Cloning and Characterization of Dfak56, a Homolog of FocalAdhesion Kinase, in Drosophila melanogaster*

(Received for publication, April 22, 1999, and in revised form, June 17, 1999)

Jiro Fujimoto‡, Kazunobu Sawamoto§, Masataka Okabe§¶, Yasumitsu Takagii**, Tohru Tezuka‡,Shingo Yoshikawa‡‡, Haruko Ryo§§, Hideyuki Okano§, and Tadashi Yamamoto‡¶¶

From the ‡Department of Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan, the§Division of Neuroanatomy, Department of Neuroscience, Biomedical Research Center, Osaka University Graduate Schoolof Medicine and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation,Osaka 565-0871, Japan, the §§Department of Radiation Biology and Medical Genetics, Osaka University Graduate Schoolof Medicine, Osaka 565-0871, Japan, the iHirohashi Cell Configuration Project, Exploratory Research for AdvancedTechnology, Japan Science and Technology Corporation, Tsukuba 300-2635, Japan, and the ‡‡Department of MolecularNeurobiology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-0006, Japan

The focal adhesion kinase (FAK) protein-tyrosine ki-nase plays important roles in cell adhesion in verte-brates. Using polymerase chain reaction-based cloningstrategy, we cloned a Drosophila gene that is homolo-gous to the vertebrate FAK family of protein-tyrosinekinases. We designated this gene Dfak56 and character-ized its gene product. The overall protein structure anddeduced amino acid sequence of Dfak56 show signifi-cant similarity to those of FAK and PYK2. Dfak56 has invitro autophosphorylation activity at tyrosine residues.Expression of the Dfak56 mRNA and the protein wasobserved in the central nervous system and the muscle-epidermis attachment site in the embryo, where Dro-sophila position-specific integrins are localized. The re-sults suggest that like FAK in vertebrates, Dfak56functions downstream of integrins. Dfak56 was tyro-sine-phosphorylated upon integrin-dependent attach-ment of the cell to the extracellular matrix. We concludethat the Dfak56 tyrosine kinase is involved in integrin-mediated cell adhesion signaling and thus is a func-tional homolog of vertebrate FAK.

Focal adhesion kinase (FAK)1 is a member of a growingfamily of non-receptor protein-tyrosine kinases (1) and wasoriginally identified as a putative substrate for the oncogenicprotein-tyrosine kinase pp60v-src (2). Accumulating data, how-ever, show that FAK tyrosine phosphorylation is induced uponadhesion of cells to the extracellular matrix through the sur-face integrin (3) and upon stimulation of cells by a variety ofother extracellular factors including those for receptor tyrosinekinases and for G-protein-coupled receptors (4–6). FAK asso-ciates with multiple cellular components including other focaladhesion-associated proteins and signaling molecules (7–11).

FAK is localized to focal adhesions and is centrally implicatedin the regulation of cell motility and adhesion. PYK2, anothermember of the FAK family of protein-tyrosine kinases (alsonamed CAKb/CadTK/RAFTK) (12–15), is isolated from mam-mals. The function of PYK2 seems to be different from that ofFAK because tyrosine phosphorylation of PYK2 is independentof cell adhesion and is not localized to focal adhesions (13).Since calcium ionophores stimulate PYK2 and chelation ofextracellular calcium ion abrogates tyrosine phosphorylation ofPYK2 in response to various stimuli, PYK2 is thought to beregulated by calcium (12, 14).

The roles of FAK in development and cell adhesion are di-rectly revealed by gene targeting experiment (16, 17). Targeteddisruption of the FAK gene in mice results in early embryoniclethality. Cells from mutant embryos have reduced mobilityand increased number of focal adhesions, suggesting that FAKis involved in the turnover of focal adhesion contacts during cellmigration. Disruption of the mouse b1 integrin subunit, one ofthe well known upstream factors of FAK, also causes embry-onic lethality in a very early stage of development (18). How-ever, because of the anomalous phenotypes, further biologicalanalysis of knockout mice remains difficult.

