- Email: [email protected]
Drosophila DPP2C1, a novel member of the protein phosphatase 2C (PP2C) family
Gene 199 (1997) 139–143
Drosophila DPP2C1, a novel member of the protein phosphatase 2C (PP2C ) family Thomas Dick, Sami M. Bahri, William Chia * Institute of Molecular and Cell Biology, National University of, Singapore, 15 Lower Kent Road, Singapore 119260 Received 22 April 1997; accepted 23 May 1997; Received by E. Boncinelli
Abstract We report the molecular cloning, chromosome mapping and developmental transcription pattern of a putative serine/threonine protein phosphatase 2C (PP2C ), DPP2C1, from Drosophila melanogaster. The 6-kb transcript of this first Drosophila PP2C gene encodes a 1428-aa deduced protein. The DPP2C1 protein contains a #330-aa PP2C-like catalytic domain flanked by extensive N- and C-terminal sequences showing no similarities to other PP2Cs. The dpp2c1 gene maps to 4E1-2 on the X chromosome, 1.5 kb upstream of the ddlc1 gene. Northern blot analyses showed that dpp2c1 transcription is developmentally regulated, accumulating maximally during early (0–6 h) and late (12–24 h) embryogensis. The presented molecular characterisation provides the basis for a genetic dissection of DPP2C1 function. © 1997 Elsevier Science B.V. Keywords: PPM; X chromosome; 4E1-2
1. Introduction Two structurally distinct serine/threonine phosphatase sequence families exist ( Klumpp et al., 1994): (1) the PPM family of Mg2+- or Mn2+-dependent PP2C-like enzymes; and (2) the PPP family which includes PP1, PP2A and PP2B (Cohen, 1994). The antagonistic roles of PPPs with respect to protein kinase-triggered regulation pathways are well established (for a review, see Cohen (1992)), but experiments aiming at the determination of the cellular functions for PP2C family members have started only very recently (Leung et al., 1994; Maeda et al., 1993; Meyer et al., 1994; Robinson et al., 1994; Shiozaki and Russell, 1995; Shiozaki et al., 1994). Numerous PP2C-like genes from various species have been cloned (Bork et al. (1996) and references herein), and studies of the crystal structure of the human PP2Calpha have provided an insight * Corresponding author. Tel.: +65 7723790; Fax: +65 7791117; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); DPP2C1, Drosophila serine/threonine specific protein phosphatase type 2C protein 1; dpp2c1, gene encoding DPP2C1; ddlc1, gene encoding the Drosophila cytoplasmic dynein light chain 1; kb, kilobase(s); kDa, kilodalton; nt, nucleotide(s); PP2C, serine/threonine specific protein phosphatase type 2C. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 3 59 - 4
into the structure of the PP2C catalytic domain (Das et al., 1996). Within the PP2C family, the #300-aa PP2C catalytic domain occurs in numerous structural contexts. For instance, PP2C from Paramecium tetraurelia ( Klumpp et al., 1994) represents one of the smallest PP2C proteins and appears to consist of essentially only the PP2C catalytic domain. The human PP2Calpha contains an additional small stretch of aa added to the C-terminus of the PP2C catalytic domain that might provide protein substrate specificity (Das et al., 1996), whereas the Arabidopsis PP2C encoded by the ABI1 gene contains a putative Ca2+-binding EF hand motif N-terminal to the catalytic domain (Leung et al., 1994; Meyer et al., 1994). Another PP2C homologue in Arabidopsis, KAPP, associates with the receptor serine/threonine kinase, RLK5, and consists of three regions: an N-terminal membrane localisation signal, a kinase interaction domain that associates with the phosphorylated receptor, and a C-terminal catalytic domain (Stone et al., 1994). Mammalian mitochondrial pyruvate dehydrogenase phosphatase contains a N-terminal Ca2+-binding site (Lawson et al., 1993). The SpoIIE phosphatase of Bacillus subtilis has 10 membrane-spanning regions preceding the catalytic PP2C domain (Duncan et al., 1995; Bork et al., 1996; Adler et al., 1997). Finally, a 300-residue region of yeast adenyl cyclase, present imme-
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diately N-terminal to the cyclase catalytic domain ( Kataoka et al., 1985), shares sequence similarity with PP2C ( Tamura et al., 1989). This domain may function to mediate ras-GTP activation of adenyl cyclase activity (Coliccelli et al., 1990; Minato et al., 1994) and is not known to possess protein phosphatase activity. We have performed an extensive molecular characterisation of the 4E1-2 cytogenetic interval of the Drosophila genome during the analysis of the cytoplasmic dynein light chain 1 gene, ddlc1 (Dick et al., 1996). In the course of these experiments, we discovered that a transcription unit neighbouring the ddlc1 gene encodes a protein, which contains a central domain showing significant homology to the PP2C catalytic domain flanked by extensive stretches of sequences not found in other PP2Cs. We named this first PP2C-like gene identified in Drosophila dpp2c1. In order to provide the basis for a genetic dissection of the biological role of this novel PP2C subtype, using the fly as a model system, we report here the cloning, chromosome mapping and developmental expression of the dpp2c1 gene.
