Cell, Vol. 66, 451-464, August 9, 1991, Copyright 0 1991 by Cell Press

The Discs-Large Tumor Suppressor Gene of Drosophila Encodes a Guanylate Kinase Homolog Localized at Septate Junctions Daniel F. Woods and Peter J. Bryant Developmental Biology Center University of California at Irvine Irvine, California 92717

Summary Mutations of the lethal(l)di large-7 (d/g) tumor suppressor gene of Drosophila cause neoplastic overgrowth of the imaginal discs. Sequencing of a near full-length cDNA predicts a protein containing a domain homologous to yeast guanylate kinase and a region homologous to SH3, a putative regulatory motif in nonreceptor protein tyrosine kinases and other signal transduction proteins. lmmunofluorescence analysis using antibodies directed against fusion peptides shows that the d/g gene product is localized in an apical belt of the lateral cell membrane, at the position of the septate junction. The results suggest that a signal transduction process involving guanine nucleotides occurs at the septate junction and is necessary for cell proliferation control in Drosophila epithelia. lntraduction Tumor suppressor genes in humans have been identified through studies of genetic changes occurring somatically in cancer cells (Ponder, 1990), and the study of these genes and their products has already led to the identification of several key points in the pathways controlling cell proliferation. Thus the retinoblastoma gene RB encodes a nuclear protein that shows cell cycle-dependent phosphorylation (Ludlow et al., 1990) and appears to control entry of the cell into S phase (Buchkovich et al., 1989; DeCaprio et al., 1989). The chromosome 11 Wilms’tumor gene WT encodes a zinc finger protein that is probably a transcription factor (Gessler et al., 1990; Call et al., 1990). The neurofibromatosis gene Nf 7 encodes a GTPase activating protein (Wallace et al., 1990; Martin et al., 1990; Xu et al., 1990a, 1990b; Ballester et al., 1990) that is probably involved in signal transduction at the inner face of the cell membrane. One of the tumor suppressor genes (DCC) that is lost during the progression of colorectal carcinoma encodes a putative cell adhesion molecule (Fearon et al., 1990). Although most of these tumor suppressor genes have been identified through their involvement with a specific tumor type, they appear to have important roles in controlling cell proliferation in many other tissues as well (Marshall, 1991). Furthermore, some tumor suppressor gene products appear to be targets for viral transforming proteins; the RB and ~53 proteins form complexes with the transforming proteins of DNA tumor viruses such as SV40, adenovirus, and papillomavirus, as part of the transforming action of these proteins (Ludlowet al., 1990; Howe et al., 1990; Lane and Benchimol, 1990). Tumor suppressor genes in Drosophila have been iden-

tified through classical genetic analysis. Germline mutations in such genes behave as recessive lethals (Gateff and Mechler, 1989), which can easily be recovered and studied using balanced-lethal genetic systems. Seven tumor suppressor genes have been identified by isolating recessive lethal mutations in which cell proliferation in imaginal discs continues beyond the normal limits. Some of these genes have previously been reported as recessive oncogenes, but since their normal alleles prevent tumor development we refer to them as tumor suppressor genes. In fivecases(/gd, Bryant and Schubiger, 1971; ~43, Martin et al., 1977; fat, Bryant et al., 1988; tud, Gateff and Mechler, 1989; dco, Jursnich et al., 1990) the overgrowing tissue is hyperplastic: it retains its epithelial structure and its ability to differentiate. In the other two cases (/g/, Gateff and Schneiderman, 1974; d/g, Stewart et al., 1972; Woods and Bryant, 1989) the mutations cause breakdown of the columnar epithelial structure of the imaginal discs and prevent them from differentiating. The disc cells become cuboidal in shape, show irregular apical-basal polarity, and develop wide intercellular spaces, suggesting a failure of cell adhesion. The imaginal discs in these mutants are therefore considered tumorous or neoplastic (Gateff and Mechler, 1989). Mutations at the /g/ locus produce malignant neuroblastomas of the larval central nervous system (Gateff and Schneiderman, 1974) in addition to imaginal disc neoplasia. The locus has been cloned and analyzed at the molecular level by Mechler et al. (1985) and Kllmbt et al. (1989). The nucleotide sequence of cDNAssuggests that the polypeptide has some similarities to the extracellular domains of the cadherin family of cell adhesion molecules (Klimbt et al., 1989), and immunocytochemical studies indicate that the product is localized to the cell surface (Merz et al., 1990). This would be consistent with the mutant phenotype that suggests defective cell adhesion. However, the absence of a transmembrane domain indicates that the gene product is not an integral membrane protein. In this paper we present a molecular genetic analysis of the d/g locus, in which recessive lethal mutations produce a neoplastic overgrowth phenotype in imaginal discs, very similar to that seen in /g/. We examine the phenotype caused by loss of the d/g product in the embryo and small somatic clones, present the nucleotide sequence of cDNAs from the d/g gene and the predicted amino acid sequence of its product, and show the localization of the gene product based on immunofluorescence studies using confocal microscopy. Our results indicate that the d/g gene product is a guanylate kinase homolog located in a small apical belt of the lateral cell membrane coinciding in position with the septate junction. This raises the possibility that control of epithelial cell proliferation involves guanine nucleotide-mediated signal transduction occurring at septate junctions. The d/g gene product appears to be required for this signal transduction process as well as for the maintenance of apical-basal cell polarity in the disc epithelium.

