Regulation by protein-tyrosine phosphatase PTP2 is distinct from that ...

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1994, p. 5154-5164 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Vol. 14, No. 8

Regulation by Protein-Tyrosine Phosphatase PTP2 Is Distinct from That by PTP1 during Dictyostelium Growth and Development PETER K. HOWARD,"12 MARIANNE GAMPER,' TONY HUNTER,2 AND RICHARD A. FIRTEL'* Department of Biology, Center for Molecular Genetics, University of Califomia, San Diego, La Jolla, Califomia 92093-0634,' and Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, San Diego, Califomia 92186-58002 Received 9 March 1994/Returned for modification 19 April 1994/Accepted 29 April 1994

We have cloned a gene encoding a second Dictyostelium discoideum protein-tyrosine phosphatase (PTP2) whose catalytic domain has -30 to 39% amino acid identity with those of other PTPs and a 41% amino acid identity with D. discoideum PTP1. Like PTP1, PTP2 is a nonreceptor PTP with the catalytic domain located at the C terminus of the protein. PTP2 has a predicted molecular weight of 43,000 and possesses an acidic 58-amino-acid insertion 24 amino acids from the N terminus of the conserved catalytic domain. PTP2 transcripts are expressed at moderate levels in vegetative cells and are induced severalfold at the onset of development. Studies with a PTP2-lacZ reporter gene fusion indicate that PTP2, like PTP1, is preferentially expressed in prestalk and anterior-like cell types during the multicellular stages of development. PTP2 gene disruptants (ptp2 null cells) are not detectably altered in growth and show a temporal pattern of development similar to that of wild-type cells. ptp2 null slugs and fruiting bodies, however, are significantly larger than those of wild-type slugs, suggesting a role for PTP2 in regulating multicellular structures. D. discoideum strains overexpressing PTP2 from the PTP2 promoter exhibit growth rate and developmental abnormalities, the severity of which corresponds to the level of PTP2 overexpression. Strains with high overexpression of the PTP2 gene grow slowly on bacterial lawns and produce small cells in axenic medium. When development is initiated in these strains, cells are able to aggregate but then stop further morphogenesis for 6 to 8 h, after which time a variable fraction of these aggregates continue with normal timing, producing diminutive fruiting bodies. These disruption and overexpression phenotypes for PTP2 are distinct from the corresponding mutant PTP1 phenotypes. Immunoprobing PTP2 mutant strains during growth and development with antiphosphotyrosine antibodies reveals several changes in the tyrosine phosphorylation of proteins in PTP2 mutant strains compared withl that in wild-type cells. These changes are different from those identified in the previously PTP1 disruption and overexpression mutant strains. Thus, although PTP2 and characterized PTP1 are nonreceptor PTPs with similar spatial patterns of expression, our findings suggest that they possess distinct regulatory functions in controlling D. discoideum growth and development.

Othesponding

Protein-tyrosine kinases (PTKs) initiate and propagate signals in the regulation of growth, morphogenesis, and differen-

which dephosphorylates Cdc2, causing entry into mitosis (7, 13) instead of merely quenching PTK signals, act as positive regulators of cellular processes. Evidence that phosphotyrosine signaling might be important in the regulation of Dictyostelium discoideum development came initially from reports of changes in protein-tyrosine phosphorylation in the transition between vegetative growth and development (40) and from the expression cloning of two developmentally regulated kinases with tyrosine phosphorylation activity (44). Next, with the discovery and analysis of a non-transmembrane PTP from D. discoideum, PTP1, we began to assess the role of pTyr signaling in this organism (19). The phenotypes observed when D. discoideum PTP1 is overexpressed or when the gene is disrupted demonstrated that protein-tyrosine phosphorylation plays a crucial role in regulating multicellular development in D. discoideum (19). PTP1 is preferentially expressed in the anterior tip at the tipped aggregate stage and later in development exhibits a spatial pattern similar to that of anterior-like cells (ALC) in the slug and culminants. Strains in which PTP1 is moderately overexpressed exhibit morphological defects in developmental structures, whereas high levels of expression of PTP1 lead to a failure of aggregation. ptpl null mutants develop at an accelerated rate but show normal morphological development. PTPs are also involved in the regulation of cell shape. When D. discoideum cells are returned to vegetative growth after initiating their developmental program, they undergo a dramatic transient cell rounding and loss of adhesion to the substratum

