-gal buffer, 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆, 2% X-gal]. The staining reaction lasted between 1 and 4 hr. The tissues were washed in X-gal buffer and mounted in mounting medium.

SDS–polyacrylamide gel electrophoresis and immunoblotting:

Twenty wing imaginal discs were taken up in 10 mM Tris–HCl, pH 6.8, 180 mM KCl, 50 mM NaF, 1 mM NaVO₄, 10 mM β-glycerolphosphate, 1% Triton X-100, and 0.1% Tween 20 and stored at –80°. An equal volume of 2x SDS sample buffer (100 mM Tris–HCl, pH 6.8, 200 mM DTT, 4% SDS, 20% glycerol, bromophenol blue) was added, and the proteins were boiled for 5 min before loading on a 10% SDS–polyacrylamide gel. Separation of mono- and diphosphorylated JNK was achieved by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) at low voltage (50–60 V). Separated proteins were transferred onto Immobilon-P PVDF membrane (Millipore, Bedford, MA). The membrane was blocked with 5% fat-free powdered milk in TBST (137 mM NaCl, 3 mM KCl, 25 mM Tris, 0.1% Tween 20) and incubated with the appropriate antibody [1:500 rabbit polyclonal -P-JNK (Promega, Madison, WI), 1:1000 rabbit polyclonal -JNK (Santa Cruz Biotechnology), 1:2000 -tubulin (Sigma, St. Louis), and 1:10,000 horseradish-peroxidase-(HRP)-conjugated secondary antibody (Sigma)]. HRP was detected with Supersignal West Pico (Pierce, Rockford, IL) and blue sensitive X-ray film (GRI).

puckered is a genetic enhancer of flapwing:

flw¹ is a weak viable allele of Drosophila PP1β with indirect flight muscle defects (RAGHAVAN 2000), but otherwise normal morphology (Figure 1, A and B) and viability. In the process of analyzing mutants for the PP1c inhibitor Inhibitor-2 at 67C10, we found that Df(3L)AC1 (67A02-67D13) genetically enhances flw¹ to complete lethality. Further analysis in the 67A–D region identified the EMS-induced mutation l(3)67BDn (LEICHT 1988) to be lethal over Df(3L)AC1 and to strongly enhance flw¹. l(3)67BDn/+ heterozygotes have normal viability and wings (not shown), whereas flw¹/Y ; l(3)67BDn/+ was semilethal (90% of pupae fail to eclose), and surviving males exhibited strong wing defects (Figure 1C) and occasional third leg malformations (Figure 1C, arrow). Further analysis revealed that l(3)67BDn had a lethal mutation in the 67B–D region (below), but meiotic recombination mapping showed that the flw enhancing mutation was located between the markers scarlet (73A03) and curled (86D03-04). Deficiency mapping between those markers placed the flw enhancer in or near to 84F02. By testing available mutants in this region, we found that two mutants in puc at 84E10-11 (puc^A251 and puc^H246), as well as l(3)j4E1, a P{lacW} insertion in the second intron of puc (Figure 1E; SPRADLING 1999), failed to complement l(3)67BDn (not shown). All these alleles showed similar mutant phenotypes to each other and l(3)67BDn when combined with flw¹ (e.g., Figure 1D), although they were generally weaker than l(3)67BDn. flw¹/Y ; puc^A251/+; for example, they had normal viability, and only 30% exhibited wing phenotypes that ranged from slight defects in the posterior part of the wing to stumps. The third leg malformation (Figure 1D, arrow) was visible only with the most severe wing phenotypes. We hypothesized that the stronger enhancement of flw¹ by l(3)67BDn was due to second-site mutations on the l(3)67BDn chromosome and extensively cleaned up the l(3)67BDn chromosome by recombination, exchanging the 67A–67D region in the process. This reverted the lethality over Df(3L)AC1, suggesting that the original l(3)67BDn chromosome carries at least one additional mutation, possibly in the 67A–67D region. Df(3L)AC1 was viable over puc^A251, which suggested that the Df(3L)AC1 chromosome does not carry an allele of puc. The new l(3)67BDn recombinant genetically interacted with flw in the same way as puc^A251 and puc^H246 (not shown). We concluded that the primary flw enhancing mutation on the l(3)67BDn chromosome was an allele of puc and called it puc^l(3)67BDn.