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Figure

1. Mitotic

Recombination

Clones

of d/gxfz

(A) Cuticle formed by a clone of d/gxl-zcells in the leg. Note the tube-like shape of the cuticle and projection of the clone out of the normal integument. Bar, 25 urn. (8) Another leg clone at the same magnification as (A). It also evaginates out of the surface of the normal cuticle and makes bristles (arrow). Both of the d!gx’-2 clones are marked with yellow and singed.

Results Mutations in d/g Disrupt Epithelial Structure in Both lmaginal Discs and Embryos During the extended larval period characteristic of d/g mutants the imaginal discs and the larval brain continue to grow beyond their normal final size. The discs fuse with each other and with the brain, and the single-layered columnar epithelium of the imaginal disc gives way to a dense mass of cuboidal cells that show disorganized apical-basal polarity except in small regions of the disc (Stewart et al., 1972; Murphy, 1974; Woods and Bryant, 1989). The discs also lose the ability to differentiate into adult cuticular structures, even after transplantation into genetically normal hosts (Stewart et al., 1972). We have investigated the ability of d/g imaginal disc cells to differentiate by examining clones of homozygous mutant cells produced by X-ray-induced mitotic recombination in an otherwise heterozygous and therefore normal fly. Large clones are never observed, but small clones that are homozygous for d/g can produce cuticle (Figure l), indicating that surrounding wild-type tissue can partially rescue the defect in differentiation orthat there is sufficient perdurance of the wild-type gene product to support a limited amount of differentiation. However, the mutant tissue evaginates out of the plane of the body surface and fails to elongate with the surrounding normal tissue, suggesting that epithelial morphogenesis isdisrupted within the clone. We have also produced homozygous d/g clones by X-ray-induced mitotic recombination in the female germline and examined the mutant embryos that are derived from these clones and that also lack zygotic d/g gene prod-

uct (Figure 2). By germband extension the d/g- embryos show abnormal segmentation in the epidermis and nervous system (compare Figures 2A and 28; also see Perrimon, 1988). The germband fails to retract, and by late embryogenesis the tissues of d/g- embryos are severely disrupted and break into clumps (Figures 2C-2E). These late embryos also appear to contain more cells than wild type (compare Figures 2C and 2D). In these d/g- embryos the epidermis forms tube-like structures rather than a continuousfolded epithelium; however, it still makes recognizable cuticular structures (Figures 2F and 2G). This result is in contrast to an earlier study in which cuticular derivatives were not found in d/g- embryos (Perrimon, 1988). Molecular Organization of the d/g Locus and Its Predicted Product We have cloned the interval containing d/g (lOB9-11; Woods and Bryant, 1989) and localized the gene to within a 20 kb region using DNA alterations found in four d/g alleles (Figure 3A). Six mRNAs that show different patterns of developmental expression are transcribed from overlapping parts of this 20 kb region, and four d/g alleles produce qualitative or quantitative changes in this set of transcripts (Woods and Bryant, 1989). Ten cDNA clones were obtained by screening cDNA libraries from four developmental stages and were completely sequenced. The nucleotide sequences of these cDNAs indicate that the d/g locus consists of at least 17 different exons that are spliced to give rise to several different mRNAs (0. F. W. and P. J. B., unpublished data). We have concentrated our initial analysis on one of the more abundant d/g transcripts, d/g-A (Figure 3A). Northern blot

Drosophila 453

Figure

Tumor

2. Embryos

Suppressor

Gene

from Germline

Clones

of d/gm52

and the (A) A d/g+ embryo at the beginning of germband retraction. (B) A dlgms2 embryo at a similar stage. Note the loss of normal segmentation formation of vesicles (arrow). (C) A late stage d/g+ embryo immediately before hatching. (D) A similarly staged d/gmsz embryo. Most of the tissue types are present but are abnormally shaped. There also appears to be an increased number of cells compared with the embryo in (C). The nerve cord fails to condense in these d/g m52embryos (see asterisk marking the neuropile). Bar in (A) is 100 urn. (E) A higher magnification of (D). Note the formation of vesicles in the epidermis that secrete cuticle (closed arrow). Cells from the nervous system extrude through the epidermis (open arrow), Bar, 10 urn. (F) Cuticle produced from an embryo like that seen in (D). The cuticle forms long tube-like structures. (G) Side view of the same embryo as(F) showing the formation of denticles on the inside of the embryo (closed arrow). The vitelline membrane is marked with the open arrow. The anterior ends of the embryo are to the right.

