Research Article 3177

IntroductionProtein kinases are a large and diverse superfamily that impactsvirtually every fundamental process in cells (Manning et al., 2002).Mechanisms that regulate these enzymes are correspondinglydiverse, including intra- and intermolecular interactions,posttranslational modification, subcellular compartmentalization,and feedback. Despite common themes in the regulation of proteinkinase activity, the molecular details vary among kinase familiesand cell context. Consideration of these differences is imperativeto fully understand the nature of substrate specificity, theconsequences of kinase misregulation, and the potential for targetedtherapeutic inhibitors and agonists. Various methodologies,including structural studies and in vitro manipulations, haveincreased our understanding of kinase biology significantly,however in vivo support for regulatory models derived from invitro studies will help to consolidate physiologically relevantregulatory mechanisms.

In this report, we examine the molecular mechanisms regulatingactivation of the Drosophila mixed-lineage kinase (MLK), encodedby the slpr locus (Stronach and Perrimon, 2002), which is amember of the tyrosine-like kinase group (Manning et al., 2002).MLKs were named for their mixed homology kinase domains,with residues matching both tyrosine and serine/threonine kinases(Dorow et al., 1993); however, biochemical assays demonstratespecificity for serine and threonine residues (Gallo et al., 1994).MLKs are mitogen-activated protein kinase kinase kinases(MAP3Ks) that phosphorylate and activate MAP2K dual-specificitykinases, which in turn stimulate MAPKs of the Jun N-terminalkinase (JNK) and p38 families (Hirai et al., 1997; Kiefer et al.,1996; Rana et al., 1996; Teramoto et al., 1996; Tibbles et al.,1996). Seven mammalian MLKs have been identified, clusteringinto three subfamilies: the core MLKs (MLK1–4), the dual leucine

zipper kinases (DLK and LZK), and the zipper sterile-a-motifkinase (ZAK) (for a review, see Gallo and Johnson, 2002). Allfamily members activate the JNK pathway when overexpressed incultured cells (Hirai et al., 1997; Liu et al., 2000; Merritt et al.,1999; Rana et al., 1996; Tibbles et al., 1996); however, theirendogenous activities and regulation in response to distinct signalshave been more difficult to discern (Craig et al., 2008). Geneticanalyses using invertebrate models have shed light on functionsfor MLK and DLK family members in vivo. For instance, ourprevious studies implicated the Drosophila MLK, Slpr, in regulatingJNK-dependent tissue morphogenesis (Polaski et al., 2006; Stronachand Perrimon, 2002), whereas the nematode mlk-1 gene is requiredfor stress response to heavy metals (Mizuno et al., 2004). BothDrosophila and C. elegans DLK genes regulate neuronal synapticstructure and function via JNK or p38 MAPKs, respectively(Collins et al., 2006; Hammarlund et al., 2009; Nakata et al.,2005). The functional link between DLKs and nervous systemdevelopment appears to be conserved in mammals as well (Hiraiet al., 2006; Itoh et al., 2009). Targeted gene disruption of murineMLK core family members has been less revealing. Mlk1, Mlk2double knockout mice appear normal, whereas Mlk3 mutant miceare viable but abnormal in some cytokine and metabolic stresssignaling pathways (Bisson et al., 2008; Brancho et al., 2005;Jaeschke and Davis, 2007). Genetic analysis of ZAK has not beenreported, although expression studies suggest a role in hypertrophicgrowth of cultured cardiomyoblasts, consistent with its expressionin heart tissue (Huang et al., 2004; Liu et al., 2000).

Core MLKs have a Src-homology 3 (SH3) domain, a kinasedomain, tandem leucine zippers (LZ) followed by a Cdc42-Racinteractive binding motif (CRIB) (Burbelo et al., 1995) and a longdivergent C-terminus (Gallo and Johnson, 2002). Maximalactivation of mammalian MLK3 protein in cultured cells is a

Regulation of mixed-lineage kinase activation inJNK-dependent morphogenesisRebecca A. Garlena*, Rebecca L. Gonda*, Alyssa B. Green, Rachel M. Pileggi and Beth Stronach‡

University of Pittsburgh, Department of Biological Sciences, Pittsburgh, PA 15260, USA*These authors contributed equally to this work‡Author for correspondence (stronach@pitt.edu)

Accepted 30 May 2010Journal of Cell Science 123, 3177-3188 © 2010. Published by The Company of Biologists Ltddoi:10.1242/jcs.063313

SummaryNormal cells respond appropriately to various signals, while sustaining proper developmental programs and tissue homeostasis.Inappropriate signal reception, response or attenuation, can upset the normal balance of signaling within cells, leading to dysfunctionor tissue malformation. To understand the molecular mechanisms that regulate protein-kinase-based signaling in the context of tissuemorphogenesis, we analyzed the domain requirements of Drosophila Slpr, a mixed-lineage kinase (MLK), for Jun N-terminal kinase(JNK) signaling. The N-terminal half of Slpr is involved in regulated signaling whereas the C-terminal half promotes cortical proteinlocalization. The SH3 domain negatively regulates Slpr activity consistent with autoinhibition via a conserved proline motif. Also, likemany kinases, conserved residues in the activation segment of the catalytic domain regulate Slpr. Threonine 295, in particular, isessential for function. Slpr activation requires dual input from the MAP4K Misshapen (Msn), through its C-terminal regulatorydomain, and the GTPase Rac, which both bind to the LZ–CRIB region of Slpr in vitro. Although Rac is sufficient to activate JNKsignaling, our results indicate that there are Slpr-independent functions for Rac in dorsal closure. Finally, expression of various Slprconstructs alone or with upstream activators reveals a wide-ranging response at the cell and tissue level.

Key words: Drosophila, JNK signaling, Dorsal closure, Kinase

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multistep process, involving GTPase binding, relief of inhibition,dimerization and autophosphorylation (Bock et al., 2000; Leungand Lassam, 1998; Leung and Lassam, 2001; Vacratsis and Gallo,2000; Zhang and Gallo, 2001). Complex multistep regulation ofkinase activation has been studied extensively for members of theRaf and Src families, revealing that autoinhibition, membranerecruitment and regulatory phosphorylation are recurring themes(Boggon and Eck, 2004; Leicht et al., 2007).

To gain a better understanding of the mechanisms cells employto engage kinases in signal transmission, we are examining thesteps of Slpr activation during Drosophila embryonic dorsal closure.Among the MAP3Ks in Drosophila, Slpr is the only core MLKprotein (Stronach, 2005), facilitating genetic analysis of itsregulation in vivo. slpr was first identified as a locus required forJNK signaling during the process of dorsal closure (Stronach andPerrimon, 2002) wherein the ectoderm on each flank of the embryois pulled toward the dorsal midline enclosing the embryo in aseamless epidermis (Harden, 2002; Jacinto et al., 2002b; Kiehartet al., 2000). Molecular markers reporting JNK signaling activityare present in cells leading the advancing epithelium, and mutationsin JNK pathway components impair dorsal closure by disruptingboth gene expression and organization of the cytoskeleton (Gliseet al., 1995; Hou et al., 1997; Jasper et al., 2001; Kaltschmidt etal., 2002; Kockel et al., 1997; Riesgo-Escovar et al., 1996; Slusset al., 1996; Stronach and Perrimon, 2002; Xia and Karin, 2004).