2. Experimental and discussion 2.1. Cloning and mapping of the dpp2c1 transcription unit Previously, we reported the analyses of the gene encoding the cytoplasmic dynein light chain 1, ddlc1 (Dick et al., 1996). ddlc1 maps to 4E1-2 on the X chromosome and was identified by the P-element induced female sterile mutation ddlc1ins1 in the promoter region of the ddlc1 gene, 40 bp upstream of its transcriptional start point. In order to characterise molecularly the flanking genomic region of the ddlc1 gene, a chromosome walk was carried out, and about 12 kb of genomic
DNA upstream of the ddlc1 gene was isolated. Within the cloned genomic region, a single transcription unit, dpp2c1, encoding a transcript of 6 kb, was detected. With the genomic DNA as the probe, cDNA clones from embryonic libraries were obtained that encompassed the coding region of the dpp2c1 transcript. The structure of the dpp2c1 transcription unit was determined by Northern and Southern blotting. A schematic summary of the organisation of the dpp2c1 transcription unit relative to the genomic DNA, the ddlc1 gene and the P-element insertion present in the ddlc1ins1 fly line is shown in Fig. 1. The structural analysis of the locus revealed that the dpp2c1 transcription unit is separated from the ddlc1 transcribed region by 1.5 kb genomic DNA. The two genes are transcribed in opposite directions. 2.2. Sequence analysis of the dpp2c1 cDNA To isolate cDNAs representing the 6-kb dpp2c1 transcript, embryonic libraries were screened. Seven cDNAs covering the DPP2C1 protein coding region were isolated. Restriction analysis and partial sequencing showed that all cDNA clones belong to the same cDNA class. The two longest, partially overlapping cDNA clones covering the complete coding region were sequenced (see Fig. 2 legend for details). The composite cDNA sequence of 5171 bp represents an almost fulllength transcript compared with the transcript size of about 6 kb. The cDNA contains a single large open reading frame from nt 745 (ATG) to 5031 ( TAG) with stop codons present in all three reading frames upstream of the putative start codon. The predicted DPP2C1 protein is 1428 aa long, with a calculated molecular mass of 155 kDa ( Fig. 2). A similarity search at the GenBank database (National Center for Biotechnology Information,
Fig. 1. Genomic region encompassing the dpp2c1 gene and the neighbouring ddlc1 gene. A restriction map of the genomic DNA is shown. The direction of transcription of the two genes is indicated by horizontal arrows. The precise intron–exon organisations have not been elucidated. The inverted triangle represents the P-element insertion site in the ddlc1ins1 line (Dick et al., 1996). Fragment no. 1 flanking the P-element was used as a probe to screen a lambda EMBL3 wild-type genomic library ( Tamkun et al., 1992), and EMBL3-LZ and EMBL3-L5 were isolated, respectively. Fragment no. 2 was used for a genomic walk, and EMBL3-L3 was isolated. No. 3 indicates the genomic fragment used for the cloning of the dpp2c1 cDNA no. 9 (see Fig. 2 legend ). Standard molecular biology techniques were carried out as described by Sambrook et al. (1989). E= EcoRI, X=XhoI, S=SalI.
T. Dick et al. / Gene 199 (1997) 139–143
Fig. 2. Amino acid sequence deduced from the nt sequence of the dpp2c1 cDNA. The cDNA sequence has been entered in GenBank under accession No. U96697. The 333-aa PP2C-like region (aa 235–567) is indicated in bold. Asterisk (*) marks stop codon. The genomic fragment no. 3 ( Fig. 1) was used to screen a 4–8-h embryonic cDNA library constructed by Brown and Kafatos (1988), and five cDNA clones were isolated. No. 9, the longest cDNA clone (3.7 kb), was sequenced. As the no. 9 cDNA did not contain a stop codon in the reading frame encoding the DPP2C1 protein, its 3∞ 0.9-kb EcoRI fragment was used for a cDNA walk using a lambda gt11 embryonic cDNA library provided by Bernd Hovemann. Two cDNA clones were isolated, and the longest clone, no. 2 (1.8 kb), was sequenced. cDNA no. 2 showed a 0.3-kb sequence overlap with cDNA no. 9. The dpp2c1 cDNA sequence derived from clone no. 2 starts at nt no. 3709. For sequence analysis, overlapping restriction fragments were subcloned into M13 and pBluescript, and universal and gene-specific primers were used. Sequences were assembled and analyzed with DNA Star software. The figure shows 60 aa per line.