tiation. Progression through the cell cycle and the induction of cell proliferation in response to growth factors are regulated by PTKs. Receptor PTKs in Drosophila melanogaster, Caenorhabditis elegans, and mice transduce extracellular morphogenic signals that direct cell fate. The need to regulate these signals is demonstrated by the ability of oncogenic forms of tyrosine kinases to elevate the levels of tyrosine-phosphorylated (pTyr) proteins in cells, leading to the unrestrained growth of neoplasia. Protein-tyrosine phosphatases (PTPs) which selectively dephosphorylate pTyr proteins have been isolated (3, 33). These have been grouped into two structural categories, those that are cytoplasmic or membrane associated and contain a single highly conserved phosphatase domain of -270 amino acids (nonreceptor) and those that span the plasma membrane and contain, in most cases, two copies of this conserved domain (receptor-like). Other more distantly related members of the family act with dual specificity upon phosphoserine and phosphothreonine as well as pTyr substrates. Little is known about the specific biological roles of these proteins, although one of the important insights is that two of the most studied PTPs, CD45, required for T-cell activation (32), and Cdc25, * Corresponding author. Mailing address: Center for Molecular Genetics, Rm. 225, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0634. Phone: (619) 534-2788. Fax: (619) 534-7073.

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that correlates with the tyrosine phosphorylation of actin (20, 39). This cell shape change and loss of adhesion is accelerated and prolonged in ptpl null cells and diminished in cells in which PTP1 is overexpressed (20). In the initial analysis of PTP1 described above, clones for two additional PTPs, PTP2 and PTP3, were isolated. To add to our understanding of the role of PTPs in regulating signaling pathways that utilize tyrosine phosphorylation, we have analyzed the gene encoding D. discoideum PTP2. A cDNA of the PTP2 gene has also been recently cloned and examined by Ramalingam and coworkers (36). Those authors reported that the PTP2-encoded protein showed the expected PTP activity when expressed in Escherichia coli, and that overexpression from the non-cell-type-specific actin 6 promoter, which is preferentially expressed during growth and early development,

resulted in cells that failed to aggregate. In this report, we show that like PTP1, PTP2 is developmentally regulated and is expressed in a population of randomly scattered cells with a distribution similar to that of ALCs. Overexpression of PTP2 from the PTP2 pronibter results in distinct growth and developmental phenotypes, with a partial arrest at the mound stage. ptp2 null cells, on the other hand, show normal temporal development but produce slugs and fruiting bodies that are distinctly larger than those of wild-type strains. The altered dosage of PTP2 in the gene knockout and overexpression strains results in changes in the wild-type pattern of pTyr proteins during growth and development. Some of these changes are distinct from those seen in PTPI mutant strains. This study demonstrates that, although PTP2 is expressed in a spatial pattern similar to that of PTP1 during multicellular development, PTP2's role in D. discoideum is distinct from that of PTP1.