In Drosophila, study of cell adhesion was started by theidentification of position-specific (PS) antigens as homologs ofvertebrate integrins (19). Several Drosophila genes encodingintegrins have been identified, including myospheroid (mys),multiple edematous wings (mew), and inflated (if), which en-code the integrin b chain (bPS integrin) (20), aPS1 integrin (21),and aPS2 integrin (19), respectively. Genetic analyses of thesemutations demonstrate that integrins are required for a widevariety of developmental processes, including germ-band re-traction, muscle attachment, and morphogenesis of the wingand eye (22). Furthermore, genetic approaches to isolate thegenes encoding possible downstream molecules of integrinshave been performed (23).

In this study, we isolated the cDNA for the Drosophila hom-olog of FAK. Characterization of the protein product stronglysuggested that Drosophila FAK is an important downstreamfactor of integrin-mediated signaling pathways, as is mamma-lian FAK.

EXPERIMENTAL PROCEDURES

Isolation of cDNA and Genomic DNA Clones—Polymerase chainreaction was performed using fully degenerate oligonucleotides corre-sponding to the amino acid sequences HRDIAAR (for the forwardprimer) and DVWAFG (for the reverse primer) as primers and 108

plaque-forming units of Drosophila embryo cDNA library (a gift from T.Todo, Radiation Biology Center, Kyoto University, Kyoto, Japan) as

* This work was supported by a grant for advanced cancer researchfrom the Ministry of Education, Science, and Culture of Japan. Thecosts of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

The nucleotide sequence reported in this paper has been submitted tothe DDBJ/GenBankTM/EBI Data Bank with accession numberD88898.

¶ Present address: Dept. of Developmental Genetics, National Insti-tute of Genetics, Mishima 411-0801, Japan.

** Present address: Dept. of Biology, Fukuoka Dental College,Fukuoka 814-0193, Japan.

¶¶ To whom correspondence should be addressed. Tel.: 81-3-5449-5301; Fax: 81-3-5449-5413; E-mail; tyamamot@ims.u-tokyo.ac.jp.

1 The abbreviations used are: FAK, focal adhesion kinase; PS, posi-tion-specific; GST, glutathione S-transferase; kb, kilobase(s).

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 41, Issue of October 8, pp. 29196–29201, 1999© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org29196

template. The amino acid sequences were located in conserved motifs ofthe kinase domain and are unique for FAK and PYK2. Amplified cDNAs(;200 base pairs) were subcloned into the pBluescript vector (Strat-agene) and sequenced with Thermosequenase (Amersham PharmaciaBiotech). Replicate filters from a Drosophila embryo cDNA library wereprehybridized for 3 h at 65 °C in 7% SDS and 0.5 M sodium phosphatebuffer (pH 8.0) containing 100 mg/ml salmon sperm DNA (Sigma) andthen hybridized to the 32P-labeled cDNA fragment for 16 h at 65 °C.After hybridization, the filters were washed with 0.23 SSC and 0.1%SDS. Positive clones were isolated and sequenced on both strands.Other molecular biological techniques were performed as describedpreviously (24). DNA and protein sequence analyses were performedusing Genetics Computer Group software packages and ClustalW andBLAST programs employing the DDBJ/GenBankTM/EBI Data Bankrunning at the Human Genome Center of the University of Tokyo(Tokyo, Japan).

Southern and Northern Blotting—Genomic DNA from wild-type (Or-egon-R strain) Drosophila melanogaster (1 mg/sample) digested withsuitable restriction enzymes was electrophoresed on 1.0% agarose gel.mRNA samples from cultured cells and Drosophila tissues were elec-trophoresed on formaldehyde-containing 1.2% agarose gel. The frac-tionated RNAs were then transferred to nylon membranes (Hybond,Amersham Pharmacia Biotech) and subjected to hybridization usingfull-length Dfak56 cDNA and the 59-portion (SacII fragment, corre-sponding to nucleotides 1–2743) of the Dfak56 cDNA as probes. Hybrid-izations were performed employing the same procedure as that used forcDNA screening.

Cells and Antibodies—Drosophila Schneider cells (S2) and deriva-tives of their integrin-expressing cells were a gift from D. L. Brower(Arizona University). BG2-c6 and 7E10 cells were described previously(25, 26). The cells were cultured at 27 °C under normal atmosphericconditions in Shields and Sang M3 medium (Sigma) supplemented with10% of fetal calf serum. Simian COS cells were cultured at 37 °C under5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10%fetal calf serum.

