nuclear import of activated d-erk by dim-7, an importin family … · we report the identiﬁcation...

INTRODUCTION

Pattern formation of a multicellular organism requires thatcells respond to inductive cues that direct their properdifferentiation. Protein tyrosine phosphorylation is a keyregulatory mechanism involved in the interpretation of theseinductive signals. Receptor tyrosine kinases (RTKs) actrepeatedly throughout Drosophiladevelopment. Some RTKs,such as Torso and Sevenless, are dedicated solely to thedetermination of larval terminal structures or the R7photoreceptor, respectively (reviewed by Perrimon et al., 1995;Raabe, 2000). By comparison, the epidermal growth factorreceptor (EGFR) is involved in the specification of the dorsalchorion, embryonic ventral cell fates, tracheal precursor cells,adult photoreceptors and wing veins, to name a few (seereviews by Perrimon and Perkins, 1997; Schweitzer and Shilo,1997).

RTKs engage an evolutionarily conserved signaling cassetteto bring about the activation of D-ERK (reviewed by VanDer Geer et al., 1994; Li and Perrimon, 1997). Ligandbinding induces receptor autophosphorylation and promotesphosphorylation of multiadapter proteins (e.g. Daughter ofSevenless) at distinct tyrosyl residues to form recognition

motifs for SH2 domains. The SH3-SH2-SH3 adapter proteinDRK (Downstream of receptor tyrosine kinase) and the proteintyrosine phosphatase (PTP) Corkscrew (CSW) are two proteinswhose SH2 domains recruit these molecules to specificphosphorylation sites. Both DRK and CSW are capable ofmediating additional heteromeric protein interactions. In someinstances, phosphorylation of CSW at Tyr666 generates adocking site for DRK. The SH3 domains of DRK bind to Sonof Sevenless (SOS), the guanine nucleotide exchange factor forRAS. SOS stimulates the conversion of RAS:GDP toRAS:GTP, which serves as a molecular switch for the ensuingactivation of a phosphorylation cascade consisting of theserine/threonine kinase RAF, the dual specific tyrosine/threonine kinase MEK and the serine kinase D-ERK (Perkinset al., 1992; Perkins et al., 1996; Allard et al., 1996; Cleghonet al., 1996; Cleghon et al., 1998; Herbst et al., 1996; Raabe etal., 1996).

The activation of D-ERK induces its movement from thecytoplasm to the nucleus where it modulates the activity of asubset of transcription factors (Chen et al., 1992; Gonzalez etal., 1993; Lenormand et al., 1993; Hill and Treisman, 1995;Robinson and Cobb, 1997; Lewis et al., 1998). Severalmolecular determinants that affect translocation have recently

1403Development 128, 1403-1414 (2001)Printed in Great Britain © The Company of Biologists Limited 2001DEV5454

The initiation of gene expression in response to Drosophilareceptor tyrosine kinase signaling requires the nuclearimport of the MAP kinase, D-ERK. However, the moleculardetails of D-ERK translocation are largely unknown. Inthis regard, we have identified D-Importin-7 (DIM-7), theDrosophilahomolog of vertebrate importin 7, and its genemoleskin. DIM-7 exhibits a dynamic nuclear localizationpattern that overlaps the spatial and temporal profileof nuclear, activated D-ERK. Co-immunoprecipitationexperiments show that DIM-7 associates withphosphorylated D-ERK in Drosophila S2 cells.Furthermore, moleskin mutations enhance hypomorphic

and suppress hypermorphic D-ERK mutant phenotypes.Deletion or mutation of moleskin dramatically reduces thenuclear localization of activated D-ERK. Directly linkingDIM-7 to its nuclear import, this defect can be rescued bythe expression of wild-type DIM-7. Mutations in theDrosophila Importin β homolog Ketel, also reduce thenuclear localization of activated D-ERK. Together, thesedata indicate that DIM-7 and Ketel are components of thenuclear import machinery for activated D-ERK.

Key words: Corkscrew, Receptor tyrosine kinase signaling, Importinsuperfamily, MAP kinase, Nuclear transport, moleskin, Drosophila

SUMMARY

Nuclear import of activated D-ERK by DIM-7, an importin family member

encoded by the gene moleskin

James A. Lorenzen 1, Scott E. Baker 2,*, Fabienne Denhez 1,‡, Michael B. Melnick 1,§, Danny L. Brower 2 andLizabeth A. Perkins 1,¶

1Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA2Department of Molecular and Cellular Biology, Department of Biochemistry, Life Sciences South Building, University of Arizona,Tucson, AZ 85721, USA*Present address: Novartis Agricultural Discovery Institute, 3115 Merryfield Row, San Diego, CA 92121, USA‡Present address: Cutaneous Biology Research Center, Massachusetts General Hospital, Building 149-3, Boston, MA 02129, USA§Present address: New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USA¶Author for correspondence (e-mail: [email protected])

Accepted 19 January; published on WWW 22 March 2001

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been described. Studies with MEK indicate that the upstreamactivator of ERK is maintained in the cytoplasm by its leucine-rich nuclear export signal (Fukuda et al., 1996). In the absenceof RTK stimulation, MEK physically associates with ERKto maintain it in the cytoplasm. Stimulation of the pathwaydissociates the ERK:MEK complex by promoting thephosphorylation of the TEY activation motif on both tyrosineand threonine although phosphorylation of the tyrosyl residueby MEK is sufficient to release ERK (Fukuda et al., 1997;Adachi et al., 1999). Dual phosphorylation of ERK inducesdimerization with either a phosphorylated or anunphosphorylated partner. With a molecular size of ~84 kDa,an ERK dimer is too large to move unassisted through thenuclear pore, which can, in principal, accommodate the passivediffusion of molecules smaller than 50-60 kDa (reviewed inCole and Hammell, 1998; Görlich, 1998; Moore, 1998).Indeed, mutations that prevent dimer formation and inhibitorsof the active transport mechanism hinder the accumulation ofERK in the nucleus (Khoklatchev et al., 1998; Adachi et al.,1999). Therefore, it is likely that an import factor exists thatspecifically recognizes the activated form of this kinase.

Nuclear import and export mechanistically mirror oneanother, with each process using specific transport factors andthe GDP/GTP-bound state of the Ran GTPase to regulate thedirectional movement of cargo proteins into or out of thenucleus (reviewed by Cole and Hammell, 1998; Görlich, 1998;Moore, 1998). To date, a large family of importin-relatednuclear transport proteins have been identified, includingimportin α and importin 7 (RanBP7). Nuclear import ofcytoplasmic cargo typically initiates with the recognition of adistinct nuclear localization signal (NLS) by either an importreceptor (e.g. importin β) or an import receptor/adapter pair(e.g. importin β/importin α). Importin β represents theprototypical import receptor: it is able to directly bindcomponents of the nuclear pore complex while simultaneouslybinding either a cargo protein or another transport factor. Incontrast, importin αappears to act only as an adapter, bridgingthe interaction between importin βand cargo proteinspossessing a ‘classical’ mono- or bipartite, basic NLS. Avariation of the import receptor/adapter theme is illustrated byimportin 7, which can function as either the adapter or thereceptor. As an adapter, importin 7 heterodimerizes withimportin β to form a functional transport complex for histoneH1, which lacks a ‘classical’ NLS (Jäkel et al., 1999). Yet,importin 7 is also capable of directly importing severalribosomal proteins without the aid of importin β (Görlich etal., 1997; Jäkel and Görlich, 1998).

We report the identification of the Drosophila melanogasterhomolog of importin 7 (DIM-7), encoded by moleskin(msk)gene, and ascribe a role for this molecule, along with theDrosophila importin β homolog Ketel (KET), in the nucleartranslocation of activated D-ERK. (DIM-7 will be used to referto the protein, molecular function or activity; moleskin(msk)will be used to refer to the gene that has previously beenreferred to as diminished 7 (http://flybase.bio.indiana.edu).)