Although the core members of the Drosophila JNK pathwayhave been identified, the mechanism of pathway activation in theectoderm during tissue closure is still poorly understood. Geneticevidence is consistent with Slpr functioning downstream of two

inputs, the Rac GTPases and the MAP4K Misshapen (Stronachand Perrimon, 2002; Su et al., 1998), yet a mechanisticunderstanding of how they trigger Slpr activity has not beenrealized. In the work reported here, we examined the regulation ofSlpr activation through a structure–function analysis of mutatedand modified forms in vivo, in the context of tissue closure. Ourdata support a model for Slpr activation that includes multipleinputs, dual regulation by the SH3 domain, and modulation ofsubcellular distribution by the C-terminal domain. Moreover, wedemonstrate a physical interaction between Slpr and Misshapen(Msn) that corroborates genetic data supporting their role in dorsalclosure. Altogether, the modified forms of Slpr constitute a broadrange of activities that define important functional domains andcontribute to our understanding of how Slpr modulates JNK-dependent morphogenesis.

ResultsSlpr structure–function analysisTo dissect the mechanisms that regulate Slpr activity in vivo, wehave generated derivative constructs that harbor mutations ordeletions of various domains and/or motifs conserved among MLKfamily members (Fig. 1). Their properties were assessed intransgenic animals in the following manner. First, to identifyessential functions, transgenes were tested for the ability to rescuesemi-viable and lethal slpr mutants (Fig. 2). Second, to indirectlyassay catalytic activity in vivo, we asked what effect transgenicprotein expression had on JNK signaling in the embryo duringdorsal closure, using the puc-lacZ reporter, pucE69 (Fig. 3). Puc-lacZ is normally evident in the leading row of ectodermal cells

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Fig. 1. Domain architecture and expression of Slpr andderived transgenic constructs. (A)Organization and positionof Slpr domains (SH3, Src homology 3; LZ, leucine zipper;CRIB, Cdc42–Rac interactive binding) and mutant alleles.Constructs are shown as black lines with mutated amino acidsnoted. The kinase activation segment, flanked by conservedresidues (underlined), shows putative Ser/Thr phosphorylationtargets (bold). The sequence of the LZ–CRIB linker shows theproline residues (bold) that were targeted for mutagenesis.(B)Alignment of the activation segments of Drosophila Slpr(Dme) and its human (Hsa) homologs. Conservedphosphorylation sites are indicated by grey boxes. Black boxesidentify experimentally determined primary phosphosites in thehuman MLKs. (C)Sequence alignment of the LZ–CRIB linkersof Slpr and its human homologs. Note the conserved prolineresidue(s) followed by a stretch of basic amino acids (greyboxes). (D)Western immunoblots of embryonic lysatesexpressing HA-epitope-tagged transgenic proteins. b-tubulinwas used as a loading control. Transgenic proteins were stablyexpressed with the exception of the Cterm (asterisk), whoselevels are reduced.

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undergoing closure and has been used extensively as a marker toevaluate JNK pathway activity. Third, to discern the impact oftransgene expression on morphogenetic processes known to requireendogenous Slpr function, dorsal and thorax closure were monitoredfor completion (Figs 2, 3). Finally, by correlating the activity of thetransgenic Slpr derivatives with their subcellular protein localizationin cells, we have identified protein domains and motifs that regulateSlpr distribution and function (Fig. 4).

Mutations in activation segment residues disrupt SlprfunctionThe catalytic domain of many kinases includes a flexible activationsegment, bounded by conserved DFG and APE motifs, which isoften targeted for auto- and trans-phosphorylation (Nolen et al.,2004). Upon modification, the segment adopts a conformation thatpromotes substrate binding and catalysis (Nolen et al., 2004). Theactivation segment of Slpr contains conserved serine and threonineresidues that are putative phosphorylation sites (Fig. 1B). If theseresidues are important for Slpr kinase structure or function, specificmutations are expected to decrease Slpr activity accordingly. Site-directed mutagenesis of all three Ser/Thr residues in the activationsegment of Slpr to unphosphorylatable alanine (TST>AAA) rendersthe protein nonfunctional, based on several criteria. First, Slpr-AAA fails to rescue slpr mutant alleles when expressed ubiquitouslywith arm-G4 (Fig. 2A). Moreover, it behaves as a dominantnegative protein with respect to stimulation of JNK pathway targetgene expression and dorsal closure (Fig. 3Ac,Bb). Importantly,the transgenic protein is expressed at levels equivalent to the wild-type form (Fig. 1D) and localizes in a subcellular patternindistinguishable from the wild-type form (Fig. 4Bc), demonstratingthat mutating the activation segment does not result in a wholesaledestabilization of the protein. Hence, these data show that Slpr-AAA is equivalent to a catalytically dead form (Slpr-KD), whichsuggests that the Ser/Thr residues are essential for function in vivo.

To pinpoint which residue is of primary importance, additionalconstructs were generated to produce proteins with two of thethree residues changed to alanine (double A mutants, e.g. TST toTAA, ASA or AAT; Fig. 1A). Slpr-TAA and Slpr-ASA and the triplealanine mutant produced similar results in the rescue, JNK signalingand dorsal closure assays (Fig. 2A, Fig. 3Ad-e,Bc-d), suggestingthat they are nonfunctional. Notably though, the Slpr-AAT mutanthad a notable effect in JNK signaling and embryonic dorsal closure(Fig. 3Af,Be). Moreover, ubiquitous expression of Slpr-AATresulted in recovery of a few rescued adults with the lethal slpr921

and slpr3P5 alleles (Fig. 2A). Indeed, many additional rescuedmales are observed as pharate adults (still in the pupal cases)among the progeny of these experimental crosses, implying that asignificant fraction of embryos survive dorsal closuremorphogenesis, whereas few if any rescued pharate males survivedwith expression of Slpr-AAA, -TAA, or -ASA. Together these dataimplicate threonine 295 as a crucial residue for Slpr-mediated JNKsignaling.

The SH3 domain of Slpr negatively regulates JNKsignalingMany kinases that are stimulated by extracellular signals (e.g. Raf,Src) are inactive until a signal exceeds an activating threshold.Commonly, inducible kinases possess their own inhibitory domainsto silence activity until the appropriate time. For mammalianMLK3, the SH3 domain is implicated in autoinhibition by bindingto a proline-containing motif between the LZ and CRIB domains

(Zhang and Gallo, 2001). However, the CRIB domain of yeastSte20 kinase is implicated in negative autoregulation of its kinasedomain, raising alternative possibilities (Lamson et al., 2002). Todetermine whether the SH3 domain of Slpr regulates its activity invivo, three constructs were generated, SH3 only, DSH3 and Slpr-AVA (P472A, P474A; Fig. 1A,C). Expression of SH3 alone inhibitedJNK signaling in the embryo (Fig. 3Aj). Nevertheless, it waspossible to recover some adults that displayed a severe cleft thoraxphenotype indicative of aberrant thorax closure and reduced JNKactivity (Fig. 3Cg) (Agnes et al., 1999; Zeitlinger and Bohmann,1999). Moreover, ubiquitous expression did not complement slprmutants, but rather reduced recovery of slprBS06 mutants relative tothe control (Fig. 2A) and failed to rescue the closure phenotype ofthe embryonic lethal slpr921 allele (Fig. 2B, supplementary material

3179Regulation of Drosophila MLK in vivo

Fig. 2. Rescue of slpr mutants by ubiquitous transgene expression. (A)Slprtransgenes (y-axis) were expressed with arm-G4 in each slpr mutantbackground (see Fig. 1 and its legend). The number of rescued slpr mutantmales relative to FM7 male siblings was plotted as a percentage ‘rescue toadulthood’. The graph is based on data (supplementary material Table S1)from crosses with at least three transgenic lines per construct. Values >100%indicate that the recovery of rescued males exceeded sibling controls.(B)Rescue of the embryonic dorsal closure phenotype of slpr921 mutants asindicated by reduction in embryos with a severe phenotype. Representativecuticles are shown on the right. Raw data are given in supplementary materialTables S2, S3. Cuticles of slpr mutant progeny were unambiguously identifiedthrough genetic linkage with the gt mutant phenotype.