MATERUILS AND METHODS General molecular biology. Standard molecular biological techniques were used for common procedures, as reviewed by Sambrook et al. (37). Molecular approaches for working with D. discoideum have also been described elsewhere (18, 29). PCR amplification of D. discoideum DNA and screening of cDNA and chromosomal libraries. Reactions were conducted by using Perkin-Elmer Cetus reagents in a Coy Tempcycler. The templates used were derived from the axenic D. discoideum KAx-3 strain and include 12- to 16-h cDNA and genomic DNA. Degenerate oligonucleotide sequences were as follows: primer 1 for D Y I N A had a degeneracy of 24 [5' CCGAAT TCGGA TCC GA(TC) TA(TC) AT(CAT) AA(TC) GC 3'], primer 2 for D F W R M I/V W had a degeneracy of 96 [5' CCGAATTCGGA TCC GA(TC) TT(TC) TGG (CA)G(TA) ATG (GA)T(ATC) TGG G 3'], and primer 3 for H C S A G V G R had a degeneracy of 7,776 [5' CCGAATTC C(TG) (GTA)CC (GAT)AC (GAT)CC (GAT)GC (GAT)(CG)(AT) (AG)CA (GA)TG 3']. The position and direction of these primers are noted as arrows above highly conserved subregions in the PTP domain in Fig. 1. Primers 1 and 3 flank N-terminal and C-terminal end points in the conserved domain, respectively, and primer 2 is nested within this region as a second N-proximal starting primer. PCR cycle conditions were 30 cycles at 92°C for 1 min, 45°C for 2 min, and 74°C for 3 min, followed by an incubation at 74°C for 15 min. PCRs using primers 1 and 3 yielded prominent products of -650 and -600 bp for genomic DNA templates and -550 and -500 bp for cDNA templates. These reaction products were amplified with primers 2 and 3, yielding prominent products -70 bp smaller than the primer 1 and 3 products for each case. The PCR products were subcloned and sequenced. A subclone that

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contained a candidate novel PTP (later termed PTP2) was used as a probe to screen a 12- to 16-h developmental XZAP cDNA library (38). Seven independent cDNAs, amongst them cDNA 6 (1.8 kb), corresponding to PTP2 were isolated, the largest of which was 2.1 kb. However, the 2.1-kb clone contained heterologous sequences at the 5' end of the cDNA and cDNA 6 was used for subsequent work. Genomic clone 1-G was isolated by probing a library derived from a partial Sau3A digest of genomic KAx-3 DNA with cDNA 6. Southern blot analysis and cloning of the PTP2 promoter region. For Southern blot analysis, the chromosomal DNA of the wild-type strain KAx-3 was digested with Asp 718 and Asp 718 in combination with several other restriction endonucleases. The cloned cDNA 5' to the internal Asp 718 site was used as a probe (800 bp). A 3.4-kb Asp 718-NsiI fragment, presumably containing the PTP2 promoter (Fig. 2), was detected and subsequently cloned directly into the Asp 718 and NsiI sites of the pGEM-7Zf vector (Promega), resulting in pMG9. lacZ reporter strains and n-gal staining. The genomic 2.7-kb HincII-Asp 718 fragment (Fig. 2), containing the PTP2 promoter sequence and 138 codons of the N-terminal PTP2 open reading frame was fused via a 3-amino-acid linker sequence to the ninth codon of lacZ of plasmid SP60-lacZ (15), replacing the SP60 promoter sequences. The construction of this translational fusion required three cloning steps. The 2.7-kb HincII-Asp 718 fragment of pMG9 was subcloned into the ClaI (filled in with Klenow polymerase) and Asp 718 sites of pGEM-7Zf. The resulting plasmid pMG10 was digested with Asp 718 and XhoI, the 5' ends were filled in with Klenow polymerase, and the plasmid was religated. In this plasmid, pMG12, only the XhoI site was regenerated by the fusion. Next, the 2.7-kb PTP2 fragment of pMG,12 was cut out with BamHI and XhoI (the XhoI site was filled in with Klenow polymerase) and cloned into the BamHI (filled in with Klenow polymerase- and BglII-digested plasmid SP60-lacZ. The resulting PTP2-lacZ plasmid pMG16 was verified by detection of the generated XhoI site at the fusion. KAx-3 cells were electroporated, and by G418R selection (see the description of culturing and development of D. discoideum strains below), stable clonal transformed lines were obtained (8, 18). Several clonal isolates were screened for ,B-galactosidase (,3-gal) activity in vegetative cells. Since the copy number of plasmid DNA integrated in transformants is variable, clonal isolates produced different levels of reporter mRNA, giving a range of weakly to strongly staining clones. When the distributions of staining cells during development in these clones were compared, they were found to be consistent among isolates, and strongly staining isolates were used for detailed characterization of the staining pattern. Staining was performed as described previously (6, 19). Creation of gene disruption and overexpression cell lines. The PTP2 gene disruption plasmid pMG7 was made by exchanging part of the catalytic PTP domain (between the Asp 718 and the HincII [HpaI] sites) with a 3.2-kb fragment containing the THYI gene. First, 1.6 kb of PTP2 genomic DNA (HpaI-Asp 718 fragment of plasmid 1-G [the Asp 718 site resides in the multiple cloning site of 1-G]) was cloned into the HincII and Asp 718 sites of pBluescript SK- (Stratagene), resulting in pMG4. pMG4 was digested with HindIII and BamHI, and a 3.2-kb HindIII-BamHI fragment containing the THY1 gene was ligated into it, resulting in pMG6. pMG6 was cut with BamHI, blunted with Klenow polymerase, and cut with XbaI, and the 0.8-kb cDNA (Asp 718 [filled in with Klenow polymerase]-XbaI fragment of cDNA 6 [using the XbaI site within the multiple cloning site of cDNA 6]) was inserted, yielding pMG7. The thymidine auxotrophic strain JH10, created by gene disruption of the THY1 locus (16, 28),