MATERIALS AND METHODS

Yeast two hybrid screensYeast two hybrid screens were performed essentially as described

(Golemis et al., 1994). Drosophilaovary or imaginal disc acid fusionlibraries (a gift from R. Brent) and the bait plasmid (pEG202) weretransformed into yeast strain EGY188. Approximately 1 milliontransformants were screened for interaction with the C terminus ofCSW by assaying for activation of a LEU2 and/or lacZreporter gene.The largest cDNA that was obtained was approximately 1 kb in lengthand encoded the C-terminal 309 amino acids of DIM-7.

Molecular biologyAll molecular biology manipulations were carried out using standardtechniques (Sambrook et al., 1989). A cDNA containing the completeopen reading frame of mskwas isolated from an ovarian lambda gt22Adirectional cDNA library (provided by P. Tolias; Stroumbakis et al.,1994). Sequence was determined from both strands using aThermoSequenase Kit (Amersham).

Expression vectors for CSW GST fusion proteins (GST-SH2domains, residues 1-198, and GST-PTP+C-term, residues 486-841)were made in pGEX-2T using the BamHI/EcoRI sites. For the yeasttwo hybrid screens, the C terminus of CSWY1229(residues 655-841)was placed into the EcoRI/XhoI sites of the vector pEG202. Forectopic expression, mskwas inserted into the NotI/XhoI site of pUAST(Brand and Perrimon, 1993). For northern analysis, mRNA fromstaged embryos was prepared with the QuickPrep Micro mRNAPurification Kit (Pharmacia). Three micograms poly-A+ RNA wasloaded per lane to a denaturing agarose gel.

Immunoprecipitation and GST pulldownDrosophila S2 cells (a gift from L. Cherbas) were maintained inShields and Sang M3 Insect media supplemented with 10% fetalcalf serum, 0.6% yeastolate, 0.3% bactopeptone and penicillin-streptomycin. Pervanadate stimulation (used to sustainphosphotyrosine levels, which were increased because of inhibitionof intracellular PTPs; Heffetz et al., 1990) was for 15 minutes at afinal concentration of 200 µM. In some instances, cells were serumstarved for 24 hours and stimulated for 5 minutes with 150 nMbovine insulin (Sigma). S2 cell and embryo lysates were typicallyprepared by Dounce homogenization in 20 mM Tris-Cl, pH 7.5,5 mM EDTA, 10% glycerol, 1% Triton X-100, 150 mM NaCl, 1 mMEGTA, 10 mM NaPi, 10 mM NaF, 7.5 TIU/ml aprotinin, 2 µg/mlleupeptin, 1 mM benzamidine, and 5 mM iodoactic acid or 1 mMvanadate. For the GST pulldown experiments, 1-5 µg of GST fusionprotein immobilized on glutathione Sepharose (Pharmacia) wasincubated overnight with 1.2 mg of Oregon Rembryonic proteinlysate.

The following antibodies were used for immunoprecipitation from1-10 mg of protein or western analysis: F1088, a rabbit polyclonalantibody raised against full-length CSW expressed as a GST fusionprotein; 1129 and 61Frat-1, polyclonal antibodies generated in rabbitand rat, respectively, against the C-terminal 160 amino acids ofDIM-7 fused to GST; ERK1/2 (Stressgen); dpERK (clone MAPK-YT, Sigma); and PY20 (Transduction Laboratories). Detection ofimmunoblotted proteins was with SuperSignal West PicoChemiluminescent Substrate (Pierce).

Drosophila strains and geneticsAll flies were raised on standard Drosophila media at 25oC.Chromosomes and mutations that are not described in the text, orbelow, can be found at FlyBase (http://flybase.bio.indiana.edu).

Isolation of msk allelesA ‘P1 filter’ (Genome Systems) containing an array of P1 genomicclones was hybridized with a probe derived from the cDNA encodingDIM-7. One positive signal was obtained that corresponded to a P1clone whose cytological position had been unambiguously assignedto the 66B6-10 region of the third chromosome (Berkeley DrosophilaGenome Project). This localization was confirmed when thirdchromosome deficiencies, Df(3)1420(Df(3L)pbl-X1) and Df(3)1541

J. A. Lorenzen and others

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1405D-Importin 7 and D-ERK nuclear import

(Df(3L)66C-G28), which overlap in the region of 66B8/9-66B10(http://flybase.bio.indiana.edu), were determined by Southernanalysis to be deleted for the msk gene.

Details of the mskalleles and the screen used to generate them willbe presented elsewhere (S. E. B., J. A. L., L. A. P., and D. L. B.,unpublished). However, it is important to note that these alleles weremapped to the overlap region of Df(3)1420and Df(3)1541and thatsequence analysis of these alleles reveals molecular lesions in the genepredicted to encode the DIM-7 protein (S. E. B., J. A. L., L. A. P.,and D. L. B., unpublished).

P-element transformationThe full-length mskcDNA was subcloned into pUAST vector (Brandand Perrimon, 1993) and, following standard protocols (Spradling,1986), injected into y w/y w; ∆2-3, Sb/In(3)TM6pre-cellular embryos.This strain constitutively synthesizes an endogenous transposase,which is present in the ooplasm. Eight independent transformantswere identified by rescue of their white eye color to near wild type.

Ectopic expression of DIM-7 and rescue experimentsThe UAS/Gal4 system of ectopic expression was used to expressDIM-7 in a subset of segments within the developing embryo. UAS-msk was constructed and transgenic flies generated as describedabove. The GAL4 driver used in these experiments was Kr-GAL4; i.e.GAL4 is expressed under the control of the Krüppel (Kr) promoter.‘Rescued’ embryos (genotype UAS-msk/Kr-GAL4; Df(3)1420 +/+Df(3)1541) were derived by crossing two stocks of genotype: UAS-msk/CyO ftz-lacZ; Df(3)1420/TM6β AbdA-lacZand Kr-GAL4/CyOftz-lacZ; Df(3)1541/TM3 ftz-lacZ. Embryos of the appropriategenotype were distinguished from their siblings by the absence of β-galactosidase staining originating from the balancer chromosomes.

In situ hybridization and immunohistochemistryIn situ hybridization of wholemount embryos using digoxigenin-labeled probes was performed (Tautz and Pfeifle, 1989). Single-stranded sense and antisense digoxigenin-containing DNA probeswere prepared by PCR labeling using appropriate primers.

For immunohistochemistry, embryos were fixed with 4%formaldehyde and immunostained (Michelson, 1994), except that theblocking step was omitted. The primary antibodies were 61Frat-1(1:300); anti-β-galactosidase (Promega, 1:500); and dpERK (cloneMAPK-YT, Sigma, 1:500). Secondary antibodies conjugated to biotin(BMB) were detected with the Vectastain Elite ABC kit (Vector Labs)in combination with TSA-Indirect (NEN).

All embryos were photographed with a Nikon FXA equipped withNomarski optics. Confocal imaging was performed on a Leica TCSSP Spectral confocal microscope. Fluorescent detection of dpERKwas done with FITC-Streptavidin (Jackson Immunoresearch, 1:2000).DNA, hence nuclei, were detected with propidium iodide (Sigma).