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Tables S2, S3). Thus, like Slpr-KD and Slpr-AAA, the SH3 domainalone has dominant negative properties, disrupting endogenousSlpr-dependent JNK activation. By contrast, expression of aconstruct lacking the SH3 domain (DSH3) stimulated JNK signalingabove that of Slpr-WT overexpression, with substantial upregulationof puc-lacZ away from the leading edge (Fig. 3Ak), epithelialpuckering at the dorsal midline (not shown) and adult thorax

phenotypes indicative of hyperactive JNK signaling (Fig. 3Cf)(Zeitlinger and Bohmann, 1999). Also, DSH3 weakly rescuesslprBS06 mutants, resulting in an ~12% increase in recovery overanimals without the transgene (Fig. 2A; supplementary materialTable S1), and substantially rescues the embryonic dorsal openphenotype of slpr921 mutants (Fig. 2B; supplementary materialTables S2, S3), consistent with activation of JNK signaling.Altogether, these data support the hypothesis that the SH3 domainof Slpr is a negative regulatory domain; however, they do notreveal whether it inhibits Slpr activity by an intramolecularinteraction.

To address this, a mutant was generated in which two prolineresidues, located between the LZ and CRIB domains of Slpr, werereplaced by alanine (PVP>AVA). These residues are in a conservedregion (Fig. 1C) important for SH3 binding in mammalian MLK3(Zhang and Gallo, 2001). If the proline residues constitute a bindingsite for SH3 domain autoinhibition, then mutations that abrogatebinding are expected to promote activation. Indeed, the Slpr-AVAprotein is remarkably active. Embryonic expression of Slpr-AVA inthe pnr-G4 domain resulted in a fivefold upregulation of puc-lacZ(Fig. 3Al, Fig. 5B) and a concomitant increase in the severity ofthe adult thorax phenotype relative to DSH3 expression (Fig. 3Cf,i),although very few of these adults were recovered. Together, thesedata corroborate results of in vitro assays that the SH3 domainfunctions in negative regulation, and the inhibitory site in theLZ–CRIB linker region is functionally conserved. In addition, ouranalysis reveals a potential positive contribution of the SH3 domain.

The Slpr C-terminus regulates protein distributionThe C-terminal half of MLK proteins is the least conserved amonghomologs although it tends to be rich in serine, threonine andproline residues (Phelan et al., 2001; Vacratsis et al., 2002). Yet,there are no recognizable protein domains to suggest a function.To investigate the function of the C-terminus of Slpr, twocomplementary constructs, SKLC-WT and Cterm, were analyzed(Fig. 1A). The constructs had neither dominant negative norconstitutive activity with respect to JNK target gene expressionand tissue morphology (Fig. 3Ag,i,Ce,h); however, SKLC-WTexpression supported rescue of slpr mutants, second only to thefull length wild-type form (Fig. 2) indicating that this fragment,lacking the C-terminus, retains function. Incorporating theactivation segment mutations to create SKLC-AAA abrogatedfunction (Fig. 2, Fig. 3Ah,Cd), demonstrating the requirement foran intact kinase. Intriguingly, examination of SKLC transgenicprotein distribution by immunofluorescence revealed a differencein subcellular localization compared with wild-type protein.Overexpressed full-length Slpr is cortically enriched, although notparticularly biased in either apical or basolateral domains [seePolaski et al. (Polaski et al., 2006) and this work, Fig.4A,Ba,b,Ca,d]. Conversely, the SKLC variants lacking the C-terminus were found to be diffusely distributed, with more extensivecytoplasmic and nuclear localization compared with the full-lengthprotein (Fig. 4Bg,h,Cb,e). Cterm itself still appeared corticallyenriched (Fig. 4Bi,Cc,f), localizing in a similar pattern to wild-typein epithelia and other cell types. Thus, Cterm promotes corticalenrichment, but otherwise has no rescue function (Fig. 2), and doesnot block endogenous signaling (Fig. 3Ai,Ch). Consistent with thisinterpretation, those Slpr variants that include the C-terminus, suchas Slpr-WT and DSH3, were largely cortical and showed littlecytoplasmic staining. By contrast, any variant that lacks the C-terminus, including SKLC, SK (not shown) and SH3, failed to be

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Fig. 3. Effect of Slpr transgene expression on embryonic JNK target geneexpression and tissue closure. (A)lacZ expression from the JNK-dependentpucE69 enhancer trap in embryos expressing the indicated Slpr transgenes withpnr-G4. Images are lateral views of three segments of stage 14 embryosfocusing on the leading edge of the dorsal ectoderm. Dominant negativeactivity reduces the number of cells with active JNK signaling and puc-lacZexpression (c,d,e,h,j). Some transgenes have no effect (g,i), whereas othersactivate signaling, increasing the intensity and extent of lacZ expression(b,f,k,l). Scale bar: 10mm. (B)Thr295 in the activation segment of Slpr kinaseis essential for function. (a-e)Dorsolateral views of stage 16 embryos stainedfor Fasciclin III, revealing the ectodermal epithelium and extent of dorsalclosure. Scale bar: 20mm. (C)The effect of expression of Slpr transgenes,using pnr-G4, on the adult thorax morphology parallels the effect of transgeneexpression in the embryo. Dominant negative transgenes produce midlinethorax clefts and widening of the space between adjacent dorsocentral bristles(a,d,g) compared with no transgene controls (b). Hyperactive Slpr variantspromote loss of midline tissue, such as the scutellum, and narrowing of theinter-bristle space (c,f,i).

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restricted to the cortex, localizing in the cytoplasm and nucleus tovarying extents (Fig. 4B), illustrated most dramatically by the SH3domain alone (Fig. 4Bj), which was found in the nucleus in mostcells by the end of embryogenesis, although the significance of thislocalization is unclear.