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National Institutes of Health; Blast programme, Altschul et al., 1990) revealed a significant degree of sequence identity between a central stretch of #330 aa of the predicted Drosophila DPP2C1 protein and various PP2Cs, defining a PP2C-like catalytic domain within the DPP2C1 protein (Fig. 2). Fig. 3 shows as an example a comparison of the PP2C-like catalytic domain of DPP2C1 with the human PP2Calpha catalytic domain. The overall sequence identity between the two sequences is 26%, a degree of identity typical for pairwise comparisons of PP2Cs ( Klumpp et al., 1994). Recently, the crystal structure of the human PP2Calpha was elucidated (Das et al., 1996). It shows that a binuclear Mn2+ ion site that indirectly co-ordinates a phosphate ion forms the catalytic site of the PP2C. Mn2+ -bound water molecules co-ordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. All of the 10 invariant residues found within the PP2C-like family (Das et al., 1996) are situated at, or close to, the metal binding sites (Das et al., 1996; Fig. 3). Mn2+ at site 1 forms direct co-ordination with the carboxylate groups of three aspartate residues: Asp60, Asp239 and Asp282, while Mn2+ at site 2 forms only one direct contact to a carboxylate side chain, that of Asp60. Three water molecules co-ordinate Mn2+ at site 1, one of which is shared with Mn2+ at site 2. The four other ligand co-ordination to the metal at site 2 are the carbonyl oxygen of Gly61 and three water molecules. Hydrogen bonds between the carboxylate side chains of Glu37 and
Fig. 3. PP2C-like domain of DPP2C1. Comparison of the aa sequences of the central 333-aa PP2C-like domain of the DPP2C1 protein (aa 235–567, see Fig. 2) and the N-terminal 291-aa catalytic domain of the human PP2Calpha is shown (Das et al., 1996; NCBI Seq ID: 247169). Amino acids that are identical with the human PP2Calpha sequence are boxed. Residues that are invariant within all PP2Cs are indicated by an X- (Das et al., 1996). Residues in the human PP2Calpha binding metal ions and phosphate ions are indicated by M- and P-, respectively. The residue numbers correpond to human PP2Calpha. The alignment was performed using DNA Star software.
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Asp38 with two of these metal-bound water molecules stabilise metal binding to the protein. In total, four of the metal-associated water molecules serve to mediate contacts between the metal ions and phosphate by forming hydrogen bonds with three phosphate oxygen atoms. Further phosphate–enzyme interactions are provided by the guanidinium side chain of Arg33. The invariance of Thr128 and Gly240 is explained by the crystal structure since both residues contribute to the structure of the metal-binding site. The side chain of Thr128 forms a hydrogen bond with the main-chain NH of Asp60, an interaction that presumably stabilizes the co-ordination of both Mn2+ ions by the side chain of Asp60. Gly240 is adjacent to the metal co-ordinating residue, Asp239 (Das et al., 1996) Fig. 3 shows that the PP2C-like domain of the DPP2C1 protein contains all metal ion binding and phosphate binding residues identified for the human PP2Calpha. Furthermore, all 10 aa invariant within all PP2Cs described by Das et al. (1996) are present in the PP2C-like domain of the DPP2C1 protein ( Fig. 3). This suggests that the central 333-aa stretch of the DPP2C1 protein represents a PP2C-like domain, putting the 155-kDa Drosophila protein into the family of PP2Clike proteins. The central PP2C-like domain is flanked by an additional 234 N- and 861 C-terminal aa, respectively, which do not show any obvious similarities to other known sequences in the databases ( Fig. 2). These extensive Nand C-terminal sequences specific to the Drosophila PP2C protein suggest that DPP2C1 represents a new PP2C subtype, where the PP2C catalytic domain is found in a novel structural context.