RESULTS

The C terminus of CSW interacts with DIM-7CSW is a multifunctional component of the RTK signalingcassette that leads to activation of DrosophilaD-ERK (Gabayet al., 1997a; Ghiglione et al., 1999). The predominantmaternal and zygotic form of CSW (CSWY1229) has tandem N-terminal SH2 domains and the catalytic domain is interruptedby ~150 non-catalytic residues termed the ‘PTP insert’ (Fig.1A). The PTP insert encodes two cysteine fingers and overall is30% serine/threonine rich. The C terminus of CSW contains aproline-rich segment and a tyrosine that, when phosphorylated,is capable of binding DRK (Perkins et al., 1992; Cleghon etal., 1998). We used the structural domains of CSW to search

for interacters using the yeast two hybrid technique. As CSWfunctions downstream of multiple RTKs during oogenesis,embryogenesis and morphogenesis (Perkins et al., 1992;Perkins et al., 1996; Allard et al., 1996), Drosophilaovary andimaginal disc yeast fusion libraries were screened (Gyuris etal., 1993; Golemis et al., 1994). With the carboxyl terminus ofCSWY1229 as the bait, a cDNA was isolated that represented30% and 63% of the positive clones from these libraries,respectively. We then obtained, from an ovarian cDNA library,a 3.8 kb cDNA with an open reading frame encoding a putative1049 amino acid protein (Stroumbakis et al., 1994). Theprotein sequence has significant identity with a family ofnuclear import factors that includes human and Xenopusimportin 7 (RanBP7) (53%), human RanBP8 (48%) and themore distantly related S. cerevisiae, Nmd5p (24%) (Görlichet al., 1997) (Fig. 1B). These proteins possess an N-terminalbinding motif for the Ran GTPase. The presence of this motifrelates these molecules to a large superfamily of Ran bindingproteins that also includes importin β. Based on thesealignments, the CSW interacter is likely to be the Drosophilahomolog of human and Xenopus laevisforms of importin 7;therefore, we designate the protein D-Importin 7 (DIM-7) inaccordance with the nomenclature suggested (Jäkel andGörlich, 1998).

CSW interacts with DIM-7 in vivoTo assess whether the interaction between DIM-7 and CSWoccurs in Drosophila, anti-CSW immunoprecipitates of S2cell lysates were analyzed for the presence of DIM-7 usingwestern blots. As shown in Fig. 2A, antibodies that recognizeendogenous CSW are able to co-immunoprecipitate theendogenous 120 kDa DIM-7. Pre-immune serumimmunoprecipitated neither CSW (data not shown) nor DIM-7 (Fig. 2A) supporting the conclusion that CSW and DIM-7associate in vivo.

To further confirm an interaction between CSW and DIM-7, affinity pull-down experiments were performed (Fig. 2B).GST fusion proteins consisting of either the SH2 domains ofCSW (GST-SH2 domains) or the PTP domain with the Cterminus (GST-PTP + C-term) were incubated with proteinlysates prepared from embryos collected from 0 to 5 hoursafter egg laying, times at which CSW is known to functionin the Torso, EGF and Heartless RTK signaling pathways(Perkins et al., 1992; Perkins et al., 1996; L. A. P. and A. M.Michelson, unpublished). Proteins pulled down by eitherGST fusion protein were analyzed for the presence of DIM-7. While the SH2 domains of CSW are incapable of bindingto DIM-7, the PTP domain with the C terminus of CSWretains endogenous DIM-7 (Fig. 2B). Together, these resultsindicate that CSW forms a complex, through its C terminuswith DIM-7 in vivo.

DIM-7 is tyrosine phosphorylated and associateswith D-ERKThe association of DIM-7 with CSW prompted a search foradditional links between DIM-7 and RTK signaling. As manysignaling molecules, including CSW (Cleghon et al., 1996), aretyrosine phosphorylated in response to ligand binding toits receptor, we asked whether DIM-7 might also bephosphorylated. Lysates were prepared from S2 cells that hadbeen left untreated and cells that had been either treated with

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1406 J. A. Lorenzen and others

CSW Y1229

A

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MEA KLTELLRATI DPNPEQRKAAEDQLAQI HKII GFVPTI LQI VMQTTVEQPVRQAGAVYLKNLI NSSWSDHEAKPGE. PI PFSI HEQDRAMI RGAI VD 99MDP TI I EALRGTMDP.. ALREAAERQLNEAHKSLNFVSTLLQI TMSEQLDLPVRQAGVI YLKNMI TQYWPDRETAPGD. I SPYTI PEEDRHCI RENI VE 97MDPNI LI EALRGTMDP.. ALREAAERQLNESHKSLHFVSTLLQI TMSEQLELPVRQAGVI YLKNMI TQYWPDREVTPGE. LPPHTI PEEDRHCI RENI VE 97MDLNRFI QALKGTI DP..K LRI AAENELNQSYKII NFAPSLLRI I VSDHVEFPVRQAAAI YLKNMVTQYWPDREPPPGEAI FPFNI HENDRQRI RDNI VE 98MDI TELLQCFACTLDHNAAVRTNAETHLKNASKVPGFLGACLDI I AADEVPENI KLSASLYFKNKI TYGWSAGARQGSNELLDSHVDPDEKPVVKDMLI K 100

AI VHA.... PELI RVQLSVCVNHII KSDFPG. RWPQVVDSI SI YLQNQDVNGWNGALVTMYQLVKTYEYKRHEERTPLNEAMNLLLPMI YQLMVRLLAEQ 194AI I HS.... PELI RVQLTTCI HHII KHDYPS. RWTAI VDKI GFYLQSDNSACWLGI LLCLYQLVKNYEYKKPEERSPLVAAMQHFLPVLKDRFI QLLSDQ 192AI MHS.... PELI RVQLTTCI HHII KHDYPN. RWTAVVEKI GFYLQSDNSACWLGI LLCLYQLVKNYEYKKPEERSPLI AAMQHFLPMLKDRYI QLLADP 192GI I RS.... PDLVRVQLTMCLRAII KHDFPG. HWPGVVDKI DYYLQSQSSASWLGSLLCLYQLVKTYEYKKAEEREPLI I AMQI FLPRI QQQI VQLLPDS 193TMVSVSKTSPRCI RVLKSALT. VII SEDYPSKKWGNLLPNSLELLANEDI TVTYVGLLCLAEI FRTYRWKNNDERQDLEELI LNYFPALLNYGANVLFQD 199

S..... EQSVLLQKQI LKI YYALTQYTLPLDLI TKEI FSQWMEI CRQVADRAVP. DSSHLDDDE. RTEFPYWKTKKWALHI MVRMFERYGSPSNVVSEKY 287S..... DQSVLI QKQI FKI FYALVQYTLPLELI NQQNLTEWI EI LKTVVNRDVPNETLQVEEDD. RPELPWWKCKKWALHI LARLFERYGSPGN. VSKEY 285S..... EQSVLI QKQI FKI FYALVQYTLPLELI NQQNLAEWI EI LKTVVDRDVPAETLQVDEDD. RPELPWWKCKKWALHI LARLFERYGSPGN. VSKEY 285S.....YY SVLLQKQI LKI FYALVQYALPLQLVNNQTMTTWMEI FRTII DRTVPPETLHI DEDD. RPELVWWKCKKWALHI VARLFERYGSAGN. VTKEY 286GKYMNNEQI GELVKLI I KI YKFVSYHDLPFTLQRSESFTPWACFFVSII QQPLPQEVLAI SDI EVRSKNPWVKCKKWALANLYRLFQRYASTSLTRKFQY 299