and polymerase chain reaction analyses show that the d/g-A transcripts are found throughout development in both larval and imaginal cells (data not shown). There are also two alternative sites for polyadenylation, giving rise to two molecular mass variants of the d/g-A transcript. The nucleic acid and predicted amino acid sequences from the 5.1 kb near full-length d/g-A cDNA, isolated from a 6-12 hr embryonic library and containing the complete open reading frame, are shown in Figure 36. The predicted

d/g-A protein consists of 960 amino acids and has a molecular mass of 102 kd. It has no hydrophobic stretches that would correspond to a signal sequence or transmembrane domain. It is rich (10.9%) in serine and has 19 potential phosphorylation sites for serine/threonine kinases (Figure 38). Homology searches (Pearson and Lipman, 1990) revealed the following five domains in the predicted protein (Figure 4; Figure 5). Amino acids 6-421 show 19.1% identity (69.8% similarity) with part of the filamentous hemag-

ii

P=,“I UY -

Figure

3. Structure

of the d/g Gene

and Predicted

14”

@-A

(5lkb)

untranslated

Proteins

(A) Molecular map of the d/g region showing the location of several DNA rearrangements and the exons from the d/g-A cDNA. The upper line is the genomic DNA; the lower lines are the exons (boxes) and introns determined from sequencing the genomic and cDNAs. HP321 is a temperaturesensitive allele. b88 is derived from a P element-induced allele (J. Fristrom, K. Fechtel, J. Moore, and J. Naetzle, personal communication), and SW was induced by diepoxybutane (R. Pearson, personal communication). V, insertions; A, deletions. b88 also contains some P element sequence flanking the deletion. The striped region of the cDNA is an opa repeat (Wharton et al., 1985). The question mark indicates exon-intron junctions not yet mapped in genomic DNA. (9) The nucleotide sequence and predicted protein sequence from the d/g-A cDNA. The nucleotide and amino acid numbers are shown on the left. Potentially phosphorylated amino acids are circled. The asterisk represents the termination codon determined from the cDNA sequence. There is approximately 1.7 kb of untranslated sequence in the cDNA before the poly(A) tail (data not shown), The poly(C) tract at the beginning of the sequence was added when the cDNA library was constructed (L. Kauvar, personal communication). The changes caused by d/gv5’ and d/g”” are shown as lines above the nucleotides that are deleted. The d/g’” deletion extends into the 3’ untranslated region.

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Figure 4. Sequence Predicted d/g Protein

Comparisons

with

the

(A) Comparison between the amino acid sequence of the predicted @-A protein and that of crk (Mayer et al., 1988) in the SH3 domain. Two dots indicate identity. one dot a conservative substitution. (B) Comparison between part of the amino acid sequence of the predicted d/g-A protein and that of yeast guanytate kinase (GUK; Berger et al.. 1999). Two dots indicate identity; one dot indicates a wnservative substitution. Double undetiine, the amino acids that, in GUK, are involved in hydrogen bonding to the phosphate group of GMP; single underline, amino acids that form direct contacts with GMP (Stehle and Schulz, 1990).

insert near the 5’ end of the gene, and the d/p8 allele shows a small deletion, also near the 5’ end (Figure 3). These results indicate that both the guanylate kinase function and the amino terminal part of the gene product are important for its tumor suppressing function.

glutinin produced by whooping cough virus (Domenighini et al., 1990), and amino acids 127-631 show 12.3% identity (64.6% similarity) to the human collagen a chain (Ramirez et al., 1990). Amino acids 297-452 represent a serinel alaninelasparagine-rich region encoded by an opa repeat (Wharton et al., 1985). Amino acids 604-662 show close homology to the SH3 (src homology 3) region (also called the A box or “modulatory” domain) found in the src family of nonreceptor protein tyrosine kinases and a variety of other proteins. The closest homology is to the v-crk oncogene (42% identity, 71% similarity, over a 59 amino acid stretch; Figure 4A; Mayer et al., 1988). Amino acids 717764 represent a PEST sequence, a motif associated with proteins that have a short half-life (Rogers et al., 1986). Amino acids 770-948, near the carboxyl end of the molecule, show 35.5% identity (77.4% similarity) with the entire published sequence (186 amino acids) of yeast guanylate kinase (Figure 46; Berger et al., 1989). Changes in Mutant Alleles To determine which parts of the predicted d/g protein are necessary for its tumor suppressor function, we have sequenced several alleles. As shown in Figure 3, the d/gy5g mutation is a 14 bp deletion in the first third of the guanylate kinase domain that changes the open reading frame and introduces a stop codon after 12 amino acids. The d/g”” alteration is a 100 bp deletion that changes the carboxyl end of the protein just outside the region with guanylate kinase homology. As shown previously (Woods and Bryant, 1989) the dlgHF32’ allele is associated with a 5.5 kb