View larger version (54K): In this window In a new window Download PPT slide   FIGURE 1.— (A–D) puc enhances flw. Adult males of (A) Oregon-R (OreR; wild type); (B) flw1/Y; (C) flw1/Y ; l(3)67BDn/+; and (D) flw1/Y ; pucA251/+. flw1/Y have wild-type wings (B), whereas introducing a mutant copy of either l(3)67BDn or pucA251 leads to severe wing defects (C and D) and occasional leg deformations (arrows). l(3)67BDn/+ and pucA251/+ appear wild type (not shown). (E) puc gene structure and mutants. Coding regions (black) and untranslated regions (lavender) are indicated. l(3)j4E1 and pucA251 are P insertions in the second intron of puc. pucJK0 is a 2.2-kb deletion.

 
To test whether the genetic interaction between flw¹ and l(3)j4E1 was due to the P insertion in puc, we remobilized the P element and tested the recovered w^– excision chromosomes for genetic interaction with puc^A251 and flw¹. While l(3)j4E1 did not complement puc^A251, we found that the majority of excisions were viable over puc^A251 and did not enhance flw¹, proving that l(3)j4E1 indeed carries a P-element-induced allele of puc and that this is responsible for the genetic interaction with flw. However, all of these excision derivatives were lethal as homozygotes, which suggests that the l(3)j4E1 chromosome carries a second lethal mutation. One w^– excision line, puc^JK0, failed to complement puc^A251 and enhanced flw¹ in the same way as the other puc alleles. Molecular analysis identified a 2.2-kb deletion with the left breakpoint in the second puc intron at the original P-element insertion site and the right breakpoint in the third exon, deleting the first 51% of the puc phosphatase catalytic domain (Figure 1E).

Since the amino acid sequences of all PP1c proteins are extremely similar (DOMBRÁDI 1993), we tested whether the genetic interaction between flw and puc is specific to flw. puc^A251 did not show any discernible mutant phenotype with Pp1-87B^87Bg-3, an allele of the major PP1c gene (not shown). Therefore, we concluded that puc specifically enhances flw.

Expression of constitutively active Hemipterous (hep^CA) enhances flw¹:

puc codes for a dual specificity phosphatase that dephosphorylates Drosophila JNK (bsk) (MARTIN-BLANCO et al. 1998). We wondered whether other members of the Drosophila JNK pathway also genetically interact with flw. The enzymatic antagonist to the JNK phosphatase Puc is the JNK kinase Hep (GLISE 1995). We expressed constitutively active Hep (UAS-hep^CA) with the wing-specific driver vg-Gal4 in the developing wing. At 25°, vg-Gal4/+ ; UAS-hep^CA/+ (vg>hep^CA) was lethal, but at 18° it was viable and exhibited some wing defects, most notably missing posterior wing-vein material, notches in the wing margins, and held-out wings (Figure 2B). This phenotype was enhanced in flw¹ hemizygous background. flw¹/Y ; vg>hep^CA wings (Figure 2D) were much smaller than the wings of their flw¹/+ female siblings (Figure 2C) and were frequently notched and blistered. Furthermore, flw¹/Y ; vg>hep^CA were only 10% viable relative to flw¹/+ ; vg>hep^CA (not shown). Thus, expression of hep^CA produced wing phenotypes that overlapped with flw¹/Y ; puc/+ in as much as posterior wing tissue was lost (see Figure 3D), and it also enhanced flw¹, even though the phenotypes of flw¹/Y ; vg>hep^CA differed slightly from flw¹/Y ; puc/+.