QKFAEWYLPTFSQGVLEVLLKI LDQYRN. RVYVSPRVLTDVLNYLKNAVSHAYTWKLI KPHMVAVI QDVI FPI MSFTDSDQELWESDPYEYI RLKFDI FE 386NEFAEVFLKAFAVGVQQVLLKVLYQYKE. KQYMAPRVLQQTLNYI NQGVSHALTWKNLKPHI QGIIQ DVI FPLMCYTDADEELWQEDPYEYI RMKFDVFE 384NDFAEVFLKAFAVGVQQVLLKVLYQYKE. KQYI APRVLQQTLNYFNQGVSHAVTWKNLKPHI QGIIQ DVI FPLMCYTDSDEDLWQEDPYEYI RMKFDVFE 384FEFSEFFLKTYAVGI QQVLLKI LDQYRQ. KEYVAPRVLQQAFNYLNQGVVHSI TWKQMKPHI QNI SEDVI FSVMCYKDEDEELWQEDPYEYI RMKFDI FE 385DEFKQMYCEEFLTQFLQVVFDQI EKWGTGQLWLSDECLYYI LNFVEQCVVQKTTWKLVGPHYNVI LQHVI FPLLKPTAETLEAFDNDPQEYI NRNMDFWD 399

DYATPVPAAQSLLHSMCKKR. KGI LPKAMATI MQI I TSPNADNK..Q KDGALHMI G....... TLADVLLKKASYRDQVESMLTTYVFPEFQNPAGHMRA 476DFI SPTTAAQTLLFTACSKR. KEVLQKTMGFCYQI LTEPNADPR.. KKDGALHMI G....... SLAEI LLKKKI YKDQMEYMLQNHVFPLFSSELGYMRA 474DFI SPTTAAQTLLFTSCSKR. KEVLQKTMGFCYQI LTEPAADPR.. KKDGALHMI G....... SLAEI LLKKKI YKDQMEFMLQNHVFPLFSSELGYMRA 474DYASPTTAAQTLLYTAAKKR. KEVLPKMMAFCYQI LTDPNFDPR.. KKDGALHVI G....... SLAEI LLKKSLFKDQMELFLQNHVFPLLLSNLGYLRA 475VGYSPDLAALALLTTCVTKRGKTTLQPTLEFMVSTLQSAVGDYNNIMLDNALQI ESCLRI FSSI I DRLI TKDSPFASEMEKFI LTYVLPFFKSQYGFLQS 499

RACWVLHYFCDVQI KNPQVLAEI MRLTTNALLTDK. ELPVKVEAAI GLQMFI SSQDEAPQYVEAQI KEI TKELLTIIR ETENEDLTNVMQKI VCTFTEQL 575RACWVLHYFCEVKFKSDQNLQTALELTRRCLI DDR. EMPVKVEAAI ALQVLI SNQEKAKEYI TPFI RPVMQALLHIIR ETENDDLTNVI QKMI CEYSEEV 573RACWVLHYFCEVKFKVDQNLQTALELTRRCLI DDR. EMPVKVEAAI ALQVLI SNQEKAKEYI VPFI RPVMQALLHIIR ETENDDLTNVI QKMI CEYSEEV 573RSCWVLHAFSSLKFHNELNLRNAVELAKKSLI EDK. EMPVKVEAALALQSLI SNQI QAKEYMKPHVRPI MQELLHI VRETENDDVTNVI QKMI CEYSQEV 574RVCDI CSKLGSMDFKDPIIT STI YEGVMNCLNNSSNSLPVELTAALALQTFI SD. DQFNMKLSEHVVPTMQKLLSLSNDFESDVI SGVMQDFVEQFAEQL 598

LPVATEI C............. QHLATTFSQVLES... E. EGSDEKAI TAMSLLNTI ETLLSVMEEHPDVLLNLHPI VI NVVGHI FQHNI TDFYEETFSLV 658TPI AVEMT............. QHLAMTFNQVI QTGPDE. EGSDDKAVTAMGI LNTI DTLLSVVEDHKEI TQQLEGI CLQVI GTVLQQHVLEFYEEI FSLA 659TPI AVEMT............. QHLAMTFNQVI QTGPDE. EGSDDKAVTAMGI LNTI DTLLSVVEDHKEI TQQLEGI CLQVI GTVLQQHVLEFYEEI FSLA 659ASI AVDMT............. QHLAEI FGKVLQS.. DEYEEVEDKTVMAMGI LHTI DTI LTVVEDHKEI TQQLENI CLRI I DLVLQKHVI EFYEEI LSLA 659QPFGVELMNTLVQQFLKLAI DLHETSNLDPDSFTNVDSI PDESDKQMAALGI LSTTI SI LLSFENSPEI LKNLEQSFYPAAEFI LKNDI EDFYRECCEFV 698

Y..D LTAKAI SPEMWQMLELI YQVFKKDG...I DYFI DI MPALHNYVTVDTPAFLSNPNRLLAI LDMCKTMLTS. SPGEDPECHAAKLMEVII LQCKG. Q 751H.. SLTCQQVSPQMWQLLPLVFEVFQQDG... FDYFTDMMPLLHNYVTVDTDTLLSDTKYLEMI YSMCKKVLTG. VAGEDAECHAAKLLEVII LQCKGRG 753H.. SLTCQQVSPQMWQLLPLVFDI FQQDG... FDYFTDMMPLLHNYVTVDTDTLLSDTKYLEMI YSMCKKI LTG. VAGEDAECHAAKLLEVVI LQCKGRG 753Y.. SLTCHSI SPQMWQLLGI LYEVFQQDC... FEYFTDMMPLLHNYVTI DTDTLLSNAKHLEI LFTMCRKVLCG. DAGEDAECHAAKLLEVII LQCKGRG 753ENSTFLLRDI TPI SWKI LELI GECNRKPDSMVSYYLSDFMLALNNI LI YGRNELKKNEFYTKI I FEI YQKAVTAEDNSLDDLRVVFDLSQELVLALDDSL 798

I DSVI HMFVELALSRLTREVQSSELRTMCLQVVI AALYYNPQLLLSI LDKMSQQNN. DSI SAHFI KQWLHDTDCFLG. I HDRKLCVLGLCTLI SLGEAKP 849I DQCI PLFVEAALERLTREVKTSELRTMCLQVAI AALYYNPHLLLNTLENLRFPNNVEPVTNHFI TQWLNDVDCFLG. LHDRKMCVLGLCALI DM. EQI P 851I DQVI PLFVEAALERLTREVKTSELRTMCLQVAI AALYYSPPLLFNTLENLRFPNNEEPVTNHFI KQWLNDVDCFLG. LHDRKI CVLGLCALI EL. EQRP 851I DQCI PLFVQLVLERLTRGVKTSELRTMCLQVAI AALYYNPDLLLHTLERI QLPHNPGPI TVQFI NQWMNDTDCFLG. HHDRKMCII GLSI LLEL. QNRP 851PQQYRERLLADVVG.. SI LTQKNELKTNVVFSVTAFNVVI SNMI TEPLI TLQYLKQ. QGCLEI FFQTWI TDYI PNYKRCYDI KLSVLALLKI I LKLESND 895

QVLSEVAGKI VPALI LLFDGLKRAYESRAQEEEEDEEEEDG.... D.... DCEEALSSDEDDMDEMAPDYLDKLAEFAKTKGNESGFEVKAEI KDDDADS 941QVLNQVSGQI LPAFI LLFNGLKRAYACHAEHENDSDDDDEA.... E.. DDDETEELGSDEDDI DEDGQEYLEI LAKQAGEDGD........... DEDWEE 934QVLNQMSSQI LPAFLLLFNGLKRAYACHAEQENDSDDDGDG.... E.. DDEDAAELGSDEDDI DEEGQEYLEI LAKQAGEDGD........... DEDWED 934PAVDAVVGQI VPSI LFLFLGLK..Q VCATRQLVNREDRSKA.... EKADMEENEEI SSDEEETNVTAQAMQSNNGRGEDEEEE........... DDDWDE 934YSVLNLEN. LVPQLGSI VTQLASRLPTALRQLANQRKEFSSSGFEEDTKWDENFLDVGDDDENDDEG. DLTEKYLELI KNRAD...........S LDFVD 982