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was electroporated with the EcoRI- and Asp 718-digested pMG7 (EcoRI and Asp 718 flank the insert). Cells were selected in HL5 medium without exogenously added thymidine. Clonal isolates were screened by Southern blot analysis. The PTP2 overexpression plasmid pMG15 carries a 4.5-kb genomic DNA insert with the PTP2 promoter, coding, and transcription termination regions (Fig. 2) and the cassette for G418R selection. The promoter driving PTP2 transcription is in the opposite orientation of the actin promoter driving Tn5 expression. Because both of these transcripts contain transcription termination sequences, as expected Northern (RNA) blots of PTP2 overexpression show a major band the size of the endogenous PTP2 transcript, with no evidence of readthrough transcription (Fig. 3). For PTP2 construction, two genomic DNA fragments were fused at the internal Asp 718 site. The generated 4.5-kb genomic DNA fragment contained the PTP2 promoter, the PTP2 coding region, and the PTP2 transcription termination signal. The construction was achieved by a threefragment ligation. Plasmid pMG1O (as described above) was cut with Asp 718 and XhoI. The 1.8-kb Asp 718-AvaI fragment of plasmid 1-G (the Aval site within the multiple cloning site was filled in with Klenow polymerase) and a 2.2-kb XhoI-Asp 718 fragment (Asp 718 was filled in with Klenow polymerase) containing the promoter and 5' coding region of the D. discoideum actin 6 gene fused in frame with the Tn5 neomycin resistance gene (29) were cloned into pMG10, yielding the overexpresser construct pMG15. KAx-3 was electroporated with closed circular pMG15 or pMG15 linearized at the unique HindIII site. Continuous selection for G418R was absolutely required to obtain stable transformants (see the description of culturing and development of D. discoideum cells below). In some cases (see Results), it was also necessary to add heatkilled E. coli B/r cells to the growth medium (23). PTP2 overexpression was analyzed by Northern blots. Western blot analysis. For the developmental time course protein studies, about 4.6 x 107 exponentially growing cells, washed free of growth media, were applied uniformly to 4-cm2 Millipore filters and allowed to develop on 12 mM Na-KPO4, pH 6.1, agar plates. Cells were collected from half a filter for each time point and lysed in boiling protein sample buffer as previously described (19). For the growth shift protein studies, exponentially growing cells were washed and incubated for starvation in shaking suspension at 150 rpm in 12 mM NaKP04 buffer, pH 6.1 (10' cells per ml). After 4 h, cells were centrifuged and resuspended in HL5 growth medium. Samples (between 1.5 x 106 and 4.0 x 106 cells, depending on cell size) were taken every 5 min and boiled in sample buffer. After 25 min, these growth-stimulated cells were washed and incubated in the starvation buffer a second time. Again, samples were collected every 5 min. Protein samples from both experiments were examined by fractionation with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Coomassie blue staining. For Western blot (immunoblot) analysis, equal amounts of protein, based on the Coomassie blue staining intensity, were size fractionated on SDS-polyacrylamide (10% acrylamide) gels, blotted to Immobilon-P membrane, and probed with anti-pTyr antiserum (24). For the developmental time courses, the detection signal used was Enhanced Chemiluminescence (Amersham). The protocol for detection was as described by the manufacturers, with the following changes: fresh 0.05% Tween 20 was added to lx Tris-buffered saline (TBST) for all incubation and wash steps, the blots were incubated in blocking solution (5% bovine serum albumin in lx TBST) overnight at room temperature, and primary and secondary antibody incubation steps were conducted with the blots in blocking solution. For the growth shift Western blots,