View larger version (59K): In this window In a new window Download PPT slide   FIGURE 2.— Overexpression of constitutively active hemipterous in the wing (vg>hepCA) enhances flw1. (A) OreR. (B) vg>hepCA. (C) flw1/+ ; vg>hepCA. (D) flw1/Y ; vg>hepCA. Wings of flw1/+ ; vg>hepCA exhibit blisters (arrow) and notched wing margins and resemble those of vg>hepCA (B and C), whereas flw1/Y ; vg>hepCA wings are much smaller (D). B–D were reared at 18°, as vg>hepCA is lethal at 25°.

 

View larger version (76K): In this window In a new window Download PPT slide   FIGURE 3.— pucA251 expression in wing imaginal discs and wing phenotypes. pucA251-lacZ expression in wing imaginal discs (A, C, E, G, I, and K) and adult wings (B, D, F, H, J, and L). (A and B) pucA251/TM6B. (C and D) flw1/Y ; pucA251/+. (E and F) flw1/Y ; bsk1/+ ; pucA251/+. (G and H) flw1/Y ; zip1/+ ; pucA251/+. (I and J) flw1/Y ; pucA251/P{sqhAA}. (K and L) pucA251/P{sqhEE}. Ectopic lacZ staining is visible in flw1/Y ; pucA251/+ wing imaginal discs (C; compare to A), and the resulting wings show various defects (D, missing posterior wing tissue). bsk1 and zip1 each abolish the ectopic lacZ staining (E and G) and the resulting wings look normal (F and H). P{sqhAA} inhibits neither the ectopic lacZ staining nor the wing phenotype of flw1/Y ; pucA251/+ (I and J). P{sqhEE} does not induce lacZ staining in a pucA251 mutant background (K), and pucA251/P{sqhEE} wings appear wild type (L).

 

basket (DJNK) mutants suppress flw⁶:

Both puc mutants and hep^CA overexpression should shift the equilibrium of nonphosphorylated (inactive) JNK and phosphorylated (active) JNK toward the latter. We therefore wondered whether hyperactivation of JNK was the molecular cause of the genetic interaction with flw¹. If so, reducing the level of JNK should reduce the amount of active JNK and might thereby have a suppressing effect on strong flw mutants. We found that two alleles of basket (bsk¹ and bsk²) both suppressed the semilethality of the strong allele flw⁶. The survival rate of flw⁶/Y is <1%, whereas the survival rate of flw⁶/Y ; bsk¹/+ was 26% (Table 1). bsk¹ also suppressed the strong and semilethal allele flw⁷ (not shown).

View this table: In this window In a new window   TABLE 1 bsk1 suppresses flw6 (from cross flw6/FM7c x bsk1/CyO, act-GFP)

 
Regulation of nonmuscle myosin activity is the only essential, nonredundant function of flw in Drosophila (VERESHCHAGINA 2004). The fact that bsk could suppress flw⁶, albeit more weakly than, for example, the flw⁶ suppressors zip or P{sqh^AA} (VERESHCHAGINA 2004), could mean that nonmuscle myosin regulation and the JNK pathway are biochemically linked. JNK could work either upstream of flw, for example, as an (indirect) activator of nonmuscle myosin, or downstream of flw, partly mediating a lethal response initiated by hyperactive Zip.

Flw is a negative regulator of puc expression in wing imaginal discs:

The allele puc^A251 is due to a P{lArB} insertion in the second intron of puc (Figure 1E) and has also been widely used as a puc enhancer trap, as the expression of the lacZ gene on the P element is under the control of a genomic puc enhancer (DOBENS 2001). X-gal staining showed that the only lacZ expression in wing imaginal discs of puc^A251 heterozygotes was background staining at the tip of the presumptive notum. However, in flw¹/Y ; puc^A251/+ wing imaginal discs, lacZ was ectopically expressed (Figure 3, A and C), suggesting that flw acts as a negative regulator of puc expression in wing imaginal discs. Since puc encodes a JNK phosphatase, we hypothesized that this upregulation of puc expression might be due to ectopic activation of JNK, which has been shown to induce apoptosis in wing maginal discs (ADACHI-YAMADA et al. 1999a). In flw¹/Y ; puc^A251/+ wing imaginal discs, lacZ was expressed in a distinct region that will later develop into the posterior part of the adult wing; this was also the area that was most affected in the mutant wings (Figure 3D).