DGDAEESVGDLNETGLESFTTPI DDEENESAI DEYWTFKEVI TALSAQDQAWYALLTSNLTPEQAKALQEVVVTADQRKAAKES. KLI EKQGGFAFPQTA1040D........ DAEETALEGYSTI I DDEDNP.. VDEYQI FKAI FQTI QNRNPVWYQALTHGLNEEQRKQLQDI ATLADQRRAAHES. KMI EKHGGYKFSAPV1023D........ DAEETALEGYTTLLDDEDTP.. I DEYQI FKAI FQKLQGRDPVWYQALTQGLNEDQGKQLQDI ATLADQRRAAHES. KMI EKHGGYKFNAPV1023E........V LEETALEGFSTPLD. LDNS.. VDEYQFFTQALI TVQSRDAAWYQLLMAPLSEDQRTALQEVYTLAEHRRTVAEAKKKI EQQGGFTFENKG1023G.......Y DAKETFDDLEEDPLTGSI LDT. VDVYKVFKESI ANLQHVDSNRYQGI LRHLTPADQELFMGI MNA.......................... 1048

VPTSFKFGS...... 1049VPSSFNFGGPAPGMN1038VPSTFNFGNPAPGMN1038VLSAFNFG. TVPSNN1037...............

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D-Importin 7 H-Importin 7 X-Importin 7 H-RanBP8 Sc-Nmd5p











D-Importin 7 H-Importin 7 X-Importin7 H-RanBP8 Sc-Nmd5p

B

Fig. 1.DIM-7 physically binds to CSW and is evolutionarily conserved. (A) The domain structure of CSWY1229: SH2 domains (blue); catalyticdomain (black); PTP insert (yellow); and proline-rich segment (red). The underlined segment indicates the region of CSWY1229 used as a bait inthe yeast two hybrid screen. (B) The amino acid sequence of DrosophilaDIM-7 and four closely related proteins from other species: humanimportin 7 (RanBP7); Xenopusimportin 7 (RanBP7); human RanBP8; and S. cerevisiae Nmd5p. Identical residues are shown with yellowbackgrounds while similar residues are shown with blue backgrounds. Amino acids in DIM-7 that match the consensus Ran binding motifs(Görlich et al., 1997) are indicated with an asterisk. The GenBank accession number for DIM-7 is AF132299.


pervanadate to inhibit tyrosine phosphatase activity (Heffetz etal., 1990) or serum starved and treated with insulin (Biggsand Zipursky, 1992). DIM-7 was immunoprecipitated andthe resulting western blot was probed with antibodies tophosphotyrosine. Endogenous DIM-7 becomes phosphorylatedon tyrosine in response to either pervanadate or insulin (Fig.2C). Interestingly, two proteins of 68 kDa and 74 kDa in theDIM-7 immunoprecipitates also become highly tyrosinephosphorylated (asterisks in Fig. 2C). The smaller protein co-migrates with a 68 kDa molecule that is immunoreactive withour DIM-7 antibodies (data not shown), suggesting that thisprotein represents either a breakdown product of DIM-7 or atruncated, possibly functional form of DIM-7.

Given these connections with RTK signaling and itspredicted cellular role as a nuclear transport protein, wehypothesized DIM-7 might function as the nuclear importprotein for the activated, doubly phosphorylated form of D-ERK, dpERK. Consistent with this hypothesis would be aninteraction between DIM-7 and D-ERK in stimulated cells. Totest this idea, endogenous DIM-7 was immunoprecipitatedfrom both unstimulated or stimulated S2 cell lysates. Thewestern blot was probed with an antibody to mammalianERK1/2 that crossreacts with both the inactive and activeforms of D-ERK. As shown in Fig. 2D, DIM-7 co-immunoprecipitates endogenous D-ERK from only stimulatedcells. It follows that DIM-7 should also be able to associatewith activated ERK. The ability to test this prediction is due tothe availability of antibodies that specifically recognize dpERK(Gabay et al., 1997a; Gabay et al., 1997b; Yung et al., 1997).Probing a similar western blot to that shown in Fig. 2D, weobserve that DIM-7 and dpERK co-immunoprecipitate (Fig.2E), confirming that endogenous DIM-7 interacts withendogenous activated D-ERK.

DIM-7 is encoded by the gene moleskin The gene that encodes DIM-7 lies in the region of overlapbetween deficiencies Df(3)1420and Df(3)1541 withincytological bands 66B8/9-66B10 (data not shown). One ormore genes essential for embryonic viability resides within thisinterval since animals with the genotype Df(3)1420 +/+Df(3)1541 die at late embryonic stages with a cuticle thatresembles wild type (data not shown). Four alleles of the mskgene were also found to map to the overlap region of Df(3)1420and Df(3)1541. Sequence analysis of msk2, msk4, and msk5

identified stop codons within the DIM-7 open reading frame.The msk5 allele possesses a nonsense mutation in codon 2,effectively making this allele a DIM-7 protein null. All of thesealleles are late embryonic or larval lethal, with embryoniccuticles being indistinguishable from wild type (data notshown). The mskalleles were identified as suppressors of adominant ‘blistermaker phenotype’ generated by gain ofintegrin function during imaginal development (Brabant et al.,1996; Brabant et al., 1998). The details of this screen and thealleles will be presented elsewhere.

msk mutations exhibit a dominant geneticinteraction with rolled allelesGiven that DIM-7 could interact with phosphorylated D-ERK,we sought to establish a genetic interaction between theseproteins. Determination of the adult wing vein is dependentupon signaling by the Drosophila EGFR and, consequently,activated D-ERK (reviewed by de Celis, 1998). Animalscarrying viable, loss-of-function mutations in the EGFR or itsdownstream effectors exhibit defects in vein formation. rolled1

(rl 1) is a viable, hypomorphic allele of D-ERK whosephenotypes include frequent thinning or disruption of wingvein L4 distal to the posterior crossvein (Fig. 3B; FlyBase –

Fig. 2.Biochemical connections betweenDIM-7 and RTK signaling. (A) Westernanalysis reveals that endogenous DIM-7is seen in CSW but not controlimmunoprecipitates. S2 cell lysates wereimmunoprecipitated with either CSWantiserum or pre-immune serum. (B) ThePTP catalytic domain + C terminus, butnot the SH2 domains, of CSW is able toassociate with endogenous DIM-7 inembryos. The SH2 domains or the PTPcatalytic domain + C terminus were fusedto glutathione S-transferase andexpressed in E. coli. The fusion proteinswere mixed with 0-5 hour embryoniclysates and assessed by western analysisfor their ability to pull down DIM-7.(C) Western analysis of endogenousDIM-7 immunoprecipitates withphosphotyrosine antibodies indicates thatDIM-7 is tyrosine phosphorylated uponcell stimulation, which is defined astreatment of S2 cells with either insulinor pervanadate, as identical results were obtained with each. Two additional endogenous tyrosine phosphorylated proteins of 68 kDa and 74kDa (asterisks) are observed to co-immunoprecipitate with DIM-7 from stimulated cell lysates. Reprobing of the blot (lower panel) indicatesthat both lanes have an equivalent amount of DIM-7. (D) Endogenous DIM-7 co-immunoprecipitates endogenous D-ERK only from stimulated(pervanadate) S2 cells. (E) Endogenous DIM-7 co-immunoprecipitates endogenous dpERK only from stimulated (pervanadate) S2 cells. DIM-7immunoprecipitates were probed on western blots with a polyclonal ERK1/2 antibody that crossreacts with D-ERK (D) or a monoclonalantibody to dpERK (E).

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http://flybase.bio.indiana.edu/). Homozygous rl 1/rl 1 fliescarrying one copy of msk2 or msk1 display a marked increasein the frequency of thinning or small gaps in L4 vein,respectively (Fig. 3D,E; Table 1). The effect of the protein nullmsk5 allele is even more dramatic as nearly 100% of the wingsexhibit large gaps in the L4 vein (Fig. 3F; Table 1).