Z-Y:: .-~-; 1 em &J

-

OPA

50aa

PEST &sx

FILAMENTOUS

SH3 pGEX

Developmental Expression of the d/g Proteins Two bacterial expression constructs were made from parts of the d/g-A cDNA that encode part or all of the SH3 domain and most of the guanylate kinase domain (Figure 5). Protein from one fusion construct was used to raise antibodies in rats, and protein from the second construct was used to raise antibodies in rabbits. Since the regions used are common to all of the transcripts, both antisera are expected to recognize the protein products of all of the transcriptvariants. We haveobtained indistinguishable results with the two antisera. We have studied the localization of the d/g proteins at different developmental stages by indirect immunofluorescence using confocal microscopy (Figures 6-9). The protein is found mainly in epithelia, where it is localized at the apical part of the lateral cell membrane. Adult Reproductive Tissues In the male reproductive tissue the apical region of the lateral cell membranes of the accessory gland and of the sperm pump(Figures 6Aand 6B) stain intensely. The testis exhibits a complex pattern of staining including specific labeling of structures within the gonial cells (Figure 6C). The oogonial cells also stain more intensely than the surrounding tissue (Figure 6D). In the ovaries the protein is

--_.

rnnH

--

GUANYLATE

KINASE

Figure 5. Homologies Protein

of the Predicted

d/g-A

See text for explanation. Below are shown the parts of the cDNA used for making bacterial expression constructs for generating antibodies.

Figure

6. Confocal

Images

Showing

Expression

of the d/g Protein

in the Adult Reproductive

Tissue

(A) An optical section from a male accessory gland. Most of the staining appears localized along the apical-lateral membrane facing the lumen. (6) Section of a male sperm pump. The arrow points to the apical side of the epithelium. (C) A section from the end of the testis. The arrow points to a labeled structure within the gonial stem cells. (D) A similar section from the end of an ovary. The arrow points to a group of gonial cells that stain more intensely with the antibodies. (E) Staining of a developing oocyte. The follicle cells are labeled along the membrane mainly at the apical-lateral junctions. The nurse cells exhibit staining throughout the cell. (F) A section of a developing oocyte. The arrow points to staining within the cytoplasm of the oocyte. The staining of the follicle cell membrane is readily apparent. Bars in (A)-(D) and (F), 10 pm; bar in (E), 25 urn.

Drosophila 457

Figure

Tumor

7. Confocal

Suppressor

Images

Gene

Showing

Expression

of the d/g Protein

in the Embryo

(A) Optical section of a cellular blastoderm stage. Note staining of cell outlines but lack of staining at cell corners. (B) Section of a germband-extended embryo showing the diffuse staining at this stage. However, cell membranes are still immunoreactive. (C) Dorsal closure. The peripheral neurons are visible at this stage (arrow). (D) An embryo slightly older than in(C). Note the more defined staining of the epidermal cells compared with(C). Both(C) and(D) are reconstructions of serial optical sections. (E) Section from an embryo the same age as (D) showing the bright labeling of the apical-lateral junctions of the developing gut (arrow). (F) A section of the ventral ganglion in a late embryo. At this stage the more intense staining of the nerve fibers is also visible. Bar in (A), 10 urn; bars in (B)-(F), 25 km.

Cdl 458

Figure

8. Confocal

Images

Showing

Expression

of the d/g Protein

in the Late Third

lnstar

Larva

(A) Serial reconstruction of optical sections of a salivary gland. The antibodies stain a thick belt-like structure at the apical-lateral (B) A reconstruction of the proventriculus. Note the staining along the cell boundaries, particularly at the corners. (C) A section from a wing disc showing the staining around the apical-lateral membrane. (D) Another section from a wing disc showing the extreme localization of staining in imaginal cells. Also note the less intense staining active cell (arrow). (E) Staining in the larval brain. (F) A higher magnification of the end of the ventral ganglion showing the more intense staining of the axons (arrow). Bars in (A)-(D) and (F), IO urn; bar in (E), 100 urn.

membrane.

of a mitotically

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Gene

Larvae In the larvae both polytene and diploid cells stain with antibodies against the d/g protein. In the salivary gland the gene product is present as a wide apical belt around each cell (Figure6A). The proventriculus also stains, particularly at the cell corners (Figure 8B). In the imaginal discs, staining is observed at the apical region of the lateral membrane, forming a narrow belt around each cell (Figures 8C and 8D). Cells in division stain less intensely than their neighbors but still have a localized ring of d/g protein near the apical cap (Figure 8D). The protein is found in specific parts of the larval brain including the neuropile of the optic lobes and the ventral ganglion (Figures 8E and 8F). Tissue dissected from a larva homozygous for a putatively null d/g allele shows patchy staining in the salivary gland (Figure 9A) and no staining along the membrane of the imaginal disc cells (Figure 9B). Discussion

Figure 9. Distribution That Are Homozygous

of the d/g Protein in Late Third lnstar Larvae for a Genetically Null d/g Mutation (m52)