Upstream input to Slpr activation: Rac1 and MisshapenThe role of Rac GTPase in JNK signaling and dorsal closure isseparableBiochemical and genetic evidence support a role for the GTPases,Rac and Cdc42, in stimulating JNK signaling via MLK proteins(Bock et al., 2000; Teramoto et al., 1996) by binding to the CRIBmotif, typically in a nucleotide-dependent fashion (Burbelo et al.,1995). Indeed, full-length Slpr and SKLC bound preferentially tothe GTP-loaded form of Rac and Cdc42 in in vitro pull downassays (Fig. 6) and although both are sufficient to upregulate JNKsignaling when constitutive active forms are overexpressed duringdorsal closure (Glise and Noselli, 1997), loss-of-function geneticdata favor Rac over Cdc42 in the regulation of endogenous JNKsignaling (Genova et al., 2000; Hakeda-Suzuki et al., 2002; Woolner

et al., 2005). Thus, we chose to examine the impact of theassociation between Slpr and Rac1 for signaling and dorsal closurein vivo. To do so, the wild-type proteins were expressedindividually, or together, using the pnr-G4, puc reporter system tomonitor JNK signaling. Expression of Rac-WT or Slpr-WT alonein embryos resulted in nearly a twofold increase in intensity andextent of puc-lacZ expression compared with embryos lacking atransgene (Fig. 5Ad,h,i,B). Coexpression led to nearly a fourfoldincrease in b-gal staining (Fig. 5Am,B). If Rac activates Slpr, thendominant negative Slpr is expected to block Rac-dependentupregulation of JNK pathway activity. Curiously, Slpr-KD did notsubstantially block ectopic puc-lacZ expression induced by Rac-WT, despite the fact that Slpr-KD alone reduces expression almostcompletely (Fig. 5Aa,g,B). The same result was observed withother dominant negative Slpr derivatives, including SH3 alone (notshown). To examine the possibility that incomplete suppression ofRac-WT by Slpr-KD simply reflects inadequate levels of Slpr, wetested whether coexpression of dominant negative bsk (JNK)transgenes with Rac-WT would block reporter expression or not.Indeed they did, contrary to our result with Slpr-KD, arguing

3181Regulation of Drosophila MLK in vivo

Fig. 4. Cortical enrichment of Slpr is perturbed byloss of the C-terminal domain. (A)Ectopic Slpr-WT-HA expression in the pnr domain is detected with anti-HA immunostaining (green) compared with twopolarized epithelial markers, Crumbs (crb; red a,a�,apical) and Discs large (dlg; red b,b�, lateral). Slpr isenriched cortically, and excluded from the nucleus, butnot restricted to a particular apicobasal subdomain.(a,b)Dorsolateral view, apical section. Arrowhead in bindicates Slpr immunoreactivity in a cell protrusionemanating from the leading edge. (a�,b�) Ventrolateralview, apical section. (a�,b�) Cross section of dorsalectoderm to visualize cell junctions; arrowheads point toCrb-positive apical junctions (a�) or Dlg-positive septatejunctions (b�). (B)Ectopic expression of indicatedtransgenes in several segments of the lateral ectodermwithin the pnr domain is detected with anti-SlprSH3 sera(a) or anti-HA immunostaining (b-l). Slpr-WT with orwithout the HA tag, the double A mutants, Cterm, DSH3,and Slpr AVA are cortically enriched (a-f,i,k,l), whereasSKLC-WT and SKLC-AAA are more diffuselydistributed (d,h). SH3 alone is detected in the cytoplasmand nuclei of cells (j). Scale bar: 20mm. (C)Slprtransgenic protein expression with arm-G4 is detected byanti-HA immunostaining (green) in slprBS06 mutant larvalbrains (a-c) and eye imaginal discs (d-f). In the absenceof endogenous Slpr protein, Slpr-WT-HA is associatedwith the cell cortex in mushroom body (a,a�) andphotoreceptor (d,d�) neurons. The Cterm has a similarcortical enrichment (c,c�,f,f�). By contrast, SKLC doesnot appear to localize at the cell membrane (b,b�,e,e�).Filamentous actin is highlighted with phalloidin as acounterstain (red, a-f). Higher magnifications of theboxed regions are shown on the right. Scale bars: 20mm.

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against inadequate levels of Slpr inhibition; however, morphologicalchanges in these cells were not similarly suppressed (supplementarymaterial Fig. S1). Thus, although JNK is necessary for transcriptionof puc, it appears not to be required for other Rac-dependentfunctions.

Given that GTPase binding to the CRIB site of MLK3 ispostulated to release SH3-mediated autoinhibition at the adjacentproline-dependent binding site (Zhang and Gallo, 2001), thenexpression of Slpr DSH3 is expected to bypass Rac-dependentactivation. To test this, DSH3 was expressed alone or with adominant negative form of Rac1 (Rac-DN) (Fig. 5Ao-q). Asdemonstrated previously, DSH3 stimulated JNK signaling (Fig.3Ak, Fig. 5Aj). Expression of Rac-DN alone weakly diminishedpuc-lacZ expression (Fig. 5Ac,B), but strongly inhibited theprogress of dorsal closure (Fig. 5Ap) as previously observed(Harden et al., 1995; Woolner et al., 2005). The Rac-DNphenotypes are clearly distinguishable from those of Slpr DSH3,

allowing for epistasis analysis. If DSH3 activity is constitutive,downstream of Rac function, then coexpression with Rac-DNshould be inconsequential, i.e. equivalent to DSH3 alone. Asshown in Fig. 5Aq, with simultaneous expression of DSH3 andRac-DN, a mixed phenotype was evident. JNK signaling wasreduced relative to that when DSH3 was expressed alone (Fig.5B), but remained hyperactivated compared with the level incontrol embryos (Fig. 5Ad,f,j,B). These data indicate thatstimulation of JNK pathway activity downstream of Rac isinsufficient to promote dorsal closure in the absence of functionalRac. In addition, maximal JNK reporter expression evoked byDSH3 also requires Rac function. Together these results implicateRac in both JNK-dependent and -independent functions,reminiscent of our data with coexpression of Rac-WT and bsk-DN, in which JNK target gene expression and changes in cellmorphology of the leading edge were distinct results of Racfunction distinguished by their sensitivity to bsk-DN suppression.

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Fig. 5. Slpr, Msn and Rac signaling modulate JNKpathway activity. (A)lacZ expression from the JNK-dependent pucE69 enhancer trap in embryos expressingthe indicated transgenes with pnr-G4. (a-n)Representative lateral views of three segments ofdorsal closure stage 14 embryos are focused on theleading edge of the dorsal ectoderm. Scale bar: 20mm.(o-q)lacZ reporter expression in embryos at early (st 14,top row) and late (st 16, bottom row) stages of closureexpressing the indicated transgenes. Lateral views areshown except in the bottom panel in o, which is a dorsalview. Arrowheads indicate the failure of dorsal closure.Scale bar: 50mm. (B)Graph of puc-lacZ reporterexpression in the indicated transgenic embryos (y-axis).Fluorescence extent and intensity from at least threeindependent samples for each genotype were quantifiedas described in the Materials and Methods, and plotted asmean area under the curve in arbitrary units (x-axis). Foldchange relative to ‘no transgene’ control (black bar) isindicated on the right.