To generate rabbit anti-Dfak56 polyclonal antibodies, glutathioneS-transferase (GST)-Dfak56 was constructed by inserting the cDNAfragment encoding amino acid residues 1–280 of the Dfak56 proteininto the pGEX-2T expression vector (Amersham Pharmacia Biotech).Expression of the GST fusion proteins in Escherichia coli BL21 wasinduced with isopropyl-b-D-thiogalactopyranoside, and the proteinswere purified with glutathione-Sepharose (Amersham Pharmacia Bio-tech) as described previously (27). A New Zealand White rabbit wasimmunized with the purified GST-Dfak56 fusion protein. Antiserumraised against Dfak56 were purified using GST and the GST-Dfak56protein bound to a HiTrap NHS-activated affinity column (AmershamPharmacia Biotech). Anti-phosphotyrosine monoclonal antibody 4G10was purchased from Transduction Laboratories, Inc.

In Situ Hybridization and Immunohistochemistry—Whole-mount insitu hybridization was conducted using digoxigenin-labeled RNA probesas described previously (28). The digoxigenin-labeled RNA probes(sense and antisense) were prepared by a standard procedure with adigoxigenin-RNA labeling kit (Roche Molecular Biochemicals) and full-length Dfak56 cDNA as a template.

Whole-mount immunohistochemistry was performed as described(29) using rabbit anti-Dfak56 antibodies at 1:100 dilution. The second-ary antibody used was horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) at 1:500 dilu-tion. Immunohistochemistry with cultured cells was performed asdescribed previously (30).

Western Blotting and Immunocomplex Kinase Assay—Cells were ly-sed in TNNE buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1%Nonidet P-40, and 2 mM EDTA) containing 1 mM sodium orthovanadate,50 units/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, and 10mM NaF. Insoluble materials were removed by centrifugation. Thelysates were separated by 7.5% SDS-polyacrylamide gel electrophoresisand transferred to a polyvinylidene difluoride membrane (Trans-Blot,Bio-Rad). The proteins reacted with anti-Dfak56 antibodies were visu-alized with a chemiluminescence detection kit (Renaissance, NEN LifeScience Products). For immunoprecipitation, the cell lysates were pre-cleared with protein A-Sepharose 4B (Amersham Pharmacia Biotech)for 1 h at 4 °C. After removing the beads, the lysates were incubated for2 h at 4 °C with anti-Dfak56 antibodies and protein-A Sepharose. Theimmune complex was washed several times with TNNE buffer and thenwith kinase buffer (20 mM HEPES (pH 7.4), 10 mM MgCl2, and 10 mM

MnCl2). Following addition of [g-32P]ATP (370 kBq, 110 TBq/mmol), theimmunoprecipitates were incubated for 30 min at 30 °C and then sep-arated by 7.5% SDS-polyacrylamide gel electrophoresis. The gels were

dried and subjected to autoradiography. Phosphoamino acid analysis ofphosphorylated proteins and two-dimensional analysis of tryptic phos-phopeptides were performed as described previously (31).

RESULTS

cDNA Cloning and Sequence Analysis of a Drosophila Hom-olog of Focal Adhesion Kinase—To identify a Drosophila FAKhomolog of protein-tyrosine kinase, we used polymerase chainreaction with degenerate oligonucleotide primers to amplifycDNAs from a Drosophila embryo cDNA library. The sequencesof the degenerate primers corresponded to the amino acid se-quences conserved in both FAK and PYK2. Amplified cDNAfragments were subcloned into plasmids and sequenced. Wesearched the DDBJ/GenBankTM/EBI Data Bank against thesesequences. A cDNA fragment was revealed to contain a novelsequence with homology to FAK and PYK2. The cDNA frag-ment was subsequently used to screen the same library. FourcDNA clones of varying sizes were isolated, and the one con-taining the longest insert (4.3 kilobase pairs) was sequenced onboth strands. The complete nucleotide sequence of the cDNApredicted an open reading frame of 1198 amino acids (Fig. 1A).The calculated molecular mass of the predicted protein was 130kDa. Since the cDNA hybridized at 56A or 56B on the secondchromosome, which was determined by in situ hybridizationwith polytene chromosomes (data not shown), we named thegene Dfak56.