Flw negatively regulates puc expression through Bsk and Zip:

The ectopic expression of puc in flw¹/Y ; puc^A251/+ suggested that flw acts upstream of puc expression. We hypothesized that flw might act upstream of JNK and tested this by introducing bsk¹ into a flw¹/Y ; puc^A251/+ background. In flw¹/Y ; bsk¹/+ ; puc^A251/+ wing imaginal discs we no longer observed ectopic lacZ expression, and the adult wings of this genotype appeared wild type (Figure 3, E and F). This argued that flw regulated puc expression through JNK in wing imaginal discs and that JNK might become hyperactivated in flw¹/Y ; puc^A251/+ flies.

We wondered in what way flw acts on JNK and puc expression. PP1c dephosphorylates many different proteins, but, for the majority of these functions, we assume that flw is redundant with the other PP1c forms, particularly Pp1-87B, which is present at much higher levels (DOMBRÁDI 1990). puc, however, genetically interacted specifically with flw and not with Pp1-87B. Since the single essential nonredundant function of Flw is inhibition of nonmuscle myosin activity, we wondered whether Flw might act on JNK and puc through nonmuscle myosin regulation. To test this, we introduced the mutant zip¹ into flw¹/Y ; puc^A251/+ background and tested for lacZ expression. Most flw¹/Y ; zip¹/+ ; puc^A251/+ wing imaginal discs did not show the ectopic expression of lacZ characteristic of flw¹/Y ; puc^A251/+ (Figure 3G). A small number of wing imaginal discs showed ectopic lacZ expression, but the staining was weaker and the affected area was always much smaller than in flw¹/Y ; puc^A251/+ wing imaginal discs (not shown). The adult wings of this genotype appeared wild type (Figure 3H). Altogether, this showed that flw regulated puc expression through zip as well as bsk and that zip, like flw, acts upstream of the regulation of puc expression.

Mbs³ enhances flw¹/Y ; puc^A251/+:

As described above, flw regulates puc expression through zip in wing imaginal discs. We wondered whether zip¹ suppressed flw¹/Y ; puc^A251/+ by compensating for a moderate hyperactivation of Zip in this mutant background. If so, we speculated that further activation of Zip in flw¹/Y ; puc^A251/+ might enhance the phenotype. Disrupting the function of the nonmuscle myosin phosphatase by mutations in the myosin phosphatase-targeting subunit should lead to increased activation of Zip. Unfortunately, no mutants have been described for the Flw-specific MYPT-75D. However, we found that the genotype flw¹/Y ; +, puc^A251/Mbs³, + is lethal (Table 2), providing further genetic evidence that hyperactivation of nonmuscle myosin can activate JNK.

View this table: In this window In a new window   TABLE 2 Mbs3 enhances flw1/Y ; pucA251/+ (from cross flw1/FM7 ; pucA251/TM6B x Mbs3/TM6B,Ub-GFP)

 

Sqh phosphorylation mutants do not alter puc expression:

Mutations in strong flw alleles lead to hyperphosphorylation of Sqh on T21 and S22, which induces hyperactivation of Zipper (VERESHCHAGINA 2004). Constructs expressing Sqh T21 and S22 phosphorylation mutants genetically interact with flw. In P{sqh^AA}, T21 and S22 are mutated to alanine; therefore Sqh^AA cannot be activated, while in P{sqh^EE} the same residues are mutated to glutamic acid, which mimics phosphorylation (JORDAN 1997). P{sqh^AA} suppresses the semilethality of flw⁶, whereas P{sqh^EE} enhances flw¹ to complete lethality, suggesting that P{sqh^AA} and P{sqh^EE} have a dominant-negative and constitutively active effect, respectively, although neither show a phenotype in a wild-type background. We used P{sqh^AA} and P{sqh^EE} to test whether hyperphosphorylation of Sqh could be the mechanism by which puc expression becomes elevated in flw¹/Y ; puc^A251/+. Both transgenes were expressed from their native promoter in a sqh⁺ background. If the ectopic expression of puc in flw¹/Y ; puc^A251/+ wing imaginal discs was due to hyperphosphorylation of Sqh, introducing P{sqh^AA} into this background might abolish the expression of puc. Conversely, expression of P{sqh^EE} might induce ectopic lacZ expression in a puc^A251 background. We found that neither was the case; there was still ectopic lacZ staining in flw¹/Y ; puc^A251/P{sqh^AA} wing imaginal discs (Figure 3I), and the resulting wings looked similar to flw¹/Y ; puc^A251/+ (Figure 3J). No ectopic lacZ expression was visible in puc^A251/P{sqh^EE} wing imaginal discs, and the wings of this genotype resembled wild type (Figure 3, K and L). Together, this suggested that hyperphosphorylation of Sqh at T21 and S22 did not cause ectopic puc expression in flw¹/Y ; puc^A251/+ wing imaginal discs.