2.3. Developmental expression analysis of dpp2c1 In order to determine the developmental expression of the dpp2c1 gene, Northern analyses with poly(A)+ RNA prepared from various developmental stages and the dpp2c1 cDNA as probe, were carried out. The results shown in Fig. 4 indicate that the level of dpp2c1 mRNA depends on the developmental stage. High levels of the dpp2c1 transcript can be detected during 0–6 h and 12–24 h of embryogenesis. During the larval stages of development, it is hardly detectable. The mRNA accumulates to appreciable levels again in pupae and adult body and head. These data show that the dpp2c1 transcript level is developmentally regulated. In-situ RNA hybridisation experiments were performed on whole-mount embryos in order to assess the spatial embryonic distribution of the dpp2c1 transcript. No specific patterns of hybridisation were observed at any stage (data not shown). Since Northern blot results clearly indicate that the dpp2c transcript accumulates to significant levels during embryonic development, these results might indicate that the dpp2c1 gene is transcribed ubiquitously in all tissues. 2.4. Conclusions (1) The present report describes the cloning and molecular characterisation of the first PP2C-like gene, dpp2c1, in Drosophila. (2) The analysis of the primary structure of the deduced DPP2C1 protein shows that the protein represents a new subtype of PP2C containing novel N- and C-terminal domains associated with a centrally located PP2C-like catalytic domain. (3) The availibility of a P-element insertion line (ddlc1ins1) harbouring a P-element close to the dpp2c1 transcription unit provides the basis for a genetic dissection of the role of this novel type of PP2C during fly development by using transposon mutagenesis.
Acknowledgement This work was supported by the Institute of Molecular and Cell Biology (IMCB), Singapore. Fig. 4. Developmental profile of dpp2c1 transcription. A Northern blot probed with 32P-labelled dpp2c1 cDNA (no. 9) is shown. Each lane contains 2 mg of Poly(A)+ RNA prepared from embryonic stages (0–6 h, 6–12 h and 12–24 h after egg deposition), larval stages (L1, first instar; L2, second instar; L3, third instar), pupae (P), adult body (B), and adult head (H ). The blot was reprobed with actin to demonstrate approximately equal loading of the RNAs in the different lanes. Fly Poly(A)+ RNA was prepared using standard methods. RNA samples were separated on formaldehyde-agarose gels and transferred to nylon membranes, hybridized, and exposed as described in Sambrook et al. (1989).
References Adler, E., Donella-Deana, A., Arigoni, F., Pinna, L.A., Stragler, P., 1997. Structural relationship between a bacterial developmental protein and eukaryotic PP2C protein phosphatases. Mol. Microbiol. 23, 57–62. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Bork, P., Brown, N.P., Hegyi, H., Schultz, J., 1996. The protein phos-
T. Dick et al. / Gene 199 (1997) 139–143 phatase 2C (PP2C ) superfamily: Detection of bacterial homologoues. Protein Sci. 5, 1421–1425. Brown, N.H., Kafatos, F.C., 1988. Functional Drosophila cDNA libraries from Drosophila embryos. J. Mol. Biol. 203, 425–432. Cohen, P., 1992. Signal integration at the level of protein kinases, protein phosphatases and their substrates. Trends Biochem. Sci. 17, 408–413. Cohen, P.T.W., 1994. Nomenclature and chromosomal localisation of human protein serine/threonine phosphatase genes. Adv. Protein Phosphate 8, 371–376. Coliccelli, J., Field, J., Ballester, R., Chester, N., Young, D., Wigler, M., 1990. Mutational mapping of ras-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase. Mol. Cell. Biol. 10, 2539–2543. Das, A.K., Helps, R., Cohen, P.T.W., Barford, D., 1996. Crystal struc˚ resoture of the protein serine/threonine phosphatase 2C at a 2.0A lution. EMBO J. 15, 6798–6809. Dick, T., Ray, K., Salz, H., Chia, W., 1996. Cytoplasmic dynein (ddlc1) mutations cause morphogenetic defects and apoptotic cell death in Drosophila. Mol. Cell. Biol. 16, 1966–1977. Duncan, L., Alper, S., Arigoni, F., Losick, R., Stragier, P., 1995. Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division. Science 270, 641–644. Kataoka, T., Broek, D., Wigler, M., 1985. DNA sequence and characterisation of the S. cerevisiae gene encoding adenylate cyclase. Cell 43, 493–505. Klumpp, S., Hanke, C., Donella-Deana, A., Beyer, A., Kellner, R., Pinna, L.A., Schultz, J.E., 1994. A membrane-bound protein phosphatase type 2C from Paramecium tetraurelia. J. Biol. Chem. 269, 32774–32780. Lawson, J.E., Niu, X.D., Browning, K.S., Trong, H.L., Yan, J., Reed, L.J., 1993. Molecular cloning and expression of the catalytic subunit of the bovine pyruvate dehydrogenase phosphatase and sequence similarity with protein phosphatase 2C. Biochemistry 32, 8987–8993. Leung, J., Bouvier-Durand, M., Morris, P.C., Guerrier, D., Chefdor, F., Giraudat, J., 1994. Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase. Science 264, 1448–1452.