The deduced amino acid sequence of the putative product ofthe Dfak56 gene showed the presence of a protein kinase do-main. Like FAK and PYK2, Dfak56 has large N- and C-termi-nal sequences flanking the kinase domain. Comparison of theamino acid sequence of the Dfak56 kinase domain with theprotein sequence data bases revealed that the kinase domain ofDfak56 is most similar to that of FAK (59% identity to humanFAK) and PYK2 (52% identity to human PYK2) (Fig. 1B). Thesequences of the N- and C-terminal regions are relatively di-vergent. The amino acid sequence alignment of the kinasedomains of these proteins is shown in Fig. 1C. Unlike thevertebrate counterparts, Dfak56 contains an additional 24-amino acid sequence near the ATP-binding site of the kinase.In the C-terminal regions, FAK and PYK2 have a conservedsequence of ;150 amino acid residues. The sequence is calledthe focal adhesion targeting sequence because it is essential forlocalization of FAK to focal adhesions (32). Dfak56 also carriesa focal adhesion targeting-like sequence (;40% identity to hu-man FAK) in the C-terminal region (Fig. 1B), suggesting thatthe focal adhesion targeting sequence is conserved as a func-tional domain.

There are tyrosine phosphorylation sites within FAK thatare conserved in PYK2. A major autophosphorylation site(Y397AEI for human FAK and Y402AEI for human PYK2) thatmediates binding to the SH2 domain of Src-like protein-ty-rosine kinases is well conserved in Dfak56 (Y430AEI). Anothertyrosine phosphorylation site (Y925ENV for FAK and Y881LNVfor PYK2) serves for binding of the GRB2 SH2 domain. Tyro-sine 954 in Dfak56 is likely to correspond to this phosphoryla-tion site, although the sequence is slightly divergent(Y954CAT). The C-terminal region of FAK has two PXXP mo-tifs, P712PKPSRP and P874PKKPPRP, which can bind to theSH3 domain. These sequences are highly conserved in PYK2,and only one of them (P772PSKPSR, corresponding toP712PKPSRP of FAK) is conserved in Dfak56. We conclude thatDfak56 is a Drosophila homolog of vertebrate FAK family pro-tein-tyrosine kinases. The sequence data did not reveal whichkinase, FAK or PYK2, is the counterpart of Dfak56.

Southern hybridization analysis strongly suggested thatDfak56 is a single-copy gene of Drosophila (Fig. 2A). Moreover,under less stringent washing conditions, no bands other than

Focal Adhesion Kinase in Drosophila 29197

those for Dfak56 were found (data not shown). Therefore, wetentatively concluded that Drosophila contains no close rela-tives of Dfak56.

Temporal and Spatial Expression of Dfak56—Northern hy-bridization analysis of Drosophila poly(A)1-selected RNAsfrom various developmental stages (embryo, larva, pupa, andadult) and cell lines showed that two mRNA species of ;5 and6 kb hybridized with the Dfak56 cDNA probe (Fig. 2B). A shorttranscript of ;1.5 kb was also detected in embryonic and larvalRNA samples. This short transcript did not hybridize to thecDNA probe corresponding to the 59-portion (including the cod-

ing region N-terminal to the kinase domain) of the Dfak56mRNA, suggesting that this transcript corresponds to the 39-portion of the Dfak56 mRNA. The expression of a short tran-script was also reported in the case of the avian FAK gene (33).

To determine the spatial expression of the Dfak56 gene, weperformed whole-mount in situ hybridization with the em-bryos. In stage 16 embryos, strong expression of the Dfak56mRNA was detected in the central nervous system and thejunction of muscle and epidermis (Fig. 3, A and B). Immuno-histochemical analysis also showed expression of the Dfak56protein in the muscle attachment (Fig. 3C). A cross-sectioned

FIG. 1. Sequence analysis of Dfak56. A, deduced amino acid sequence of Dfak56. B, schematic representation of the similarity to human FAKand PYK2 proteins. The percentages of amino acid (a.a.) identities are indicated for the kinase domain and the focal adhesion targeting (FAT)sequence of FAK and Dfak56. C, amino acid sequence alignment of the kinase domain of the Dfak56 gene product with those of the human FAKand PYK2 proteins. Amino acids conserved among the three proteins are shaded. The Dfak56 gene product has a unique insertion of 24 residuesnear the ATP-binding site of the kinase (Lys513).