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Genetic analysis has implicated misshapen (msn) in JNK signalingduring dorsal closure (Su et al., 1998). Msn is a MAP4K familykinase, and Slpr is predicted to be a substrate. To determine whetherMsn promotes JNK signaling during dorsal closure in collaborationwith Slpr, transgenes encoding the wild-type proteins were expressedindividually or together using puc-lacZ as a readout for JNKsignaling. Expression of Msn-WT or Slpr-WT resulted in a 1.4- to 2-fold upregulation of the reporter (Fig. 5Ae,i,B), whereas coexpressionresulted in 2.6-fold upregulation, which is less than an additiveeffect (Fig. 5Ai,B); however, Slpr-KD did suppress Msn-dependentJNK signaling substantially (Fig. 5Ab), consistent with expectedepistasis. These results suggest that although Msn might activateslightly more JNK signaling when Slpr is unlimited, Msn cannotmaximally activate Slpr in the absence of additional input. Rac is thelikely candidate for that input. Moreover, these results suggest acontingent order, where Rac activation of Slpr is prerequisite forfurther activation by Msn. To test this, Rac-WT and Msn-WT werecoexpressed (Fig. 5Ak). Indeed, puc-lacZ staining was greater thanfor Rac alone, or Msn plus Slpr (3.4- vs 1.9- and 2.6-fold,respectively; Fig. 5B), but still less than for Rac and Slpr together(3.9-fold), suggesting a requirement for both Msn and Rac to activateSlpr. Maximal puc upregulation upon coexpression of Msn and Racmight not be possible owing to limited endogenous Slpr.

Msn CNH domain interacts with SlprIf Msn activates JNK signaling via Slpr, then Msn is predicted tophysically interact with Slpr, yet this has not been examineddirectly. Drosophila Msn contains an N-terminal kinase domain, aproline-rich central region, and a highly conserved regulatory citronhomology (CNH) domain (Fig. 7A). One mammalian MAP4Kfamily member, the hematopoietic progenitor kinase, interacts withMLK3 via the SH3 domain to activate JNK signaling (Kiefer etal., 1996; Leung and Lassam, 2001). Alternatively, another MAP4K,NCK-interacting kinase (NIK), mediates JNK pathway activationthrough direct binding of its C-terminus to the MAP3K MEKK1(Su et al., 1997). Thus, although binding of the Slpr SH3 domainto centrally located proline motifs in Msn is a reasonableexpectation, genetic rescue experiments with msn derivativessuggest that the central region is expendable for dorsal closure,

whereas the kinase and C-terminus are essential (Su et al., 2000).To examine possible interactions, binding assays were conductedwith GST-Msn fusion proteins and in-vitro-translated Slprfragments. Tandem SH3 domains of the adapter protein Dock wereincluded as a positive control for Msn binding. Fig. 7 shows thatthe central proline-rich region of Msn pulled down Dock, asexpected; however, the Slpr SKLC and DSH3 fragments boundspecifically to the GST–Msn–CNH derivative lacking the prolinemotifs but encompassing the C-terminal domain, whereas Dockdid not (Fig. 7B). None of the fragments bound to GST alone.These data demonstrate that Msn and Slpr interact with each other,but not via a proline motif–SH3 interaction. To further narrowdown the binding site for Msn–CNH on Slpr, we tested two shorterconstructs, SK and LC, constituting two halves of the larger SlprSKLC fragment. Specific binding was retained with the LCfragment, but not the SK (Fig. 7C), indicating that the conservedregulatory C-terminus of Msn interacts with a region of Slpr,encompassing the LZ, linker, and CRIB regions (aa 405–516).

DiscussionNegative regulation of Slpr functionSrc family tyrosine kinases are autoinhibited through intramolecularinteractions mediated by their SH2 and SH3 domains (Boggon andEck, 2004). Raf Ser/Thr kinases are also subject to inhibitionthrough intramolecular interactions between N- and C-terminal

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Fig. 6. Slpr interacts preferentially with Rac-GTP and Cdc42-GTP. GSTpull-down assay using the GTPases Rac and Cdc42, loaded with either GDP orGTP as indicated, and labeled Slpr WT or Slpr SKLC proteins from a rabbitreticulocyte lysate (35S-input). The Coomassie-Blue-stained gel (upper panels)and autoradiograph (lower panel) show expression of the fusion proteins andlabeled input protein that is specifically bound, respectively.

Fig. 7. The C-terminal CNH domain of Msn interacts in vitro with theLZ–CRIB region of Slpr. (A) Schematic domain organization of Msnshowing the kinase domain, proline-rich region (PXXP), and regulatory C-terminal domain (also known as the CNH domain). Black lines represent GSTfusion protein fragments with the amino acids indicated. Dock binds within thecentral region (dotted line). (B,C) GST pull-down assay with Msn fragmentsand labeled Dock, Slpr SKLC and Slpr DSH3 proteins from a wheatgermlysate (35S-input) (B), or unlabeled Slpr SK and LC proteins from in vitrotranslation (C). The Coomassie-Blue-stained gels (upper panels) andautoradiograph or immunoblot (lower panels) show expression of the fusionproteins and input protein that is specifically bound. Molecular mass markers(kDa) are labeled.

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sequences (Wellbrock et al., 2004). Similarly, inhibition of MLK3activity requires SH3 binding to a short peptide linker between theLZ and CRIB domains (Zhang and Gallo, 2001). Introduction of aY52A substitution in the SH3 domain of MLK3 increases kinaseactivity by 2- to 2.5-fold, whereas a mutation in the putativebinding site (P469A) abrogates SH3 binding and increases catalyticactivity 4-fold (Zhang and Gallo, 2001). Our results with DSH3and Slpr-AVA recapitulate these data in vivo using puc-lacZ as asurrogate for JNK signaling in the embryo (Fig. 3, Fig. 5B)consistent with the SH3 domain playing a negative role in Slpractivation by intramolecular inhibition (Fig. 8). If the SH3 domainbound elsewhere in Slpr or to a negative regulatory partner,mutating the prolines in the LZ–CRIB linker is not expected torelieve inhibition. This is not what we observed. Furthermore,elevation in signaling capacity of the proline substitution mutantrelative to the SH3 deletion might indicate an additional positiverole for the SH3 domain in MLK regulation (Zhang and Gallo,2001). For example, once autoinhibition is relieved, the SH3domain could recruit a positive regulator. Although our in vitrobinding assays suggest that the SH3 domain is not responsible forbinding the MAP4K Msn directly, relief of SH3-mediated inhibitioncould uncover the binding site for Msn in the vicinity of the PVPmotif consistent with our mapping mapping of the interaction withMsn to the LC region of Slpr (Figs 7, 8).

Complementing the observations that the SH3 domain of Slprplays a negative regulatory role, we found that expression of theSH3 domain alone produced dominant negative phenotypes. Severalmechanisms could account for this. The isolated SH3 domain couldbind to the PVP site in endogenous Slpr preventing dimerization andactivation; however, the SH3 is expected to be displaced upon Rac-GTP binding to the CRIB motif allowing further activation ofendogenous Slpr. This scenario is inconsistent with our observationsthat JNK signaling is strongly downregulated by expression of theSH3 domain. The ectopic SH3 domain might simply be so abundantthat it outcompetes endogenous activated Rac. Alternatively, theSH3 domain of Slpr might interfere with endogenous Slpr activityby titrating an input or effector molecule. Although this is consistent

with our data that SH3 alone is epistatic to Msn (not shown), Msnbinds in vitro to Slpr independently of the SH3 domain. Thus, analternative SH3 binding partner remains a formal possibility. Finally,the SH3 domain might inhibit JNK signaling downstream ofendogenous Slpr, but upstream of the transcriptional regulation ofpuc-lacZ, which might account for the curious result that over thecourse of dorsal closure progression, the SH3 transgenic proteinbecame increasingly localized to the nucleus.