(A) A serial reconstruction of a salivary gland. Compare with the one shown in Figure 8A. Note the lack of a complete belt of staining around each cell (arrow). The nucleus also stains (star). (6) A wing imaginal disc. Note the absence of staining of the cell membrane [compare with Figure 9C). Bars, 10 urn.

detected at the lateral membranes of the follicle cells around the developing oocyte and in the nurse cell cytoplasm (Figure 6E). The d/g protein appears to be deposited within the oocyte cytoplasm (Figure 6F). Embryos In the embryo the first organized staining is at the cellular blastoderm stage (Figure 7A). At this stage d/g protein is found at the apical region of the forming lateral cell membrane, around each cell with the exception of the corners. By germband extension the staining is more diffuse within the cells but still predominantly along the membrane (Figure 78). At the time of dorsal closure, staining within the larval epidermis becomes restricted along the cell boundaries (compare Figures 7C and 7D). The developing peripheral neurons also stain (Figures 7C and 7D). Localized staining of the apical region of the lateral membrane can also be seen in the developing gut (Figure 7E). Other internal tissues stain less intensely with the antibodies. The axon bundles stain more intensely than the rest of the developing larval brain (Figure 7F).

Our results indicate that one of the major products of the d/g gene is a 102 kd protein that includes a region of guanylate kinase homology and is specifically localized to an apical belt of the lateral cell membrane in many epithelial tissues. This zone of the lateral cell membrane is occupied by septate junctions, structures in which regularly spaced electron-dense septa partition the intracellular space. They are thought to be the invertebrate equivalent of tight junctions (Noirot-Timothee and Noirot, 1980). Our working hypothesis is that the gene product is involved in signal transduction events occurring at this part of the cell and that this signal transduction is necessary for cell proliferation control in mitotically active tissues. The part of the predicted gene product that shows similarity to filamentous proteins might represent a structural component of septate junctions, necessary for epithelial integrity. d/g+ Is Required for Cell Proliferation Control, Cell Polarity, Cell Adhesion, and Differentiation in Both lmaginal Discs and Embryos In imaginal discs, d/g mutations interfere with proliferation control, apical-basal cell polarity, cell adhesion, and the ability of cells to differentiate. Some, but not all, of these abnormalities are also seen in embryos deprived of both maternal and zygotic d/g+. lmaginal discs show excess cell number (Woods and Bryant, 1989) and the mutant embryos show an apparent accumulation of excess cells in both the nervous system and epidermis, suggesting loss of proliferation control. Disruption of apical-basal polarity is shown by the morphology of imaginal disc cells and by the production of neuroblasts to both sides of the embryonic ectoderm, rather than only to the basal side as in normal embryos (Perrimon, 1988). In both imaginal discs and embryos there are indications of a failure in cell adhesion; the tube-like shape of the embryonic epidermis before cuticle formation suggests that d/g mutations disrupt the continuity of the epithelial sheet, and mitotic recombination clones in imaginal disc derivatives show similar abnormalities. A function for d/g in cell adhesion has already been suggested (Perrimon, 1986) based on phenotypic

Cell 460

analysis, The ability of epidermal cells to differentiate is lost in the imaginal discs of d/g as shown by the failure to recover cuticular structures after transplantation of mutant discs into wild-type larval hosts (Stewart et al., 1972) and the failure to recover large mitotic recombination clones that would be homozygousfor the mutation although small clones of abnormal cuticle can be recovered. The d/g product seems to be less critical for differentiation in the embryonic ectoderm, since some of the expected cuticular elements and most of the differentiated tissue types are present in the mutant embryos even when they lack both maternal and zygotic d/g product. The d/g Proteins Are Localized in the Apical Part of the Lateral Cell Membrane Our antibody localization of the d/g product shows that it is mainly localized in an apical belt of the lateral cell membrane in most tissues where it is expressed except in the nervous system. In the nervous system it is located mainly along the axons. From the predicted protein sequence d/g-A appears to be a cytoplasmic protein, since it has no signal sequence or transmembrane domain; therefore we assume that it is attached to the inner surface of the cell membrane or to the cortical cytoskeleton. Both adherens junctions and septate junctions are found in apical belts of the lateral cell membrane, the adherens junction being apical to the septate junction (Poodry and Schneiderman, 1970; Eichenberger-Glinz, 1979). The d/g staining coincides with the apical-basal position of the septate junctions. The anti-d/g antibodies stain the cell boundaries as early as blastoderm, long before septate junctions are detectable (Eichenberger-Glinz, 1979). However, during subsequent embryonic development the intense and clearly localized staining of the d/g protein becomes evident during dorsal closure, the same time that septate junctions first become frequent and large (Eichenberger-Glinz, 1979). In the salivary gland from late third instar larvae the d/g antibodies stain a wide belt-like structure, a pattern resembling the distribution of the septate junctions in this tissue (Wiener et al., 1990). Therefore, we conclude that the d/g product is either an integral part of or is associated with septate junctions. The function of septate junctions is unknown. Since invertebrate epithelia have septate junctions but no tight junctions, whereas the converse is true of vertebrate epithelia, it has been suggested that the two junction types have similar roles (Noirot-Timothee and Noirot, 1980). Tight junctions are thought to provide a transepithelial barrier and to restrict mobile membrane proteins to either the apical or the basal-lateral membrane domain, thus maintaining apical-basal cell polarity (Rodriguez-Boulan and Nelson, 1989). Breakdown of tight junctions by treatment of cells with proteases or chelating agents leads to loss of apical-basal polarity and intermixing of apical and basal-lateral membrane proteins (Ziomek et al., 1980; Pisam and Ripoche, 1978; Herzlinger and Ojakian, 1984). It is possible that septate junctions have a similar function (Noirot-Timothee and Noirot, 1980) even though they are basal to the adherens junctions whereas tight junctions are apical. If this is the case, an effect of d/g mutations