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Maeda, T., Tsai, A.Y., Saito, H., 1993. Mutations in a protein tyrosin phosphatase gene (PTP2) and a protein serine/threonine phosphatase gene (PTC1) cause synthetic growth defect in Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 5408–5417. Meyer, K., Leube, M.P., Grill, E., 1994. A protein 2C involved in ABA signal transduction in Arabidopsis thaliana. Science 264, 1452–1455. Minato, T., Wang, J., Akasaka, K., Okada, T., Suzuki, N., Kataoka, T., 1994. Quantitative analysis of mutually competitive binding of human raf-1 and yeast adenylyl cyclase to ras proteins. J. Biol. Chem. 269, 20845–20851. Robinson, M.K., van Zyl, W.H., Phizicky, E.M., Broach, J.R., 1994. TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation. Mol. Cell. Biol. 14, 3634–3645. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Shiozaki, K., Akhavan-Niaki, H., McGowan, C.H., Russell, P., 1994. Protein phosphatase 2C, encoded by ptc1+, is important in the heat shock response of Schizosacharomyces pombe. Mol. Cell. Biol. 14, 3742–3751. Shiozaki, K., Russell, P., 1995. Counteractive roles of protein phosphatase 2C (PP2C ) and a MAP kinase kinase homolog in the osmoregulation of fission yeast. EMBO J. 14, 492–502. Stone, J.M., Collinge, M.A., Smith, R.D., Horn, M.A., Walker, J.C., 1994. Interaction of a protein phosphatase with an Arabidopsis serine–threonine receptor kinase. Science 266, 793–795. Tamkun, J., Deuring, R., Scott, M., Kissinger, M., Pattatucci, A., Kaufman, T., Kennison, J., 1992. Brahma, a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF/SW12. Cell 68, 561–572. Tamura, S., Lynch, K.R., Larner, J., Fox, J., Yasui, A., Kikuchi, K., Suzuki, Y., Tsuiki, S., 1989. Molecular cloning of rat type 2C (IA) protein phosphatase mRNA. Proc. Natl. Acad. Sci. USA 89, 1796–1800.
Drosophila DPP2C1, a novel member of the protein phosphatase 2C (PP2C ) family Thomas Dick, Sami M. Bahri, William Chia * Institute of Molecular and Cell Biology, National University of, Singapore, 15 Lower Kent Road, Singapore 119260 Received 22 April 1997; accepted 23 May 1997; Received by E. Boncinelli
Abstract We report the molecular cloning, chromosome mapping and developmental transcription pattern of a putative serine/threonine protein phosphatase 2C (PP2C ), DPP2C1, from Drosophila melanogaster. The 6-kb transcript of this first Drosophila PP2C gene encodes a 1428-aa deduced protein. The DPP2C1 protein contains a #330-aa PP2C-like catalytic domain flanked by extensive N- and C-terminal sequences showing no similarities to other PP2Cs. The dpp2c1 gene maps to 4E1-2 on the X chromosome, 1.5 kb upstream of the ddlc1 gene. Northern blot analyses showed that dpp2c1 transcription is developmentally regulated, accumulating maximally during early (0–6 h) and late (12–24 h) embryogensis. The presented molecular characterisation provides the basis for a genetic dissection of DPP2C1 function. © 1997 Elsevier Science B.V. Keywords: PPM; X chromosome; 4E1-2
1. Introduction Two structurally distinct serine/threonine phosphatase sequence families exist ( Klumpp et al., 1994): (1) the PPM family of Mg2+- or Mn2+-dependent PP2C-like enzymes; and (2) the PPP family which includes PP1, PP2A and PP2B (Cohen, 1994). The antagonistic roles of PPPs with respect to protein kinase-triggered regulation pathways are well established (for a review, see Cohen (1992)), but experiments aiming at the determination of the cellular functions for PP2C family members have started only very recently (Leung et al., 1994; Maeda et al., 1993; Meyer et al., 1994; Robinson et al., 1994; Shiozaki and Russell, 1995; Shiozaki et al., 1994). Numerous PP2C-like genes from various species have been cloned (Bork et al. (1996) and references herein), and studies of the crystal structure of the human PP2Calpha have provided an insight * Corresponding author. Tel.: +65 7723790; Fax: +65 7791117; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); DPP2C1, Drosophila serine/threonine specific protein phosphatase type 2C protein 1; dpp2c1, gene encoding DPP2C1; ddlc1, gene encoding the Drosophila cytoplasmic dynein light chain 1; kb, kilobase(s); kDa, kilodalton; nt, nucleotide(s); PP2C, serine/threonine specific protein phosphatase type 2C. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 3 59 - 4