Hyperactivation of the EGFR pathway with the gain-of-function rolledSevenmaker(rlSEM) mutation promotes theformation of ectopic veins (Brunner et al., 1994; Fig. 3G).Using this allele, we observe that removal of one copy of msk2

or msk5 is sufficient to partially suppress the rlSEMectopic veinphenotype (Fig. 3H,I).

The dominant enhancement of D-ERK loss-of-functionand the dominant suppression of D-ERK gain-of-functionphenotypes by mutations in the mskgene support a role forDIM-7 in the EGFR pathway.

DIM-7 has a dynamic subcellular distributionThe RNA expression pattern of msk was examined throughoutdevelopment by northern analysis and during embryogenesisby in situ hybridization of wholemount embryos. A prominent4.4 kb msk transcript is expressed both maternally andzygotically throughout the life cycle (Fig. 4A) and a ubiquitousdistribution of this RNA is observed at all stages ofembryogenesis (data not shown).

DIM-7 antiserum revealed that, like the RNA, the protein is

expressed ubiquitously; however, its subcellular distribution iseither predominantly cytoplasmic or predominantly nuclear.The nuclear localization pattern is highly dynamic throughoutembryogenesis (Fig. 4B-F,J,L) bearing a striking overlap to thenuclear profile of dpERK (Fig. 4G-I,K,M; Gabay et al., 1997a;Gabay et al., 1997b).

Torso is the first RTK used during Drosophilaembryogenesis and is necessary for the proper specificationof terminal cell fates. Localized production of the Torsoligand at the poles results in the graded activation of thereceptor and, consequently, D-ERK in precellular blastodermstage embryos (Fig. 4G). DIM-7 is seen in nuclei throughout


Fig. 3.mskmutations dominantly modify wing vein phenotypes associated with rolled alleles. Compared with a wing from a wild-type adult(A), a wing from a homozygous rolled1 (rl 1) adult exhibits frequent thinning (B, arrow) or occasionally small gaps (not shown) in vein L4 distalto the posterior crossvein. A msk/+wing (msk5/+ shown, C) appears wild type with a small percentage having an L5 vein that fails to reach thewing margin. Introduction of one copy of the msk1 (D), msk2 (E) or msk5 (F) mutant allele in a rl1/rl 1 background dominantly enhances thefrequency and size of the gap in the L4 vein. The gain-of-function rolledSevenmaker(rlSEM) mutation induces the formation of ectopic veinmaterial (G). Introduction of one copy of either the msk2 (H) or the msk5 (I) allele in a rlSEMbackground is able to partially suppress the ectopicvein phenotype generated by the rolled gain-of-function mutation.

Table 1. Effect of mskmutations on the rolled1 wing veinphenotype

Genotype n* Wild type Thin Gaps

rl 1/rl 1 104 58% 35% 8%rl 1/rl 1; msk2/+ 108 25% 67% 8%rl 1/rl 1; msk1/+ 158 37% 39% 23%rl 1/rl 1; msk5/+ 119 0% 4% 96%msk1/+ 100 91% 0% 9%‡msk2/+ 100 85% 0% 15%‡msk5/+ 100 89% 0% 11%‡

*n is the number of wings scored. ‡Flies of this genotype exhibited gaps in wing vein L5 only.

http://flybase.bio.indiana.edu/


the embryo at this developmental period suggesting a role forDIM-7 in the nuclear import of cargo proteins in addition todpERK. At these early stages, DIM-7 is seen at the cell cortex(Fig. 4C) suggesting a role for DIM-7 at the periphery of thecell also.

The EGFR is used reiteratively throughout embryogenesis(reviewed in Perrimon and Perkins, 1997; Schweitzer andShilo, 1997). During gastrulation, EGFR-dependent dpERKcan be detected in the ventral ectoderm as two stripes on eitherside of the invaginating mesoderm (Fig. 4H). Similarly, nuclearDIM-7 immunostaining is found in the ventral ectoderm at thistime (Fig. 4E). As the ventral ectoderm pattern perdures intostages 7 and 8, embryos exhibit nuclear DIM-7 (Fig. 4F) anddpERK (Fig. 4I) along the dorsal folds, cephalic furrow andventral midline. Activation of the EGFR in subsequentembryonic stages produces dpERK positive cells in thetracheal placodes (Figs 4K, 5A,E) and again in the ventralectoderm within three to four cell rows on either side of theventral midline (data not shown). We also observe DIM-7 in

the nuclei of cells comprising the tracheal placodes (Fig. 4J)and in nuclei along the ventral midline.

While the EGFR is necessary to initially establish trachealcell fates (Wappner et al., 1997), the gene breathless (btl),encoding one of two known Drosophila FGF receptors, isessential for proper migration of tracheal cells (Klämbt et al.,1992; Reichman-Fried et al., 1994; Lee et al., 1996). BTL-dependent dpERK immunostaining can first be detected atstage 11 in the tracheal pits (Figs 4M, 5I,M) just as migrationof these cells is initiated. At this time, we see a prominentnuclear subcellular localization of DIM-7 in these sametracheal cells (Fig. 4L).

Our comparative analysis of the DIM-7 and dpERK nuclearaccumulation patterns are consistent with a role for DIM-7 inseveral RTK signaling pathways essential for embryogenesis.

Nuclear dpERK is reduced in embryos deleted forDIM-7The finding that in stimulated cells antibodies to DIM-7 are

Fig. 4. DIM-7 is expressedthroughout development in apredominantly cytoplasmic orpredominantly nuclear pattern thatoverlaps that of dpERK.(A) Developmental northernanalysis of msk reveals a major4.4 kb transcript to be maternallyprovided (E0-1) and expressedthroughout all remainingdevelopmental stages examined.A larger, predominantly maternal,transcript is also observed in theE0-1 hour lane. Lanes are markedaccording to the specific stage:numbers during embryonic (E)stages refer to hours of developmentafter egg lay; first, second and thirdinstar larval (L) stages are indicatedas L1, L2 and L3, respectively; earlypupae (EP) refers to 0-24 hours afterpupation. All lanes have beencontrolled for RNA loading (notshown). (B-M) Developmentallystaged embryos immunostained withan antibody to DIM-7 (B-F,J,L) or todpERK (G-I,K,M). In the confocalimages (D-M), DNA has beenlabeled with propidium iodide (red).The embryos depicted are at early(B,C), mid (G) and late (D)blastoderm stages, stage 7 (E, H),stage 8 (F,I), stage 10 (J,K) andstage 11 (L,M) of embryonicdevelopment. In C, enlarged from B,the arrow marks a DIM-7-positivenucleus, and the arrowhead indicatesa cell cortex that displays DIM-7immunostaining. Note that thedynamic nuclear distribution ofDIM-7 strikingly overlaps thepattern observed for dpERK(compare B and D with G; compareE with H; compare F with I; compare J with K; and compare L with M). At blastoderm stages, DIM-7 is observed in all nuclei suggestingthat DIM-7 may have multiple import substrates, in addition to dpERK, during this developmental period.