Regulation of Slpr subcellular distribution by theC-terminal regionFollowing the CRIB domain of Slpr is a long C-terminal region,constituting half of the total protein sequence. Extensivephosphorylation of the C-termini of MLK2 and MLK3 suggestthat this region might serve a regulatory role as a substrate forproline-directed kinases engaged in feedback or crosstalk control(Phelan et al., 2001; Vacratsis et al., 2002). Thus, it was surprisingto find that the SKLC-WT transgenic protein, lacking the C-terminus of Slpr, rescued slpr mutants to adulthood nearly as wellas the epitope-tagged full-length version (Fig. 2A), indicating thatthe C-terminus is nonessential during development. Though SKLCoverexpression in a wild-type background did not hyperactivateJNK signaling, it did not inhibit signaling either (Fig. 3), perhapsproviding an appropriate intermediate level of signaling for rescue.

Detection of the tagged forms of Slpr using immunofluorescencedid reveal a role for the C-terminus in proper protein localization.For example, those Slpr derivatives retaining the C-terminus wereenriched at the cortex of epithelial cells, whereas those lacking itwere predominantly cytoplasmic or nuclear, with variablelocalization at the cortex, perhaps reflecting dimerization withendogenous protein (Fig. 4). Supporting this, localization oftransgenic protein in imaginal tissues dissected from slpr mutantlarvae lacking endogenous protein revealed minimal corticalstaining of the C-terminally truncated SKLC. Moreover, if the C-terminus is responsible for cortical enrichment, then it is predictedto localize at the cortex on its own. This prediction is borne out inseveral cell types, including embryonic ectoderm and larval brainand retina (Fig. 4). Thus, although the C-terminus is important fordirecting the correct subcellular localization of Slpr, it is notabsolutely required for function, given that mutant animalsexpressing the SKLC form, lacking the localization region, survivenearly as well as those expressing the full-length form (Fig. 2). Itremains to be determined how the C-terminal region of Slpr directsits localization.

Activating Slpr signalingMany kinases require phosphorylation within their kinase domainsto become maximally active. For example, mutations in theactivation loop can render a kinase nonfunctional or hyperactivewhile leaving the active site intact (Nolen et al., 2004). Theactivation segment of Slpr is highly conserved, with three out offour putative phosphoacceptor residues being identical to humancounterparts. Intriguingly, human MLK1 and MLK3 appear torely on different threonine residues for primary activatingautophosphorylation. Specifically, an MLK1 T312A mutant (Fig.1B) is nearly inactive in vitro toward its substrate MKK4, with~1% of the wild-type kinase efficiency, and also in vivo, asmeasured by JNK activation upon MLK1 transfection (Durkin etal., 2004). MLK3, by contrast, is nonfunctional when the firstthreonine (T277) is mutated to alanine, although S281 plays anauxiliary role; the equivalent residue T285 (to MLK1 T312) was

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Fig. 8. Model describing interactions between Slpr, Rac and Msn. TheSKLC region of Slpr supports signaling functions whereas the C-terminal halfis responsible for subcellular localization. The PVP motif in the LZ–CRIBlinker region of Slpr is involved in SH3-mediated inhibition, which is relievedby interaction of the LZ–CRIB region with active Rac and the CNH domain ofMsn to activate JNK signaling, gene expression and dorsal closure. Active Racalso participates in additional JNK-independent functions with other effectorsto facilitate dorsal closure.

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not tested (Leung and Lassam, 2001). Finally, phosphorylation ofthe ultimate threonine residue (T290) in the activation segment ofanother MAP3K family member Cot (also known as Tpl2 andMAP3K8) is necessary but not sufficient for kinase activity invitro (Luciano et al., 2004). To determine whether the three residuesin the activation segment of Slpr are required for function, weanalyzed alanine substitution mutants and implicate the terminalthreonine residue (T295) as essential based on the ability tostimulate signaling in vivo (Fig. 3) and partially rescue slpr mutants(Fig. 2). Although these data clearly demonstrate a crucial role forT295, they do not indicate whether it is phosphorylated. Based onnumerous kinase structures, T295 of Slpr is near the C-terminalanchoring motif of the activation segment, placing it in the P+1loop, which contains residues contacting the peptide substrate(Nolen et al., 2004). Hence, this residue in Slpr might promote theintegrity of the catalytic active site or stabilization of substratebinding, in addition to (or rather than) being a primingphosphorylation site. However, similar mutations in Cot and MLK1do not interfere with the structural organization of the kinasedomain in so far as protein binding interactions and limited proteasedigestion patterns are unaltered (Durkin et al., 2004; Luciano et al.,2004). Generation of a phosphomimetic form of Slpr will help toaddress the role of T295 phosphorylation.

Cumulative evidence supports a role for Rac and Cdc42 in theactivation of mammalian MLK proteins, stimulating their kinaseactivity and downstream JNK signaling (Bock et al., 2000;Teramoto et al., 1996; Vacratsis and Gallo, 2000). The interactionis direct, via the CRIB motif in the MLKs, and requires the GTP-bound forms of Rac or Cdc42, not Rho (Bock et al., 2000; Burbeloet al., 1995; Nagata et al., 1998). Genetic analyses in Drosophilahave implicated Rac family proteins (Rac1, Rac2, Mtl) and Cdc42in the process of dorsal closure (Hakeda-Suzuki et al., 2002;Harden et al., 1995; Harden et al., 1999; Woolner et al., 2005), butproposed models suggest Rac GTPases are upstream of JNKsignaling whereas Cdc42 acts downstream (Genova et al., 2000;Hakeda-Suzuki et al., 2002; Ricos et al., 1999; Woolner et al.,2005). Functionally, Rac proteins regulate actin cytoskeleton-basedstructures in the leading cells of the dorsal ectoderm, including asupracellular cable linking adhesive junctions between cells tomaintain tension across the advancing epithelium (Jacinto et al.,2002a; Kiehart et al., 2000), and protrusions important forintercellular interactions across the midline during the zipperingphase of closure (Hakeda-Suzuki et al., 2002; Jacinto et al., 2000;Woolner et al., 2005). The loss of these structures in compoundRac mutants resembles the phenotype of JNK pathway mutants;moreover, upregulation of JNK signaling partially rescues Racmutant phenotypes suggesting that Rac proteins act upstream todirect JNK-dependent deployment of the actin cytoskeleton(Woolner et al., 2005).

Slpr is the best candidate linking Rac to the JNK pathway. slprmutants dominantly suppress a Rac-induced phenotype in thedeveloping Drosophila eye tissue (Stronach and Perrimon, 2002)and direct interactions between Slpr and active Rac aredemonstrable (Fig. 6) (Neisch et al., 2010). Using the reagentsdeveloped in this study, we explored further whether Rac and Slprcooperate during dorsal closure, as would be expected from existingmodels. Our coexpression studies demonstrate an additive effecton JNK target gene expression by Slpr and Rac-WT, but curiously,dominant negative forms of Slpr (SH3 and Slpr-KD) are ineffectiveat blocking ectopic signaling induced by Rac-WT (Fig. 5). Thereason for this is unclear, but raises a few possibilities. For example,

endogenous Rac might regulate JNK signaling primarily by anSlpr-dependent mechanism, but in the context of forcedoverexpression, might evoke Slpr-independent means to stimulateJNK. If so, convergence at the level of JNK is consistent with theevidence that dominant negative Bsk (JNK) is fully capable ofblocking Rac-induced ectopic puc-lacZ expression (supplementarymaterial Fig. S1). Alternatively, Rac might activate a pool ofendogenous Slpr that is ‘resistant’ to inhibition by dominantnegative Slpr. This could occur if Rac facilitates membraneassociation or dimerization of endogenous Slpr more effectivelythan dimerization with an inactivating kinase-dead form. Testingwhether Rac induction of JNK signaling is sensitive to endogenousSlpr levels might address this possibility.