into the structure of the PP2C catalytic domain (Das et al., 1996). Within the PP2C family, the #300-aa PP2C catalytic domain occurs in numerous structural contexts. For instance, PP2C from Paramecium tetraurelia ( Klumpp et al., 1994) represents one of the smallest PP2C proteins and appears to consist of essentially only the PP2C catalytic domain. The human PP2Calpha contains an additional small stretch of aa added to the C-terminus of the PP2C catalytic domain that might provide protein substrate specificity (Das et al., 1996), whereas the Arabidopsis PP2C encoded by the ABI1 gene contains a putative Ca2+-binding EF hand motif N-terminal to the catalytic domain (Leung et al., 1994; Meyer et al., 1994). Another PP2C homologue in Arabidopsis, KAPP, associates with the receptor serine/threonine kinase, RLK5, and consists of three regions: an N-terminal membrane localisation signal, a kinase interaction domain that associates with the phosphorylated receptor, and a C-terminal catalytic domain (Stone et al., 1994). Mammalian mitochondrial pyruvate dehydrogenase phosphatase contains a N-terminal Ca2+-binding site (Lawson et al., 1993). The SpoIIE phosphatase of Bacillus subtilis has 10 membrane-spanning regions preceding the catalytic PP2C domain (Duncan et al., 1995; Bork et al., 1996; Adler et al., 1997). Finally, a 300-residue region of yeast adenyl cyclase, present imme-
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diately N-terminal to the cyclase catalytic domain ( Kataoka et al., 1985), shares sequence similarity with PP2C ( Tamura et al., 1989). This domain may function to mediate ras-GTP activation of adenyl cyclase activity (Coliccelli et al., 1990; Minato et al., 1994) and is not known to possess protein phosphatase activity. We have performed an extensive molecular characterisation of the 4E1-2 cytogenetic interval of the Drosophila genome during the analysis of the cytoplasmic dynein light chain 1 gene, ddlc1 (Dick et al., 1996). In the course of these experiments, we discovered that a transcription unit neighbouring the ddlc1 gene encodes a protein, which contains a central domain showing significant homology to the PP2C catalytic domain flanked by extensive stretches of sequences not found in other PP2Cs. We named this first PP2C-like gene identified in Drosophila dpp2c1. In order to provide the basis for a genetic dissection of the biological role of this novel PP2C subtype, using the fly as a model system, we report here the cloning, chromosome mapping and developmental expression of the dpp2c1 gene.
2. Experimental and discussion 2.1. Cloning and mapping of the dpp2c1 transcription unit Previously, we reported the analyses of the gene encoding the cytoplasmic dynein light chain 1, ddlc1 (Dick et al., 1996). ddlc1 maps to 4E1-2 on the X chromosome and was identified by the P-element induced female sterile mutation ddlc1ins1 in the promoter region of the ddlc1 gene, 40 bp upstream of its transcriptional start point. In order to characterise molecularly the flanking genomic region of the ddlc1 gene, a chromosome walk was carried out, and about 12 kb of genomic
DNA upstream of the ddlc1 gene was isolated. Within the cloned genomic region, a single transcription unit, dpp2c1, encoding a transcript of 6 kb, was detected. With the genomic DNA as the probe, cDNA clones from embryonic libraries were obtained that encompassed the coding region of the dpp2c1 transcript. The structure of the dpp2c1 transcription unit was determined by Northern and Southern blotting. A schematic summary of the organisation of the dpp2c1 transcription unit relative to the genomic DNA, the ddlc1 gene and the P-element insertion present in the ddlc1ins1 fly line is shown in Fig. 1. The structural analysis of the locus revealed that the dpp2c1 transcription unit is separated from the ddlc1 transcribed region by 1.5 kb genomic DNA. The two genes are transcribed in opposite directions. 2.2. Sequence analysis of the dpp2c1 cDNA To isolate cDNAs representing the 6-kb dpp2c1 transcript, embryonic libraries were screened. Seven cDNAs covering the DPP2C1 protein coding region were isolated. Restriction analysis and partial sequencing showed that all cDNA clones belong to the same cDNA class. The two longest, partially overlapping cDNA clones covering the complete coding region were sequenced (see Fig. 2 legend for details). The composite cDNA sequence of 5171 bp represents an almost fulllength transcript compared with the transcript size of about 6 kb. The cDNA contains a single large open reading frame from nt 745 (ATG) to 5031 ( TAG) with stop codons present in all three reading frames upstream of the putative start codon. The predicted DPP2C1 protein is 1428 aa long, with a calculated molecular mass of 155 kDa ( Fig. 2). A similarity search at the GenBank database (National Center for Biotechnology Information,