FIG. 2. Southern and Northern blotanalyses of the Dfak56 gene. A, Dro-sophila adult genomic DNAs (1 mg each;Oregon-R strain) were digested by restric-tion enzymes, blotted, and probed withDfak56 cDNA. Standard molecular sizesare indicated in kilobase pairs. B,poly(A)1-selected RNAs (5 mg each) fromDrosophila cell lines (BG2-c6, derivedfrom the central nervous system; and7E10, derived from body fluid cells) andfrom embryonic, larval, pupal, and adult(male and female) flies were subjected toNorthern blotting and probed withDfak56 cDNA. Transcripts of ;5 and ;6kb were observed in all samples, and ashort ;1.5-kb transcript was observedonly in the embryo and larva using full-length Dfak56 cDNA as a probe (left pan-el). This short transcript was not detectedwith the 59-probe (right panel).

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view of the immunostained image suggests that Dfak56 isexpressed predominantly in the epidermal cells, but less or notat all in the muscular cells (Fig. 3D).

Subcellular Localization of Dfak56—We examined the sub-cellular localization of the Dfak56 protein using the Drosophilaneuronal cell line BG2-c6 (25). The BG2-c6 cells expressed theDfak56 mRNA (Fig. 2B). They also expressed integrins, andformation of integrin clusters was observed in cells with severalfocal adhesion proteins, including p21-activated kinase anda-actinin (30). Immunofluorescent staining of the BG2-c6 cellswith anti-Dfak56 antibodies produced a focal adhesion-likepattern (Fig. 4A). The nucleus was also stained with theseantibodies. In some populations, the edges of the cells were alsostained. Staining with anti-phosphotyrosine antibody produceda pattern that overlapped with that of anti-Dfak56 antibodies,except for the nucleus (Fig. 4B). Staining with anti-phosphoty-rosine and anti-bPS integrin antibodies (Fig. 4C) showed colo-calization of tyrosine-phosphorylated proteins with integrin.These data suggested that Dfak56 is a component of focaladhesion in Drosophila.

Kinase Activity of Dfak56—We tested for protein kinaseactivity and phosphorylation of Dfak56 with mutational anal-ysis. By site-directed mutagenesis, an amino acid substitutionat residue 430 (tyrosine to phenylalanine) was introduced(Y430F mutant). This residue corresponds to the major auto-phosphorylation site in vertebrate FAK (tyrosine 397). We alsosubstituted methionine for lysine at residue 513, a possibleATP-binding site essential for tyrosine kinase activity (K513Mmutant). The cDNAs for the wild-type Dfak56 protein and themutant proteins constructed in expression plasmids were thenintroduced in simian COS cells. Cell lysates were immunopre-cipitated with anti-Dfak56 antibodies, and the precipitateswere subjected to in vitro kinase assay.

Autophosphorylation of the Dfak56 protein was observed inthe wild-type protein and the Y430F mutant, but not in theK513M mutant (Fig. 5A). The Dfak56 kinase activity observedwas not attributable to co-immunoprecipitated kinases becausethe lysine at residue 513 of Dfak56 was essential for the kinaseactivity. Phosphoamino acid analysis of in vitro phosphorylatedDfak56 detected phosphorylated tyrosine only (Fig. 5B), con-firming that Dfak56 is a protein-tyrosine kinase. Furthermore,the incorporated radioactivity of the phosphorylated Y430Fmutant was approximately half that of wild-type Dfak56 (Fig.5A), suggesting that the tyrosine at residue 430 is a majorautophosphorylation site. This was confirmed by two-dimen-

sional phosphopeptide mapping of the wild-type and Y430Fmutant proteins (Fig. 5C). A major spot in the peptide map ofwild-type Dfak56 (arrow) was not observed in that of the Y430Fmutant.