The level of phospho-JNK is elevated in a flw¹/Y ; puc^A251/+ mutant background:

As shown in Figure 3E, introducing bsk¹ into a flw¹/Y ; puc^A251/+ background abolished ectopic puc-lacZ staining in wing imaginal discs. On a molecular level, this could mean that JNK is ectopically activated in flw¹/Y ; puc^A251/+ flies and activates puc-lacZ expression. JNK is activated by phosphorylation on T181 and Y183, which are located in a conserved loop of the kinase subdomain VIII (corresponding to T183 and Y185 in mammalian JNK1) (DERIJARD 1994). With an anti-P-JNK antibody raised against dually phosphorylated mammalian JNK that cross-reacts with Drosophila phosphorylated (phospho-) JNK (TATENO 2000), we examined phospho-JNK levels in extracts from wing imaginal disc. As a control, we used MS1096-Gal4/+ ; UAS-hep^CA/+ (MS1096>hep^CA), which drives constitutively active Hep in wing imaginal discs, resulting in high levels of phospho-JNK (Figure 4A). In flw¹/Y ; puc^A251/+ extracts, there were elevated levels of phospho-JNK, but not in flw¹/+ ; puc^A251/+ and wild type. We also noted the presence of a band of slightly faster mobility in MS1096>hep^CA, flw¹/Y ; puc^A251/+, flw¹, and puc^A251/TM6B, but not in wild type (Figure 4A). We assume that this band corresponds to mono-phosphorylated JNK, to which the antiphospho-JNK antibody has a low cross-reactivity. Only dually phosphorylated JNK is active (ANDERSON 1990). Taken together, JNK is ectopically activated in flw¹/Y ; puc/+ wing imaginal discs, but not in flw¹ and puc^A251/TM6B.

View larger version (32K): In this window In a new window Download PPT slide   FIGURE 4.— (A) JNK is ectopically activated in flw1/Y ; pucA251/+ wing imaginal discs. antiphospho-JNK detects dually phosphorylated (active) JNK in the control MS1096>hepCA and flw1/Y ; pucA251/+, but not in flw1/+ ; pucA251/+, flw1/Y, pucA251/TM6B, and wild type (OreR). A band of slightly faster mobility, probably corresponding to monophosphorylated JNK, can be detected in MS1096>hepCA, flw1/Y ; pucA251/+, flw1/Y, and pucA251/TM6B. (B and C) Model of interaction between Flw and JNK in wild type (B) and flw1/Y ; puc/+ (C). Active signaling is indicated by solid arrows, nonactive signaling by shaded arrows. Zip, Flw, and Mbs presumably act upstream of Hep. (B) Inhibition of Zip through Flw and Mbs, as well as wild-type puc, prevent aberrant activation of JNK (Bsk). (C) Failure of Zip inhibition through flw1, and of JNK dephosphorylation through a puc mutation, lead to elevated levels of phosphorylated and activated JNK in flw1/Y ; puc/+, inducing high expression of puc. di-P-JNK, diphosphorylated JNK; mono-P-JNK, monophosphorylated JNK.