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able to co-immunoprecipitate D-ERK, in combination with theobservations that the nuclear distribution of DIM-7 resemblesthat of dpERK, prompted us to examine the nuclearlocalization of dpERK in embryos lacking DIM-7 function.Transheterozygous embryos in which both copies of the mskgene are deleted or mutated (genotypes:Df(3)1420 +/+ Df(3)1541 or msk5/msk5, respectively) were generated.Compared with wild type embryos,mutant embryos exhibited dramaticallyreduced nuclear dpERK in the twoembryonic tissues that we examined.The EGFR pathway controls nucleardpERK in the tracheal placodes of stage10 embryos (Gabay et al., 1997a;Wappner et al., 1997) and the BTL RTKpathway controls nuclear dpERK inthe tracheal pits of stage 11 embryos(Gabay et al., 1997b) (compare Fig.5A,E with 5B,F,G and Fig. 5I,M with

5J,N,O). The number of dpERK stained nuclei in eachhemisegment was reduced approximately fivefold at bothstages (Fig. 5T). Consistent with the knowledge that msk isheavily maternally contributed (Fig. 4A), we credit the residualnuclear distribution of dpERK to the perdurance of maternally


Fig. 5.Nuclear localization of dpERK isrescued by expression of wild type msk.(A-R) Embryos at stages 10 (A-H,Q) and11 (I-P,R) were immunostained for dpERK(A,B,D-J,L-R) or β-galactosidase (C,K).The DNA marker shown in the confocalimages (E,G,M,O) is propidium iodide(red). In panel E, at stage 10, D-ERKactivation in the tracheal placodes isdependent upon the EGF RTK (Gabay etal., 1997a; Wappner et al., 1997), and, atstage 11, D-ERK activation in the trachealpits is dependent upon the BTL RTK(Gabay et al., 1997b). Reduced numbers ofdpERK-positive nuclei are seen in embryosin which both copies of the mskgene aredeleted or mutated (B,F,G,J,N,O). Df1/Df2indicates the genotype Df(3)1420 +/+Df(3)1541(B,D,J,L). Significantly, ectopicexpression of a wild-type UAS-msktransgene within a domain defined by theKr promoter driving expression of GAL4(C,K) restores the ability of dpERK toaccumulate in the nucleus (D,H,L,P-R).The boxed regions demarcated in H,P areshown in Q,R, respectively. (S1 and S2) Agraphical representation of the number ofdpERK positive nuclei in each trachealplacode from stage 10 (S1, n≥5) and ineach tracheal pit from stage 11 (S2, n≥9)msk5/msk5 rescued embryos ectopicallyexpressing a wild type UAS-msk transgene(genotype UAS-msk/KrGAL4; msk5/msk5).(T) Histogram indicating the number ofdpERK positive nuclei per hemisegment inembryonic stage10 tracheal placodes andstage 11 tracheal pits in wild type (WT;stage 10, n=40; stage 11, n=38); Df(3)1420+ /+ Df(3)1541(Df1/Df2; stage 10, n=102;stage 11, n=60), msk5/msk5 (stage 10,n=101; stage 11, n=114); ketRX1/ketRX1

(RX1; stage 10, n=50; stage 11, n=73) andketRX41/ketRX41(RX41; stage 10, n=10;stage 11, n=10).


contributed DIM-7 that has not been removed from theseembryos.

DIM-7 rescues nuclear localization of dpERK To unequivocally attribute the diminished nuclear localizationof dpERK to the removal of DIM-7, we used the UAS/GAL4system (Brand and Perrimon, 1993) to ectopically express DIM-7 (UAS-msk) within a broad domain defined by the expressionof GAL4 under the control of the Krüppelpromoter (Kr-GAL4).As assayed in Kr-GAL4/UAS-lacZembryos, this promoter isable to drive the expression of lacZat these developmentalstages, which is relatively late for Kr (Figs 5C,K). Alternatively,the β-galactosidase protein may be able to perdure into theselate stages and our experiment would depend upon the DIM-7protein sharing this same property. However, in all embryos ofgenotype UAS-msk/Kr-GAL4; Df(3)1420 +/+ Df(3)1541orUAS-msk/Kr-GAL4; msk5/msk5, we observe a marked increasein the number of dpERK immunostained nuclei that could beseen in the tracheal placodes (Fig. 5D,H,Q,S1) and around theinvaginating tracheal pits (Fig. 5L,P,R,S2) that lie within the Kr-GAL4domain.

These data indicate that loss of nuclear dpERK in thetracheal placodes and pits of Df(3)1420 +/+ Df(3)1541ormsk5/msk5 embryos is due to the deletion or mutation of themskgene and firmly implicate a role for DIM-7 in the nuclearimport of dpERK following activation by the EGFR and BTLsignaling pathways.

Nuclear translocation of dpERK is reduced in ketelmutant embryosIn our initial efforts to elucidate the molecular mechanism by

which DIM-7 transports dpERK into the nucleus, we testedwhether the Drosophilahomolog of importin β, KET, mightalso constitute a component of the transport machinery. Thisscenario is a possibility since importin 7 can form a functionalheterodimer with importin βto transport histone H1 (Jäkel etal., 1999). For two loss-of-function alleles of ket (ketRX41andketRX1; Erdélyi et al., 1997), we tested whether or not the abilityof dpERK to translocate from the cytoplasm to the nucleus isaffected. Homozygous mutant embryos for both alleles of ketexhibit dramatically reduced nuclear dpERK in the trachealplacodes of stage 10 embryos (Fig. 6A,B) and in the trachealpits of stage 11 embryos (Fig. 6C,D). The number of dpERKstained nuclei in each hemisegment was decreased 14-fold atstage 10 and 12-fold at stage 11 (Fig. 5T). This finding suggeststhat, like DIM-7, the importin βhomolog KET plays a role inthe nuclear import of dpERK following activation by the EGFRand BTL signaling pathways.

DISCUSSION

The activation of ERK represents the focal point of a conservedsignaling module used by a diverse array of extracellularstimuli (reviewed by Herskowitz, 1995; Marshall, 1995; Liand Perrimon, 1997; Robinson and Cobb, 1997; Lewis et al.,1998). The potency and duration of ERK activation and itsaccompanying translocation to the nucleus can profoundlyaffect the fate of a cell. This is apparent in PC12 cells wherethe decision to proliferate or differentiate depends upon thenumber and duration of receptors stimulated (Dikic et al.,1994; Traverse et al., 1994). Throughout development, cellsrespond to spatial and temporal signals and must interpretgradients to produce qualitative differences in gene expression.In Drosophila, the terminal system or Torso RTK signalingpathway illustrates one example whereby quantitativedifferences in D-ERK activity generate distinct cell fates(Ghiglione et al., 1999). Distinct quantitative levels of D-ERKactivity inside a cell may be achieved within the RTK pathwayby modulating D-ERK phosphorylation. Moreover, it isapparent that mechanisms exist to control the localization ofERK activity by either regulating its retention in the cytoplasmand/or its nucleocytoplasmic shuttling. In this regard, nucleartranslocation of dpERK is not always a compulsoryconsequence of RTK signaling. In Drosophila asphotoreceptors are recruited into the developing retina, dpERKis held in the cytoplasm for up to several hours followingEGFR and Sevenless RTK signaling. This raises the possibilitythat import is differentially controlled relative to D-ERKphosphorylation and/or dimerization (Kumar et al., 1998).

Interest in the active transport of dpERK also partly stemsfrom the observation that dimer formation is a commonproperty of mammalian MAP kinase family members.Presumably, D-ERK shares this property as the residuesinvolved in dimer formation have been conserved (J. A. L. andL. A. P., unpublished). Although monomeric dpERK can enterthe nucleus passively, it has been shown that import of dimericERK is an active process (Khokhlatchev et al., 1998; Adachiet al., 1999). We have clarified mechanistic issues in thenuclear relocalization of dpERK through the identification ofDIM-7, the Drosophilahomolog of importin 7 and member ofthe importin superfamily of nuclear transport proteins. DIM-7

Fig. 6.Nuclear dpERK is reduced in ket mutant embryos. Thenumber of dpERK-positive nuclei is reduced as shown in twotracheal placodes (Stage 10; A,B) or tracheal pits (Stage 11; C,D)from four homozygous ket/ket mutant embryos. For comparison withwild-type controls see Fig. 5A (stage 10) and Fig. 5I (stage 11). Aquantitative analysis of this data is shown in Fig. 5T. The arrowsindicate cells where dpERK appears to be excluded from the nucleus.