Phosphorylation of Dfak56 upon Cell Attachment to the Ex-tracellular Matrix—Drosophila S2 cells and stable transfec-tants (34) expressing Drosophila PS integrin chains of aPS1 andbPS (called PS1 cells) or aPS2 and bPS (called PS2 cells) werecultured on plastic dishes. Among them, only PS2 cells couldattach to a plastic dish in serum-containing medium because ofthe cross-reactivity of aPS2bPS integrin to vertebrate vitronec-tin and fibronectin (34). Using these cell lines, the levels ofphosphotyrosine in the Dfak56 protein were examined follow-ing integrin-dependent cell attachment to the extracellularmatrix. As shown in Fig. 6, the Dfak56 protein was tyrosine-phosphorylated in PS2 cells that attached to the surface of thedish. This indicated that Dfak56 is tyrosine-phosphorylatedupon integrin-dependent cell attachment to the extracellularmatrix. Note that tyrosine-phosphorylated proteins of 50–60kDa were associated with the Dfak56 protein (Fig. 6). Thesephosphoproteins were also present in the anti-Dfak56 precipi-tates of PS1 cells or suspended PS2 cells, but not in precipitatesof parental S2 cells. This suggests that the interaction betweenDfak56 and these phosphoproteins requires the presence ofintegrins and is enhanced by cell attachment to the extracel-lular matrix.

DISCUSSION

We have identified a Drosophila homolog of vertebrate focaladhesion kinase and termed it Dfak56. Dfak56 shares both

FIG. 4. Subcellular localization of Dfak56 by immunofluores-cence. BG2-c6 cells were grown on glass coverslips. After fixation, thelocalization of the Dfak56 protein (A) and phosphotyrosine (B) wasvisualized by immunofluorescence. In C, the cells were stained forphosphotyrosine (red) and for the bPS integrin subunit (green), showingthe colocalization of tyrosine-phosphorylated proteins and the bPS inte-grin subunit.

FIG. 3. Expression of Dfak56 in Dro-sophila embryos. Whole-mount RNA insitu hybridization (A and B) and immuno-histochemistry (C and D) were performedwith stage 16 embryos. A shows wholeembryos, and B and C show magnifiedviews of the surface of the embryo. Dshows a cross-section of the embryo. e,epidermis; m, muscle cells. Dfak56 is ex-pressed at the muscle attachment sites(arrowheads).

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sequence and structural homology with the vertebrate FAKfamily of protein-tyrosine kinases (FAK and PYK2). We dem-onstrated that Dfak56 is tyrosine-phosphorylated following in-tegrin-dependent cell attachment to the extracellular matrix.This property is the unique functional feature that distin-guishes FAK from PYK2. Thus, we conclude that Dfak56 is afunctional homolog of vertebrate FAK rather than PYK2. How-ever, tyrosine phosphorylation of FAK and PYK2 occurs inresponse to a number of stimuli, and some of them overlap(stimuli for the G-protein-coupled receptor, e.g. bradykinin)(35–37), suggesting that the biochemical function of FAK andPYK2 is partly interchangeable. Furthermore, the similarity ofthe kinase domains between FAK and PYK2 (50–60% aminoacid identity) is almost the same as that between Dfak56 andFAK or PYK2. Therefore, we strongly suspect that Drosophila

has only one FAK-related gene and that the ancestor geneduplicated and diverged in vertebrates during evolution.

In chicken embryonic fibroblasts, the FAK gene is alterna-tively transcribed to generate another short mRNA that en-codes a C-terminal portion of the FAK protein named FAK-related non-kinase (33). Ectopic expression of FAK-related non-kinase in fibroblasts blocks the formation of focal adhesions onfibronectin, acting as an inhibitor of FAK (38). We observed arelatively short, 1.5-kb transcript corresponding to the 39-por-tion of the Dfak56 mRNA only in embryo and larva (Fig. 2B).This mRNA might encode a FAK-related non-kinase-like pro-tein. If so, Dfak56-mediated cell adhesion processes may beregulated by the expression of this FAK-related non-kinase-like protein in embryogenesis.