on septate junctions could account for the loss of apicalbasal cell polarity seen in the mutant imaginal discs. Septate junctions are also thought to function in adhesion (Noirot-Timothee and Noirot, 1980), so an effect of d/g on these structures could also account for the extensive loss of cell contact seen in mutant imaginal discs. The nondividing polytene cells of the larva are apparently not affected by d/g mutations (Poodry and Woods, 1990) even though they express large amounts of the d/g protein. A possible explanation is that some of the d/g protein in these cells is maternal or synthesized on maternal transcripts. This would account for the defective development of the larval epidermis seen in mutant embryos derived from mutant germline clones. The maternal contribution may be insufficient to rescue the imaginal disc cells in the same larvae because the imaginal disc cells depend on zygotic, rather than maternal, d/g product during growth. Furthermore, it seems likely that the d/g protein is resynthesized during each mitosis of imaginal disc cells. When larvae carrying the temperature-sensitive allele ,-JgHF321 are grown at the permissive temperature until the imaginal discs terminate cell division at their normal final size, then additional culture at the restrictive temperature does not lead to neoplasia. However, if the discs are still undergoing cell division when the larvae are shifted to the restrictive temperature, they become neoplastic (D. Sponaugle, personal communication). Similar results have been reported for /g/ (Hanratty, 1984). This could be explained if the d/g protein is broken down during mitosis and resynthesized in each cell cycle; cells that lacked functional d/g protein after cell division would then become neoplastic. In support of this argument most of the d/g protein appears to be lost from dividing cells (see Figure 8D). The PEST sequence that is found in d/g-A may function as a signal for degradation during each cell cycle. Structure and Function of the Predicted d/g-A Protein Our homology searches have revealed several domains of homology within the predicted d/g protein. The N-terminal half of the d/g-A protein shows limited homology but high similarity to two filamentous proteins, filamentous hemagglutinin and collagen a chain. These high similarity values over a long region suggest similar physical properties rather than true sequence homology, and the obvious possibility is that this region of the molecule has a filamentous or fibrous conformation. It seems likely that it forms part of the structure of the septate junction. Amino acids 604-662 of the predicted d/g-A protein show clear homology to the SH3 domain found in nonreceptor protein tyrosine kinases of the src family (Pawson, 1986) and various other proteins, many of which are involved in signal transduction. These include the cab/ and v-c& products (Mayer et al., 1988) a-spectrin (Wasenius et al., 1989) phospholipase C II (Stahl et al., 1988), neutrophil NADPH oxidase (two separate SH3 domains; Lomax et al., 1989), GTPase activating protein (GAP; Trahey et al., 1988), and the predicted products of the yeast genes cdc25 (Broek et al., 1987; Rodaway et al., 1989) and fus1 (Rodaway et al., 1989; Trueheart et al., 1987; McCaffrey