Fig. 1. Genomic region encompassing the dpp2c1 gene and the neighbouring ddlc1 gene. A restriction map of the genomic DNA is shown. The direction of transcription of the two genes is indicated by horizontal arrows. The precise intron–exon organisations have not been elucidated. The inverted triangle represents the P-element insertion site in the ddlc1ins1 line (Dick et al., 1996). Fragment no. 1 flanking the P-element was used as a probe to screen a lambda EMBL3 wild-type genomic library ( Tamkun et al., 1992), and EMBL3-LZ and EMBL3-L5 were isolated, respectively. Fragment no. 2 was used for a genomic walk, and EMBL3-L3 was isolated. No. 3 indicates the genomic fragment used for the cloning of the dpp2c1 cDNA no. 9 (see Fig. 2 legend ). Standard molecular biology techniques were carried out as described by Sambrook et al. (1989). E= EcoRI, X=XhoI, S=SalI.
T. Dick et al. / Gene 199 (1997) 139–143
Fig. 2. Amino acid sequence deduced from the nt sequence of the dpp2c1 cDNA. The cDNA sequence has been entered in GenBank under accession No. U96697. The 333-aa PP2C-like region (aa 235–567) is indicated in bold. Asterisk (*) marks stop codon. The genomic fragment no. 3 ( Fig. 1) was used to screen a 4–8-h embryonic cDNA library constructed by Brown and Kafatos (1988), and five cDNA clones were isolated. No. 9, the longest cDNA clone (3.7 kb), was sequenced. As the no. 9 cDNA did not contain a stop codon in the reading frame encoding the DPP2C1 protein, its 3∞ 0.9-kb EcoRI fragment was used for a cDNA walk using a lambda gt11 embryonic cDNA library provided by Bernd Hovemann. Two cDNA clones were isolated, and the longest clone, no. 2 (1.8 kb), was sequenced. cDNA no. 2 showed a 0.3-kb sequence overlap with cDNA no. 9. The dpp2c1 cDNA sequence derived from clone no. 2 starts at nt no. 3709. For sequence analysis, overlapping restriction fragments were subcloned into M13 and pBluescript, and universal and gene-specific primers were used. Sequences were assembled and analyzed with DNA Star software. The figure shows 60 aa per line.
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National Institutes of Health; Blast programme, Altschul et al., 1990) revealed a significant degree of sequence identity between a central stretch of #330 aa of the predicted Drosophila DPP2C1 protein and various PP2Cs, defining a PP2C-like catalytic domain within the DPP2C1 protein (Fig. 2). Fig. 3 shows as an example a comparison of the PP2C-like catalytic domain of DPP2C1 with the human PP2Calpha catalytic domain. The overall sequence identity between the two sequences is 26%, a degree of identity typical for pairwise comparisons of PP2Cs ( Klumpp et al., 1994). Recently, the crystal structure of the human PP2Calpha was elucidated (Das et al., 1996). It shows that a binuclear Mn2+ ion site that indirectly co-ordinates a phosphate ion forms the catalytic site of the PP2C. Mn2+ -bound water molecules co-ordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. All of the 10 invariant residues found within the PP2C-like family (Das et al., 1996) are situated at, or close to, the metal binding sites (Das et al., 1996; Fig. 3). Mn2+ at site 1 forms direct co-ordination with the carboxylate groups of three aspartate residues: Asp60, Asp239 and Asp282, while Mn2+ at site 2 forms only one direct contact to a carboxylate side chain, that of Asp60. Three water molecules co-ordinate Mn2+ at site 1, one of which is shared with Mn2+ at site 2. The four other ligand co-ordination to the metal at site 2 are the carbonyl oxygen of Gly61 and three water molecules. Hydrogen bonds between the carboxylate side chains of Glu37 and
Fig. 3. PP2C-like domain of DPP2C1. Comparison of the aa sequences of the central 333-aa PP2C-like domain of the DPP2C1 protein (aa 235–567, see Fig. 2) and the N-terminal 291-aa catalytic domain of the human PP2Calpha is shown (Das et al., 1996; NCBI Seq ID: 247169). Amino acids that are identical with the human PP2Calpha sequence are boxed. Residues that are invariant within all PP2Cs are indicated by an X- (Das et al., 1996). Residues in the human PP2Calpha binding metal ions and phosphate ions are indicated by M- and P-, respectively. The residue numbers correpond to human PP2Calpha. The alignment was performed using DNA Star software.