 

In this study, we show that the nonmuscle myosin phosphatase flw interacts genetically with components of the JNK signaling pathway. The proteins JNK and PP1β (Flw), as well as the mechanisms of JNK signal transduction and nonmuscle myosin activation, are highly conserved between Drosophila and humans. This suggests that our findings of Drosophila PP1β regulating JNK through nonmuscle myosin may be relevant for similar processes in human cells and tissues.

bsk (JNK) mutants suppressed the semilethality of the strong allele flw⁶, while puc and constitutively active hep^CA enhanced the weak viable allele flw¹, resulting in severe wing defects. The level of diphosphorylated (active) JNK was elevated in a flw¹/Y ; puc^A251 mutant background. This, together with the finding that puc expression was upregulated in wing imaginal discs of flw¹/Y ; puc^A251/+, implies that flw can act as a negative regulator of JNK. This was further supported by the fact that puc expression was not upregulated in flw¹/Y ; bsk¹/+ ; puc^A251/+ wing imaginal discs, where reduced amounts of overall JNK (Bsk) protein probably compensate for elevated activity of JNK in flw¹/Y ; puc^A251/+. A possible difficulty with using puc^A251 [or puc^E69, another frequently used puc enhancer trap (MARTIN-BLANCO et al. 1998)] as a reporter of puc expression is that both lines are also puc mutants, and puc probably regulates its own expression through a negative feedback loop involving JNK (Figure 4B). However, we confirmed in an independent assay with an anti-P-JNK antibody that JNK was indeed ectopically activated in flw¹/Y ; puc^A251/+. Wing imaginal disc extracts from both flw¹ and puc^A251/+ showed somewhat elevated levels of monophosphorylated, but not diphosphorylated, JNK (Figure 4A). In flw¹/Y ; puc^A251/+, we suggest that dephosphorylation of JNK fails to such an extent that JNK is substantially diphosphorylated and thereby activated (Figure 4C). Since it has been shown that activation of JNK in wing imaginal discs induces apoptosis (ADACHI-YAMADA et al. 1999a,b), it is likely that the flw¹/Y ; puc/+ wing phenotype is due to increased death of cells with aberrantly activated JNK.

A zip mutant suppressed the upregulation of puc in wing imaginal discs as well as the adult wing phenotype of flw¹/Y ; puc^A251/+. This shows that nonmuscle myosin acts upstream of JNK and mediates an activating signal on JNK in a flw¹/Y ; puc/+ mutant background (Figure 4B); this is also consistent with our finding that a mutant in the myosin phosphatase-targeting subunit Mbs enhanced flw¹/Y ; puc^A251/+ to lethality. Drosophila encodes two myosin phosphatase-targeting subunits, Mbs and MYPT-75D. Mbs binds both Flw and Pp1-87B, while MYPT-75D binds Flw specifically (VERESHCHAGINA 2004). Unfortunately, we were not able to test for genetic interaction between flw¹/Y ; puc^A251/+ and MYPT-75D because no MYPT-75D mutants have been described. We also found that a Rho1 mutant abolished ectopic lacZ staining in flw¹/Y ; puc^A251 wing imaginal discs (not shown). Rho1 is an activator of Rho-dependent kinase, which phosphorylates and activates myosin light chain, as well as phosphorylating and inhibiting Mbs (DERIJARD 1994; MIZUNO 1999). Furthermore, both zip and Rho1, in combination with other genetic interactors, have been reported to show a malformed third-leg phenotype that resembles that of flw¹/Y ; puc/+ (HALSELL 1998; BAYER 2003).

Dorsal closure and wound healing depend on both nonmuscle myosin and JNK activity, but a clear genetic or molecular interaction between these pathways has not been previously demonstrated. This is the first study that shows that disruption of nonmuscle myosin can induce activation of JNK, although the mechanism of signal transduction is not clear. The single essential and nonredundant function of flw is the inhibition of nonmuscle myosin activity, presumably by dephosphorylating Sqh at T21 and S22. However, our results suggest that hyperphosphorylation of Sqh at these residues may not explain the elevated expression of puc in flw¹/Y ; puc^A251/+ flies. Additional mechanisms may exist to regulate nonmuscle myosin assembly and activity through phosphorylation (e.g., NISHIKAWA 1984; IKEBE 1990; MURAKAMI 1998), and the complex Flw/Mbs may have other targets in the actomyosin network in addition to Sqh. For example, moesin and focal adhesion kinase are potential targets for mammalian myosin phosphatase (FUKATA 1998; FRESU 2001). Interestingly, overexpression of the focal adhesion protein tensin (blistery) in Drosophila wing imaginal discs activates JNK and induces apoptosis (LEE et al. 2003).