MOL. CELL. BIOL.

the detection signal used was 125I-protein A and analysis was conducted as described previously (19). Culturing and development of D. discoideum strains. The wild-type strain, KAx-3, that is capable of growth in axenic medium, is used in these studies. Cells were grown, and clones were isolated as described previously (11). To obtain high expression of PTP2 (overexpression) and a strong a-gal staining for PTP2-lacZ, it was necessary to keep the cells under continuous G418R selection. Clonal isolates were obtained as follows. One day after electroporation, G418 at a final concentration of 10 ,ug/ml was added to the plate. The following day, the cells were resuspended, mixed with G418R E. coli B/r (pUC4K) (21), and plated on agar plates containing G418 (50 ,ug/ml). Cells from single plaques were grown as previously described, and the analysis of morphology of strains was conducted as described elsewhere (16). The growth of clonal isolates was tested on a lawn of Klebsiella aerogenes cells. Vegetatively growing cells were washed and concentrated in 12 mM Na-KPO4 buffer, 1 ,ul of these suspensions (1.0 x 104 to 3.0 x 104 cells) was spotted on a previously plated K aerogenes lawn, and plaque sizes were compared 4 or 5 days later. Nucleotide sequence accession number. The GenBank nucleotide accession number for PTP2 is L15420.

RESULTS Isolation and sequence analysis of PTP2. To search for other PTPs that might be involved in the regulation of D. discoideum development, we conducted a PCR screen of genomic DNA and cDNA. Three nested degenerate oligonucleotides, corresponding to highly conserved sequences in the conserved region of PTPs were used as PCR primers (Fig. 1). Sequence analysis of the cloned PCR products led to the identification of PTP1 and two novel PTPs, PTP2 and PTP3. Here, we describe the analysis of PTP2. The PTP2 PCR product was used as a probe to isolate one genomic clone as well as seven cDNA clones from a XZAP library made from RNA isolated from cells at 12 to 16 h of development (38). A fragment from one of these cDNAs was used to isolate two additional genomic clones. The open reading frame of PTP2 was determined by sequencing portions of these clones. A 377-amino-acid protein of 43 kDa is predicted from the sequence data, and the sequence is identical to the derived amino acid sequence reported by Ramalingam and coworkers (36). The genomic sequence reveals the presence of four introns, with three located in the catalytic region (Fig. 2). The conserved PTP region of PTP2 is flanked by an N-terminal region of 51 amino acids and a C-terminal region of 31 amino acids. Inspection of these regions for functional domains or homology to other proteins yielded no significant matches. Figure 1 shows a comparison of the PTP2 conserved region sequences with those of other PTPs. PTP2 shares 41% amino acid identity with D. discoideum PTP1 and 39% amino acid identity to human T-cell PTP. A 58-amino-acid insert with 10 aspartic acids and 12 asparagines is located at amino acid 24 of the conserved domain. Two other PTPs contain an insert in this region. CD45 PTP domain II possesses an aspartic acidrich insert of 20 amino acids at amino acid 33 of the PTP domain, and PTP2 of Saccharomyces cerevisiae contains an insertion of 30 nonacidic amino acids in this region. Developmental and spatial expression of PTP2. To establish the temporal transcription pattern of PTP2 during D. discoideum development, RNA was isolated from cells at 4-h intervals and subjected to Northern analysis (Fig. 3A). A single PTP2 transcript of -2.5 kb was expressed at moderate levels in