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Asp38 with two of these metal-bound water molecules stabilise metal binding to the protein. In total, four of the metal-associated water molecules serve to mediate contacts between the metal ions and phosphate by forming hydrogen bonds with three phosphate oxygen atoms. Further phosphate–enzyme interactions are provided by the guanidinium side chain of Arg33. The invariance of Thr128 and Gly240 is explained by the crystal structure since both residues contribute to the structure of the metal-binding site. The side chain of Thr128 forms a hydrogen bond with the main-chain NH of Asp60, an interaction that presumably stabilizes the co-ordination of both Mn2+ ions by the side chain of Asp60. Gly240 is adjacent to the metal co-ordinating residue, Asp239 (Das et al., 1996) Fig. 3 shows that the PP2C-like domain of the DPP2C1 protein contains all metal ion binding and phosphate binding residues identified for the human PP2Calpha. Furthermore, all 10 aa invariant within all PP2Cs described by Das et al. (1996) are present in the PP2C-like domain of the DPP2C1 protein ( Fig. 3). This suggests that the central 333-aa stretch of the DPP2C1 protein represents a PP2C-like domain, putting the 155-kDa Drosophila protein into the family of PP2Clike proteins. The central PP2C-like domain is flanked by an additional 234 N- and 861 C-terminal aa, respectively, which do not show any obvious similarities to other known sequences in the databases ( Fig. 2). These extensive Nand C-terminal sequences specific to the Drosophila PP2C protein suggest that DPP2C1 represents a new PP2C subtype, where the PP2C catalytic domain is found in a novel structural context.
2.3. Developmental expression analysis of dpp2c1 In order to determine the developmental expression of the dpp2c1 gene, Northern analyses with poly(A)+ RNA prepared from various developmental stages and the dpp2c1 cDNA as probe, were carried out. The results shown in Fig. 4 indicate that the level of dpp2c1 mRNA depends on the developmental stage. High levels of the dpp2c1 transcript can be detected during 0–6 h and 12–24 h of embryogenesis. During the larval stages of development, it is hardly detectable. The mRNA accumulates to appreciable levels again in pupae and adult body and head. These data show that the dpp2c1 transcript level is developmentally regulated. In-situ RNA hybridisation experiments were performed on whole-mount embryos in order to assess the spatial embryonic distribution of the dpp2c1 transcript. No specific patterns of hybridisation were observed at any stage (data not shown). Since Northern blot results clearly indicate that the dpp2c transcript accumulates to significant levels during embryonic development, these results might indicate that the dpp2c1 gene is transcribed ubiquitously in all tissues. 2.4. Conclusions (1) The present report describes the cloning and molecular characterisation of the first PP2C-like gene, dpp2c1, in Drosophila. (2) The analysis of the primary structure of the deduced DPP2C1 protein shows that the protein represents a new subtype of PP2C containing novel N- and C-terminal domains associated with a centrally located PP2C-like catalytic domain. (3) The availibility of a P-element insertion line (ddlc1ins1) harbouring a P-element close to the dpp2c1 transcription unit provides the basis for a genetic dissection of the role of this novel type of PP2C during fly development by using transposon mutagenesis.
Acknowledgement This work was supported by the Institute of Molecular and Cell Biology (IMCB), Singapore. Fig. 4. Developmental profile of dpp2c1 transcription. A Northern blot probed with 32P-labelled dpp2c1 cDNA (no. 9) is shown. Each lane contains 2 mg of Poly(A)+ RNA prepared from embryonic stages (0–6 h, 6–12 h and 12–24 h after egg deposition), larval stages (L1, first instar; L2, second instar; L3, third instar), pupae (P), adult body (B), and adult head (H ). The blot was reprobed with actin to demonstrate approximately equal loading of the RNAs in the different lanes. Fly Poly(A)+ RNA was prepared using standard methods. RNA samples were separated on formaldehyde-agarose gels and transferred to nylon membranes, hybridized, and exposed as described in Sambrook et al. (1989).
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