Drosophila 461

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et al., 1987). Some actin-binding proteins (yeast ABP-1 , Drubin et al., 1990; Acanthamoeba myosin I, Jung et al., 1987; Drubin et al., 1990) have also been shown to contain SH3 domains. The d/g SH3 domain shows the greatest similarity to that of the v-crk oncogene (42% identity, 71 O/O similarity, over a 59 amino acid stretch). Three possible functions, which are not mutually exclusive, have been ascribed to the SH3 domain. First, it may modulate the activity of the catalytic domain of the same protein. This idea is based mainly on the finding that a deletion or change of several specific amino acids in the SH3 domain can activate transforming ability of c-src and c-abl (Jove and Hanafusa, 1987; Potts et al., 1988; Jackson and Baltimore, 1989). Second, the SH3 domain may be a localization signal. The d/g product is localized at the cell surface, and for all of the other known SH3 domaincontaining proteins there is either direct or indirect evidence for a submembrane location (Broek et al., 1987; McCaffrey et al., 1987; Jung et al., 1987; Jove and Hanafusa, 1987; Jackson and Baltimore, 1989; Wasenius et al., 1989; Drubin et al., 1990). Third, the SH3 domain may be a site of interaction with the actin filaments of the cortical cytoskeleton. In several cases (ABP-1 , Drubin et al., 1988; and myosin I, Adams and Pollard, 1989) there is evidence that the protein interacts with the actin filaments of the submembrane (cortical) cytoskeleton. The SH3 domaincontaining region of one of these proteins (myosin I protein of Acanthamoeba) defines a domain directly involved in ATP-independent actin binding (Jung et al., 1987; Drubin et al., 1990). That the SH3 domain is involved in interactions with the cytoskeleton is also suggested by the fact that mutations in this region of src cause transfected cells to take on a fusiform rather than round shape (Jove and Hanafusa, 1987). Activation (by phosphorylation) of phospholipase C II, which is located at the cell cortex, is associated with alterations in the cytoskeleton and cell morphology (Wahl et al., 1989). lmaginal discs from d/g larvae show a disorganized actin cytoskeleton (M. Walter, personal communication). We conclude that the SH3 domain of d/g might mediate interaction between this protein and the cortical cytoskeleton and that this interaction may modulate its activity. A sequence of 179 amino acids near the C-terminus of the predicted d/g-A protein shows 35.5% identity (77.4% similarity) with the entire sequence (186 amino acids) of yeast guanylate kinase (Berger et al., 1989) an enzyme that catalyzes the reaction 5’GMP + ATP * GDP + ADP. Furthermore, most of the amino acids known to be important for GMP binding in guanylate kinase are conserved in d/g-A. All 4 of the amino acids that, in the yeast enzyme, are involved in hydrogen bonding to the phosphate group of GMP (Tyr-50, Tyr-78, Arg-38, and Arg-41; Stehle and Schulz, 1990) are conserved in the d/g-A product, and the other amino acids that form direct contacts with GMP (Stehle and Schulz, 1990) are either matched (Glu-69) or conservatively substituted (Ser-34 to Pro, Ser80 to Thr, Asp-100 to Ser) in d/g-A. Therefore, it seems very likely that the d/g-A product is a functional guanylate kinase. This region of the d/g-A product also shows homology (26.4% identity and 75.7% similarity) with vaccinia

kin&e Figure 10. The cGMP and awd

Cycle

and the Possible

Modes

of Action

of d/g

See text for explanation.

virus gene A57R (data not shown; Goebel et al., 1990), suggesting that A57R might encode a previously unidentified nucleotide kinase. The d/g protein might serve a dual role as part of the structure of the septate junction and as a guanylate kinase enzyme. Our mutant sequencing shows that one d/g allele has a deletion in the catalytic domain, indicating that the catalytic activity is required for the tumor suppressing function of the protein. It is unlikely that the mutations cause neoplasia simply by interfering with the structure of septate junctions, since septate junctions are still present in neoplastic discs from some d/g alleles (D. F. W. and P. J. B., unpublished data), and dig- embryos show visible defects before the formation of the septate junctions (see Figure 2; Eichenberger-Glinz, 1979). The Protein Homologies and the Location of d/g Protein Suggest a Role in Signal Transduction Guanylate kinase catalyzes the first step in the “cGMP cycle” (Figure 10; Hall and Kuhn, 1986) which provides for the recycling of guanine nucleotides into cGMP via GTP. The effect of d/g mutations on cell proliferation could be explained by their potential effects on GDP and GTP levels, which control the activity of the protein p21ras, known to be involved in proliferation control (Bos, 1989). Thus, GDP levels, at least at the septate junctions, are expected to be reduced in d/g-A mutants. This could lead to reduced loading of p21 r*sproduct with GDP and perhaps increased loading of GTP from cellular sources other than the recycling pathway, causing activation. Recessive mutations in the Drosophila gene awd, for which the vettebrate homolog appears to be the metastasis suppressor gene nm23 (Liotta and Steeg, 1990) cause underdevelopment of imaginal discs (Dearolf et al., 1988). The awdgene

Cdl 462

encodes a nucleoside diphosphate kinase (Biggs et al., 1990) which converts nucleoside diphosphates into triphosphates. GDP levels would be expected to be elevated and GTP levels depressed in awd mutants. The GTP:GDP ratios would then be expected to be low in awd and high in d/g, at least at the septate junctions. Since the ~21”” is activated by high GTP:GDP ratios, the effects of the altered GTP:GDP ratios on ~21”” activation could account for the excess cell proliferation in d/g and inhibited proliferation in awd. The awd mutation may also act by reducing GTP available for microtubule polymerization, leading to arrest of cell division at metaphase (Biggs et al., 1990). A prediction from the above hypothesis is that activated ~21”” should produce an overgrowth phenotype similar to that seen in d/g mutants. Transfected ras genes with an activating mutation, under the control of a heat shock promoter, do produce defects in imaginal disc differentiation in response to short heat shocks (Bishop and Corces, 1988) but overgrowth has not been reported. The same mutant genes under the control of aras promoter produce much less severe phenotypes such as disorganized ommatidia, resembling the phenotype caused by some weak d/g alleles (D. F. W. and P. J. B., unpublished data). This suggests either that an increased GTP:GDP ratio may be more strongly activating than the ras mutations so far tested, or that additional G proteins may be involved in the effects of dig. Our results raise the possibility that the d/g product may control cell proliferation by regulating the supply of guanine nucleotides to membrane-bound components of a signal transduction pathway that are localized at the septate junctions, possibly p2l”“orother G proteins. The analysis of other genes identified by imaginal disc overgrowth mutations may lead to the isolation of additional components of this pathway. Experimental