VOL. 14, 1994 PTP2 PTP1 PYPI PTPT DLARI CD45I CD45II

DICTYOSTELIUM REGULATION OF TYROSINE PHOSPHORYLATION

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(See Materials and Methods for details.) Parentheses denote restriction sites destroyed by the cloning steps. A, Asp 718; Bn, BstNI; H, HincII; H/p, Hincll and HpaI; N, NsiI.

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FIG. 1. Amino acid sequence comparison of the PTP conserved domain of PTP2 with representative PTPs. The three sets of chevrons mark the position and direction of the degenerate oligonucleotide primers used for the PCR-directed isolation of PTP2. Alignments were performed by hand and at the National Center for Biotechnology Information using the BLAST (version 1.3) network service (1). PTP2 (36) is shown with amino acids 52 to 346 without the acidic insert (located between the I and S after 25 amino acids). PTP1 (19) is the previously characterized 521-amino-acid PTP from D. discoideum (GenBank accession number L07125). Shown are amino acids 117 to 468. Pypl (30) is a 550-amino-acid PTP from Schizosaccharomyces pombe (GenBank accession number M63257). Shown are amino acids 291 to 534. PTPT (4) is a nonreceptor PTP, closely related to PTP1B, of 415 amino acids isolated from a human T-cell library (PIR accession number A33899). Shown are amino acids 38 to 275. DLARI is a transmembrane-spanning PTP from D. melanogaster (42) of 2,029 amino acids (PIR accession number A36182). Shown is the domain I sequence of the two catalytic domains (amino acids 1493 to 1728). CD451 is the first domain of CD45 leukocyte common antigen, a transmembrane-spanning two-catalytic-domain PTP (35). (PIR accession number A29449). Shown are amino acids 510 to 748. CD45II is the second domain of CD45. Shown are amino acids 801 to 1064.

growing cells. This transcript accumulated to maximal levels at 8 h and persisted through the remainder of development. Analysis of cDNA clones did not reveal evidence of alternative splicing, and the derived amino acid sequence of the cDNA

clone isolated from a 12- to 16-h library was identical to that isolated by Ramalingam and coworkers (36) from a cDNA library made with RNA from vegetatively growing cells. To identify the cell types expressing PTP2, a plasmid containing a translational PTP2-lacZ reporter gene fusion, fused in frame to 2.3 kb of 5' untranslated and upstream PTP2 sequence, was transformed into the D. discoideum wild-type strain KAx-3 (Fig. 2; also see Materials and Methods). Vegetatively growing and developing cells of two independent clonal isolates were fixed and analyzed for ,B-gal activity at each morphological stage (every 3 h). The panels of Fig. 4 depict a subset of these stages. Strong ,B-gal staining was detected in -10% of growing cells, while other cells showed little or no staining (panel A). Scattered staining cells were found in the early aggregate (panel B) and at early mound stage (9 h) prior to tip formation. As the tip emerged from the mound (12 h), staining remained in the aggregate but was observed as well in cells in the forming tip (data not shown). With the formation of the first finger (14 h) and in the slug (panel C), staining was seen in cells scattered throughout the organism and not concentrated in the tip. During early and late culminant stages (panels D and E, respectively), staining was primarily localized to the lower and upper cups of the differentiating fruiting body, as well as to a few cells scattered in the stalk. Mature fruiting bodies (26 h) showed very light staining in the cups on the top and bottom of the spore mass and in the stalk tube (data not shown). Expression of PTP2 in a scattered population of cells and within the cup region is similar to the spatial localization of ALCs (41) and to that observed for PTP1 (19) (see Discussion). Phenotype ofptp2 null strains generated by gene disruption. To evaluate the function of PTP2, a strain containing a disrupted PTP2 gene was constructed by using the THY1 gene to interrupt the coding sequence (Fig. 2; also see Materials and Methods). Electroporation was used to introduce this disrup-