Dfak56 expression is mainly observed in the embryonic cen-tral nervous system and the muscle attachment process. Inmuscle attachment, PS integrins and tiggrin, the ligand foraPS2bPS integrin, are expressed. Mutants defective for theseproteins show improper formation of muscle attachment, re-sulting in embryonic lethality (39, 40). In vertebrate neurons,FAK is highly expressed and localized to a growth cone (41, 42),suggesting that FAK plays a role in neural development andaxon guidance. Recently, the role of integrins in axon guidancein Drosophila was determined (43). Null mutations in eitherthe aPS1 or aPS2 subunit gene caused widespread axon path-finding errors that could be rescued by supplying the wild-typeintegrin subunit to the mutant nervous system. Another novela integrin subunit gene, Volado, was identified to mediateolfactory learning of Drosophila (44). These data suggest thatthe integrin-mediated signal transduction pathway is impor-tant for the function of the central nervous system. Given thesimilar expression patterns and the close functional relationbetween integrins and FAK in vertebrates, Dfak56 may alsoparticipate in neural functions of Drosophila.

One of the most effective strategies for the study of signaltransduction pathways is to identify novel upstream or down-stream factors of the known players in the signaling cascade.The genetics of Drosophila are suited for the large-scale screen-

FIG. 5. Kinase activity of Dfak56. A, cell lysates of 5 3 106 COScells (control; lane 1) and COS cells transfected with wild-type Dfak56(lane 2), the K513M mutant (lane 3), and the Y430F mutant (lane 4)were precipitated with anti-Dfak56 antibodies, and washed immuno-precipitates were subjected to in vitro kinase assay. To assess the levelof expression, 0.02 volumes of the lysates used for immunoprecipitationwere subjected to Western blotting and probed with the same antibod-ies. Standard molecular masses are indicated in kilodaltons. B, shownare the results from the phosphoamino acid analysis of the phosphoryl-ated Dfak56 protein. The autophosphorylated wild-type Dfak56 proteinwas extracted from the gel and hydrolyzed. The resulting phos-phoamino acids were subjected to two-dimensional electrophoresis atpH 1.9/3.5 and autoradiographed. The positions of phosphoserine (pS),phosphothreonine (pT), and phosphotyrosine (pY) are indicated. C,shown are the results from phosphopeptide mapping of the Dfak56proteins. The autophosphorylated wild-type and Y430F mutant Dfak56proteins were extracted from the gel, digested with trypsin, and sub-jected to two-dimensional phosphopeptide mapping. Asterisks show theorigin of electrophoresis.

FIG. 6. Tyrosine phosphorylation of the Dfak56 protein uponintegrin-dependent cell attachment to the extracellular matrix.S2 cells (106) and derivatives of their transfectants were seeded on 6-cmplastic dishes and cultured overnight. The next day, the suspended cellswere shaken off and corrected. The cells attached to the dish (only forPS2 cells) were washed and lysed on the dish. The cell lysates (100 mgof protein) were immunoprecipitated (IP) with anti-Dfak56 antibodies,subjected to Western blotting, and probed with anti-phosphotyrosineantibody (pY). To assess the amount of Dfak56 protein in the celllysates, 10 mg of proteins were blotted and probed with anti-Dfak56antibodies. Standard molecular masses are indicated in kilodaltons.

Focal Adhesion Kinase in Drosophila29200

ing to isolate the suppressor or enhancer if a visible mutant ofthe interested gene is available. Using this system, a number ofthe novel molecules implicated in the receptor protein-tyrosinekinase-mediated signal transduction pathways have been iden-tified (45–47). In the locus of the Dfak56 gene, polytene band56A to 56B, we could not find mutant alleles for the Dfak56gene in the FlyBase data base. However, if we could isolate themutants of Dfak56, the genetics of Drosophila would allowanalysis of the cell adhesion signaling, and the findings wouldrelate back to studies of higher organisms.

In conclusion, we cloned Dfak56, a Drosophila homolog of theFAK gene, and characterized its product by determining itsexpression, kinase activity, and signaling through integrin-mediated cell attachment. Future genetic studies would facili-tate the analysis of the roles of FAK family proteins in vivo.

Acknowledgments—We thank T. Todo for the cDNA library and D. L.Brower for the integrin-expressing cell lines.

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Focal Adhesion Kinase in Drosophila 29201