Two important questions remain unanswered regarding our results on the activation of JNK through nonmuscle myosin. First, what is the molecular pathway from nonmuscle myosin to JNK? Several mechanisms have been identified that activate JNK and induce apoptosis in wing imaginal discs. For example, mutations in the caspase inhibitor DTraf1 (Drosophila tumor necrosis factor receptor-associated factor 1), as well as inhibition of DTraf1 by overexpression of Hid (head involution defective), Rpr (reaper), or Grim, induces JNK-mediated apoptosis (KURANAGA 2002; RYOO 2004), possibly through Msn (misshapen) or Ask1 (apoptosis signal-regulating kinase 1) (LIU 1999; KURANAGA 2002). Other factors involved in inducing apoptosis and activating JNK are Eiger (Drosophila tumor-necrosis factor superfamily ligand) (IGAKI 2002), its receptor Wengen (KAUPPILA 2003), Src42A (TATENO 2000), the serine C-palmitoyltransferase Lace (ADACHI-YAMADA et al. 1999b), Blistery (Drosophila Tensin) (LEE et al. 2003), and Decapentaplegic and Wingless (ADACHI-YAMADA et al. 1999a). It is not clear how these factors signal to JNK or whether they act in a single pathway, let alone whether any of them interact with nonmuscle myosin. It is likely that there are several independent ways of activating JNK, and it has been suggested that induction of apoptosis through JNK activation is a regulatory mechanism to eliminate abnormally developing cells in wing imaginal discs (reviewed in ADACHI-YAMADA and O'CONNOR 2004). This leads directly to our second question: Are our results of nonmuscle myosin signaling to JNK significant in a developmental context? The fact that bsk¹ suppressed the semilethality of flw⁶ indicates that the interactions that we have uncovered are not confined to our main experimental model of ectopic JNK activation in wing imaginal discs. The obvious system for studying possible interactions between nonmuscle myosin and JNK would be dorsal closure, which depends on both the coordinated assembly and the contraction of the actomyosin cytoskeleton and on activation of JNK (reviewed in JACINTO 2002). So far, there is no evidence that dorsal closure is affected in flw mutant embryos; however, there is a maternal contribution of flw that may conceal embryonic phenotypes. Actomyosin and JNK do not promote dorsal closure completely independently from each other; for example, the expression of many components of the actomyosin cytoskeleton is upregulated in response to JNK (JASPER 2001). Also, actin and myosin failed to accumulate along the leading edge of the epidermis in the puc mutant background (MARTIN-BLANCO et al. 1998). Both findings would place the actomyosin cytoskeleton downstream of JNK, whereas our genetic data place flw and zip upstream of JNK and puc. It is possible, however, that actomyosin acts both upstream and downstream of JNK during dorsal closure. Because of the conserved nature of the components involved, it is likely that our finding that nonmuscle myosin can signal to and activate JNK is relevant to furthering our understanding of processes like dorsal closure and wound healing in Drosophila and humans.

The authors thank Karen Clifton for technical assistance, Roger Karess for providing P{sqh^AA} and P{sqh^EE}, and the Bloomington Stock Center for providing the mutant fly strains. We also thank Helen White-Cooper and Esther Duperchy for help and advice and critical reading of this manuscript. This work was supported by grants from the United Kingdom Biotechnology and Biological Sciences Research Council and the United Kingdom Medical Research Council. D.B. is a Todd Bird Research Fellow at New College, Oxford.

¹ Present address: Abbott Laboratories Global Pharmaceutical Regulatory Affairs, Abbott Park, IL 60064-6157.

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