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exhibits several properties that establish its participation inRTK signaling. These include a physical interaction withCSW and dpERK, and our finding that DIM-7 is tyrosinephosphorylated in stimulated cells. Furthermore, wedemonstrate that alleles of msk, the gene that encodes DIM-7,dominantly interact with hypomorphic and hypermorphicalleles of D-ERK.

The primary structure of DIM-7 originally suggested that itmight play a role in nuclear transport. In addition to exhibitingsignificant sequence identity with its Xenopusand humanhomologs, DIM-7 also possesses a conserved Ran-bindingdomain. This latter property is a hallmark of nuclear transportproteins that display a RanGDP versus RanGTP regulatedinteraction with their cargo proteins (reviewed in Cole andHammell, 1998; Görlich, 1998; Moore, 1998). In addition tophysically binding to phosphorylated D-ERK, we observe thatDIM-7 and dpERK have overlapping nuclear localizationpatterns in developing tissues subject to RTK regulation. Thismade dpERK an attractive candidate cargo for DIM-7. Toaddress this possibility during embryogenesis and in theabsence of functional DIM-7, we assayed tracheal placodesand tracheal pits for defects in the accumulation of dpERK.Significantly, embryos in which the genomic interval encodingthe mskgene is deleted or mutated exhibit a fivefold reductionin the number of dpERK-positive nuclei. Importantly anddemonstrating that DIM-7 is essential for nuclear uptake ofdpERK, expression of wild-type DIM-7 in a msk mutantbackground restores dpERK nuclear accumulation. Thesefindings reinforce the idea that not only is dpERK activelytransported through the nuclear pore complex but also thatDIM-7 functions as either the transport receptor and/or adapterfor dpERK.

Finally, we have implicated KET in the nuclear import ofdpERK. As vertebrate importin 7 and importin β form anabundant heterodimeric complex, we asked whether theDrosophila homolog of importin β, KET participated in thedpERK transport mechanism. Supporting a model wherebyDIM-7 and KET function together in the nuclear transport ofdpERK (see below), we find that nuclear localization ofdpERK is impaired in homozygous ketmutant embryos.

Although there are other possibilities, we envision twomodels by which DIM-7 could function in the nuclear import

cycle of dpERK. In one, DIM-7 and KET could functiontogether in the same import cycle, where DIM-7 and KETserve as the import adapter and import receptor, respectively(Fig. 7A). Alternatively, DIM-7 and KET could functionindependently of each other in separate import cycles thatcould be, at least partially, redundant (Fig. 7B). However, it isunlikely that DIM-7 and KET serve totally redundant functionsfor two reasons. First, each individual locus when deleted (msk)or mutated (mskand ket) reduces substantially the number ofdpERK-positive nuclei, and, second, both msk and ketmutations, alone, are lethal.

In the literature there appears to be an increasing number oftransport receptors with complex cargo specificities. Ifimportin 7 is the functional vertebrate homolog of DIM-7, thenthis transport factor can import at least three differentproteins, dpERK, histone H1 and rpL23a. An additional pointconcerning specificity of DIM-7 regards its nearest homolog,Nmd5p, in Saccharomyces cerevisiae. Nmd5p is essential forthe nuclear import of HOG1, a p38 MAP kinase familymember that is activated in response to osmotic stress.Interestingly, the movement of HOG1 into the nucleus does notrequire the importin βhomolog, RSL1 (Ferrigno et al., 1998).This work raises the possibility that DIM-7 might also mediatethe nuclear transport of one or more otherDrosophila MAPkinase family members, D-p38a (Mpk2), D-p38b (Mpk34C)(Han et al., 1998) and D-JNK (JUN kinase; Riesgo-Escovar etal., 1996; Sluss et al., 1996). The combinatorial use of differenttransport factors may provide a means for specific recognitionof the various MAP kinase family members. For example,DIM-7 alone may bind and import D-p38; however,recognition of dpERK may require the simultaneous pairing ofDIM-7 and KET. Determining the mechanism(s) employed toestablish recognition between a cargo and its transport receptorwill require a precise molecular dissection of the interactionsbetween several transport receptor/cargo pairs.

PerspectiveCSW was first demonstrated to have a positive function duringembryogenesis in the Torso RTK pathway (Perkins et al.,1992). In this pathway CSW serves two functions. First, theadapter protein DRK does not bind Torso; instead, CSWfunctions as an adapter linking Torso to RAS (Cleghon et al.,


RanGAPRanBP1

dpERK

dpERK

DIM-7KET

dpERK

DIM-7KET

DIM-7KET

KETKET

RanGTP

RCC1

RanGTP

dpERK

RanGTP

DIM-7Ran

GTP

DIM-7

RanGTP

Nuclear Import

Cytoplasm Nucleus

NPC

A

dpERK

DIM-7

dpERK

DIM-7

dpERK

KET

DIM-7KET

B

Fig. 7.The proposed roleof DIM-7 and KET in thenuclear import of activatedD-ERK. NPC, nuclearpore complex. (See text forfurther details.)


1998). Second, CSW is able to dephosphorylate the Torsoautophosphorylation site (Cleghon et al., 1998) that bindsRasGAP (Feldmann et al., 1999). The work presented in thispaper suggests a third function for CSW as an adapter tofacilitate the physical interaction of DIM-7 with its importcargo dpERK. When two, or three, of these CSW functions areused within one signaling module, interpreting the epistaticrelationships of CSW with various signaling componentscould become problematic. For example, previous epistasisexperiments have suggested that CSW carries out its functioneither upstream or downstream of RAS1 and/or D-RAF (Lu etal., 1993; Allard et al., 1996; M. Johnson Hamlet and L. A. P.,unpublished). Now it appears these differences could simplyreflect the differential use of the various signaling capabilitiesof CSW within a given RTK pathway.

Whether or not the association of DIM-7 with CSWconstitutes part of a regulatory process at the level of thereceptor that governs D-ERK redistribution has yet to bedetermined. However, it appears that nuclear import is afundamental mechanism used by cells to modulate incomingsignals throughout development. It is expected, then, that thedevelopment of reagents to modulate the nuclear entry ofspecific molecules may have profound effects for controlingboth disease and oncogenic states.

For reagents we thank R. Brent, L. Cherbas, and P. Tolias. For flystocks we thank the Bloomington Stock Center, A. Michelson, N.Perrimon, R. Saint, J. Szabad and C.-t Wu. We are grateful to S. Lewis(Berkeley DrosophilaGenome Project) for advice on interpreting ourP1 grid results, to A. Michelson for advice on immunostaining, to C.Arnold for injecting UAS-msk, to F. Ausubel and A. Aballay forexcellent training and use of their confocal microscope, and to S.Miller for his role in the generation of the msk alleles. Superbtechnical assistance was provided by S. Kim, J. Manchester and M.Yamane. We also thank T. Clarke, P. Donahoe, N. Perrimon andmembers of the Perkins lab for helpful discussions, and M. JohnsonHamlet, A. Michelson and N. Perrimon for critical reading of themanuscript. J. A. L. has been aided by Postdoctoral Fellowships fromthe NIH (5F32GM17901-03) and the American Cancer SocietyMassachusetts. S. E. B. was supported by an NIH postdoctoralfellowship (5F32GM19149). L. A. P is supported by a Fellowshipfrom Margaret H. Walter and her daughters in honor of Carl W.Walter, MD, by the National Science Foundation (IBN9723509) andby the Department of Surgery at the Massachusetts General Hospital.

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nuclear import of activated d-erk by dim-7, an importin family … · we report the identiﬁcation...

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