Although numerous studies support a positive role for Racupstream of JNK signaling in dorsal closure, it is also curious thatRac-DN does not have a more dramatic negative effect on JNKsignaling, given the expression results in a severe tissue-closuredefect, confirming the inhibitory action of the dominant negativetransgene (Fig. 5) (Raymond et al., 2001; Woolner et al., 2005).One explanation is that the two phenotypic outcomes, geneexpression and tissue closure, require different Rac effectors (JNK-dependent and JNK-independent; Fig. 8) or unequal contributionsof the Rac family members (Hakeda-Suzuki et al., 2002; Nolan etal., 1998). Alternatively, the puc-lacZ reporter might not berepresentative of all JNK pathway target genes, such as thoserequired for cytoskeletal remodelling. In other words, incompleteloss of puc-lacZ expression as a result of Rac-DN does not precludereduction in levels of expression of other genes below a pointnecessary to sustain dorsal closure. That said, ectopic JNKactivation by Slpr DSH3 was insufficient to rescue the dorsalclosure defects resulting from diminished Rac function (Fig. 5),further supporting a Slpr- and JNK-independent role for Rac indirecting tissue closure. Protein kinase N is a good candidate tomediate JNK-independent signals from Rac-GTP (Lu andSettleman, 1999), although other effectors are possible. Altogether,our results are not entirely consistent with a simple epistaticrelationship positioning Rac GTPase upstream of Slpr activation,especially in light of the results of similar experiments with Msn,which do support Msn as an upstream input. Cumulative dataimplicate Rac in multiple pathways.

The discovery of a role for Msn MAP4K in dorsal closuremorphogenesis led to speculation that Msn and Rac might convergeon a MAP3K to activate downstream JNK signaling in theDrosophila embryo (Su et al., 1998). Identification of Slpr as aMAP3K required for dorsal closure provided a candidate. Here wedemonstrate binding between the C-terminal domain of Msn andthe LZ–CRIB region of Slpr, independent of the SH3 domain ofSlpr (Fig. 7). This result explains the genetic requirement for thekinase and C-terminal domains of Msn to rescue dorsal closuredefects of msn mutants, while the proline-rich central domain isdispensible (Su et al., 2000). Moreover, as expected of adownstream transducer, dominant negative Slpr derivatives doblock Msn-dependent upregulation of JNK signaling (Fig. 5).Although coexpression of Msn and Slpr does not activate JNKsignaling to the extent seen with Rac and Slpr together, there is anenhancement relative to each alone. This result might reflectlimiting levels of active Rac, consistent with a requirement for Racas a primary positive input and Msn a secondary activating input.Indeed, previous studies using transfected mammalian cellsdemonstrated that Rac-DN inhibits JNK activation by Msn (Su etal., 1998). The binding site on Slpr for Msn is limited to the LZ,

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PVP-containing linker, and CRIB regions where Rac and the SH3domain of Slpr also interact. Thus, it is possible that Rac binding(and relief of SH3 inhibition) subsequently exposes the interactionsite for the Msn CNH domain. Interestingly, the C-terminal CNHdomain of the mammalian Msn orthologue, NIK, interacts withanother MAP3K, MEKK1, to elicit maximal activation of SAPKpathway activity (Su et al., 1997). Notably, the CNH domain ofNIK, and MIG-15 in C. elegans, interacts with the cytoplasmic tailof b-integrin receptors (Poinat et al., 2002). Although thisinteraction has yet to be probed in Drosophila, the implication ofJNK signaling being linked to integrin function in tissue closure isnot unprecedented (Becker et al., 2000; Homsy et al., 2006;Kadrmas et al., 2004) and might provide additional insights intoregulation of dorsal closure morphogenesis and JNK pathwayactivation in future studies.

Graded Slpr activity correlates with JNK reporterexpression and tissue morphologyThe structure–function analysis of Slpr benefits from the ability todetect different levels of JNK pathway activity via reporter geneexpression in individual cells and within the whole tissue andorganism. Our results in the embryo revealed that modified formsof Slpr have distinct and wide-ranging activities, from dominantnegative to strongly hyperactive, implying that downstreamsignaling components are not limiting for signal transduction, ateither the cell or tissue level. Also, the remarkably extensive rangeof transgene activities in the embryo, correlates precisely with thebroad profile of thorax morphologies in the adult. These datademonstrate first, that the two processes, dorsal closure and thoraxclosure, are similarly sensitive across a wide range of Slpr activity;and second, that the plasticity in response at the cellular level ismirrored in the developmental flexibility at the organismal level,seen in the extreme range of thorax morphologies. These datamight also indicate that graded changes in levels of Slpr-dependentJNK signaling in epithelial tissues results in concomitant changesin signaling output, rather than a binary response at a thresholdlevel of pathway activity. Future work will investigate theunderlying cellular mechanisms responsible for signaling outputand altered tissue morphology.

Materials and MethodsSlpr transgenic constructsAll UAS–Slpr constructs described here were cloned into the pUASp vector (Rorth,1998). UAS–Slpr-WT and UAS–Slpr-KD have been described previously (Polaski etal., 2006). UAS–Slpr-WT-HA (aa 1–1148) is equivalent to UAS–Slpr-WT except forthe addition of two copies of the hemagglutinin epitope tag (HA) at the C-terminus.The HA tag is present in all subsequent constructs described here. UAS–SKLC-WT(aa 1–516) is a C-terminally truncated form of Slpr comprising only the SH3, kinase,LZ, and CRIB domains. UAS–SH3 (aa 1–114) is truncated after the SH3 domain.Both of these inserts were generated by standard PCR amplification, followed byrestriction-enzyme digestion and ligation into the appropriate vector. Alloligonucleotide primers used for cloning are listed in supplementary material TableS4.

Mutant (AAA, TAA, ASA, AAT, AVA, aa 1–1148) and N-terminally truncated formsof Slpr (DSH3 aa 110–1148, Cterm aa 537–1148) were generated using site-directedmutagenesis by overlap extension (SOEing) (Ho et al., 1989). For example, Slpr-AAA was made using overlapping mutagenic primers to change residues (T287,S291, and T295) in the kinase activation segment to alanine. Two regions wereindependently amplified and the products mixed, to generate, by overlap extension,the template for a second PCR amplification using the outermost original primers.The same strategy was employed for UAS–SKLC-AAA, replacing the wild-typefragment for the mutant fragment. Constructs were verified by dsDNA sequencing.P-element transformation was performed by Genetic Services, Inc (Sudbury, MA).

Western blot analysisAll UAS–Slpr-HA transgenic flies were crossed to arm-G4 flies and eggs werecollected overnight at 25°C. After dechorionating and homogenizing in Laemmli

sample buffer (SB), embryonic protein lysates were separated using SDS-PAGE,transferred to PVDF membrane, and probed with mouse anti-HA antibody (1:1000,Covance, 16B12). Mouse anti-b-tubulin (1:300, DSHB, E7) was used as a loadingcontrol. Low-molecular-mass transgenic proteins including SH3 were transferred innonstandard buffer, lacking SDS (25 mM Tris, 190 mM glycine, 20% methanol).