Procedures

Mitotlc Recombination Clones Homozygous somatic and germline clones (Perrimon, 1988) of d/g weregenerated by irradiating heterozygousywsnd/gX”9K7237female larvae with 1000 rads of y rays at 48 hr after egg laying and allowing them to develop into adults. The K7237 mutation is a germlinedependent dominant female sterile mutation that blocks development of heterozygous germ cells (perrimon and Gans, 1983). For the somatic clones, patches of d/g- adult cuticle were identified using the yellow marker mutation, and the flies carrying them were dissected and mounted in euparai. For the germline clones the adult females were crossed to males carrying either the deficiency for the d/g region DA622 or a genetically null allele (d/g”“); D. F. W. and P. J. B., unpublished data). The resulting embryos were collected and allowed to develop at 25°C. The embryos were then dechorionated and, for cuticle preparations, mounted in 85% lactic acid in ETOH. They were observed under phase contrast. For histological analysis the dechorionated embryos were fixed, and the viteliine membrane was removed (Dequin et al., 1984). They were then embedded in JB-4. and 3 vrn sections were cut and stained with methylene blue. Those embryos that were rescued by zygotic d/g+ from the father (Perrimon, 1988) served as the controls. Molecular Techniques Isolation of the genomic DNA and d/g cDNAs has been previously described (Woods and Bryant, 1989). The sequence was determined from plasmid subclones using Sequenase (US Biochemicals). For the cDNAs both strands were sequenced, and the exon-intron junctions

were determined by sequencing the genomic DNA using primers derived from the cDNA sequence. The open reading frame was determined and analyzed with the SNAP program (kindly provided by G. Gutman), and the amino acid sequence was used to search the GenBank database using the FASTA program (Pearson and Lipman, 1990). To sequence the guanylate kinase domain from the d/g alleles primers were made to amplify by poiymerase chain reaction (Mullis and Faloona, 1987) the genomic region containing these exons, about 1 kb of sequence. The amplified products were subcloned into the Bluescript plasmid (Stratagene), and two independent isolates for each genotype were sequenced. immunological Techniques A cDNA fragment encoding amino acids 631-919 was fused to trpE (PATH; Dieckmann and Tzagoioff, 1985). and another fragment encoding amino acids 455-919 was fused to the glutathione S-transferase gene (pGEX; Pharmacia) to create fusion genes, which were then expressed in bacteria. Protein from the trpE fusion was extracted from the bacteria and separated on SDS-PAGE. After staining with Coomassie blue, the appropriate band was cut out, and the protein was electroeluted and lyophilized. For the giutathione S-transferase fusion the bacteria were sonicated in 1% Nonidet P-40, PBS, and the fusion protein was affinity purified with a glutathione-Sepharose column and lyophilized. The purified fusion proteins were used to raise antibodies (Pocono Farms) in either rats (trpE) or rabbits (giutathione S-transferase). The sera were affinity purified with an agarose column coupled with the glutathione S-transferase fusion peptide. For immunoiocaiization, embryos were prepared as described by Dequin et al. (1984). For later stages the tissue was hand dissected in Ringer solution and fixed in 4% formaldehyde, PBS. The tissue was then blocked in 5% BSA, 0.5% Nonidet P-40, PBS for 1 hr at 22OC. The affinity-purified primary antibodies were diluted 1:lOO with the blocking solution and incubated with the tissue overnight at 4OC. The tissue was washed three times with blocking solution and incubated for 1 hr at 22OC with FITC-labeled secondary antibody (Jackson Laboratory) that had been preabsorbed with embryos and diluted to 1:40. After washing three times with blocking solution and twice with PBS the tissues were mounted in 80% glycerol, 100 mM Tris (pH 8.5) 5% n-propyi gailate and observed with the Bio-Rad MRC-600 confocal microscope. Acknowledgments We thank John Moore, Jim Fristrom, Kim Fechtei, and Jeanette Naetzle for providing information on the b88 mutant, Rebecca Pearson for providing the SW allele, and David Drubin and Hidesaburo Hanafusa for providing us with their unpublished results and helpful discussions. We also thank Jo Wen Wu for her useful insights and Jack Zeineh for sequencing some of the exon-intron junctions. This work was supported by grant DCB-8917449 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

January

8, 1991; revised

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GenBank

Accession

The accession M73529.

number

Number for the sequence

reported

in this

paper

is