Drosophila stocksChromosome location and Bloomington stock numbers are as follows: arm-G4 (II)BL#1560, pnrMD237/TM3Ser1{UAS–y+} (pnr-G4) BL#3039 (Calleja et al., 1996),pucE69 (puc-lacZ) (III) (Ring and Martinez Arias, 1993), UAS–Slpr-KD (II and III),UAS–Slpr-WT (II), slpr3P5, slpr921, slprBS06 (X) (Polaski et al., 2006), UAS–Rac1.L(Rac-WT) (II) BL#6293, UAS–Rac1.N17 (Rac-DN) (III) BL#6292 (Luo et al., 1994),UAS–msn.S (III) BL#5946 (Su et al., 1998), UAS–bskK53R (bsk-DN) (III) BL#9311.

Genetic rescueRescue of the embryonic dorsal closure phenotype of slpr921 mutants using arecombinant chromosome with the gtX11 allele was performed as described previously(Stronach and Perrimon, 2002).

For rescue of slpr mutants to adulthood, we crossed y93j w1118 slpr*/FM7;arm-G4females with w1118/Y;UAS–slpr-Tg males for each slpr allele and raised the progenyat 21±0.5°C for moderate transgene expression. Mutant and FM7 males in theprogeny were counted to quantify relative eclosion rate as an indication of transgenerescue. To avoid inadvertently including non-FM7 males that arise from non-disjunction of the maternal X chromosome, we counted only yellow–, non-FM7males.

ImmunofluorescenceEmbryos were collected overnight, fixed with 4% formaldehyde in PEM buffer (100mM Pipes, 2 mM EGTA, 1 mM MgSO4) and processed for indirectimmunofluorescence (Patel, 1994). Larval tissues were dissected from third instarmale animals that did not express FM7-linked GFP. Primary antibodies were rabbitanti-b-galactosidase at 1:1000 (Cappel), mouse anti-b-galactosidase at 1:1000(Promega), mouse anti-phosphotyrosine 4G10 at 1:1000 (Upstate Biotech), mouseanti-HA 16B12 at 1:500 (Covance), affinity purified rabbit anti-Slpr at 1:400 (Polaskiet al., 2006), mouse a-Crb Cq4 at 1:20, mouse anti-Dlg 4E3 at 1:800, mouse anti-FasIII 4G9 at 1:50 (DSHB). Texas-Red–phalloidin was used to visualize F-actin. Allfluorophore-conjugated secondary antibodies were used at 1:200 dilution (JacksonImmunoresearch).

Image capture and processingAdult flies were imaged using a Leica DFC300F camera mounted on a Leica MZ16stereomicroscope. Images of stained embryos and larval tissues were obtained bylaser confocal scanning microscopy (Bio-Rad Radiance 2000) using a Nikon E800compound microscope. Figures were assembled with Adobe Photoshop.

Quantification of puc-lacZ expressionCropped images of three segments of immunostained stage 14 embryos wereimported into ImageJ software. Equivalent rectangular regions of interest wereselected. A vertical profile plot of average grey values (y-axis) across the selection(x-axis) was generated for each region and the numerical values were imported intoMicrosoft Excel. The MODE (most common grey value) for each image wassubtracted as background. The resulting values were used to calculate area under thecurve (excluding negative values). The average of three to six areas per genotypewere determined and plotted graphically. Error bars indicate the standard deviation(s.d.). Fold change was calculated relative to the ‘no transgene’ control.

In vitro binding assaysGST-Rac and GST-Cdc42 constructs were described previously (Lu and Settleman,1999). To generate additional GST fusion protein constructs, Msn sequences werecloned into the pGEX-4T-1 vector (GE Healthcare). Msn-PRO encodes aa 321–768of the 1102 aa isoform C. Msn-CNH encodes aa 767–1102 of isoform C. The Msnbinding region of Dock, consisting of three SH3 domains (aa 141–398 of isoformsB and C), was cloned into the pET16b HIS-tag vector (Novagen) and used as apositive control in GST pull-down assays (Ruan et al., 1999). Slpr constructs usedfor in vitro synthesis were cloned into the pcDNA3.1 vector (Invitrogen). Dock andSlpr proteins were synthesized in rabbit reticulocyte or wheatgerm lysates usingTNT® Coupled Transcription/Translation Systems (Promega). For expression of GSTfusion proteins, freshly diluted overnight cultures of E. coli BL21 cells were grownfor 1 hour and induced with 0.2 mM IPTG (isopropylthiogalactoside), then switchedto growth at 21°C for 3 hours. After centrifugation, pellets were resuspended inphosphate–BME buffer [P-BME; 1� phosphate-buffered saline, 0.1 mM PMSF(phenylmethylsulphonyl fluoride), 0.001 mM EDTA, 0.013 mM b-mercaptoethanol]and lysed with 0.5 mg/ml lysozyme, incubated on ice for 30 minutes, repeatedlyfrozen and thawed, and sonicated. The homogenate was clarified by centrifugationand incubated with glutathione–agarose beads (50–100 mg/assay) overnight at 4°C.Beads were washed with P-BME buffer, blocked in 5% filtered BSA (in P-BMEbuffer) and washed again. At this point, GST-Rac and GST-Cdc42 were incubatedwith the appropriate nucleotide, either GDP or GTPgS. For the binding assay, 2–8ml of in vitro translated protein lysates were incubated with the GST-beads in TIF

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buffer (150 mM NaCl, 20 mM Tris pH 8.0, 1 mM MgCl2, 0.1% NP40, 10%glycerol) overnight at 4°C. After washing six times with excess TIF buffer, beadswere boiled in 2� SB for SDS-PAGE, western transfer, and autoradiography orimmunoblotting.

We thank John Malta, James Matous and Stephanie Polaski fortechnical contributions to the rescue experiments, site-directedmutagenesis and DNA cloning, respectively. We acknowledge theDevelopmental Studies Hybridoma Bank (DSHB) developed underthe auspices of the NICHD and maintained by The University of Iowa,Department of Biology, Iowa City, IA 52242 for antibodies describedin Materials and Methods. This work is supported by funding from theNIH (HD045836) to B.S. Deposited in PMC for release after 12months.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/123/18/3177/DC1

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SummaryKey words: Drosophila, JNK signaling, Dorsal closure, KinaseIntroductionResultsSlpr structure-function analysisMutations in activation segment residues disrupt Slpr functionThe SH3 domain of Slpr negatively regulates JNK signalingThe Slpr C-terminus regulates protein distributionUpstream input to Slpr activation: Rac1 and MisshapenThe role of Rac GTPase in JNK signaling and dorsal

Msn CNH domain interacts with Slpr

Fig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.DiscussionNegative regulation of Slpr functionRegulation of Slpr subcellular distribution by the C-terminal regionActivating Slpr signalingGraded Slpr activity correlates with JNK reporter expression and tissue

Fig. 7.Fig. 8.Materials and MethodsSlpr transgenic constructsWestern blot analysisDrosophila stocksGenetic rescueImmunofluorescenceImage capture and processingQuantification of puc-lacZ expressionIn vitro binding assays

Supplementary materialReferences