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PlTP2 overexprcssor FIG. 7. Developmental time courses of pTyr proteins for wild-type and PTP2 mutant strains. Cells growing vegetatively (0 h) were starved to induce development, and cells were harvested at 2-h intervals thereafter until the completion of development. Aggregation occurs between 6 and 8 h, the aggregate forms a tip by 11 h, slugs form by 15 h, initiation of culmination occurs by 18 h, and fruiting bodies form by 24 h. Equal amounts of total protein were loaded for all time points, and all three 10% gels were processed in parallel with the same ECL (Amersham) detection reagents (see Materials and Methods for details). Size standards are indicated in kilodaltons on the right.

of the cycle itself or developmental fate decisions made at the onset of development. ptp2 null and PTP2-overexpressing strains show distinct growth and developmental phenotypes. The phenotypes of the overexpressing strains and the expression of PTP2 in vegetative cells suggest a role for PTP2 during growth. However, we cannot exclude the possibility that overexpression of PTP2 leads to nonspecific dephosphorylation of pTyr residues and a nonspecific effect on growth and development. During multicellular development, the null phenotype leads to an increase in the size of slugs and culminants, suggesting a role for PTP2 in regulating morphogenesis. This difference in size is evident only when cells are plated at lower densities. The absence of PTP2 may reduce the number of signaling cells that direct aggregate formation, and a reduced number of signaling cells would generate larger morphological structures. A reduced

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PTP2 overexpressor FIG. 8. Changes in the pTyr pattern in response to nutrient shift in wild-type and PTP2 mutants. Vegetative wild-type and PTP2 mutant cells were harvested, washed free of nutrients and starved in buffer (Na-KPO4, pH 6.1) (S) for 4 h to initiate development at a density of 10i cells per ml. A time point sample was taken at t = 0, and cells were then collected by centrifugation and resuspended in growth medium (HL5) at a density of 5 x 106 cells per ml, collected every 5 min, and rapidly lysed in boiling sample buffer. After 30 min of growth stimulation (G), cells were collected by centrifugation, rinsed in the starvation buffer, and resuspended at 107 cells per ml. The t = 0 time point sample was collected at the first rinse, and cells were collected every 5 min thereafter as before. Equal amounts of protein were loaded, and all three 10% gels were processed in parallel with 125I-labeled protein A (see Materials and Methods for details). Size standards are indicated in kilodaltons on the right.

density of plated cells might be required to uncover this phenotype. The overexpression phenotype suggests that PTP2 can dephosphorylate pTyr residues on proteins important for the ultimate differentiation of mounds to subsequent more complex structures. It is noteworthy that this stage of develop-

VOL. 14, 1994

DICTYOSTELIUM REGULATION OF TYROSINE PHOSPHORYLATION

ment is one in which the differentiation of prestalk and prespore cell types occurs (25, 48). The observation that the phenotypes of ptp2 null and PTP2-overexpressing cells differ from the corresponding PTP1 mutants (19) suggests that PTP1 and PTP2 exert different biological influences despite similar spatial patterns of expression during the multicellular stages. This conclusion is supported by the different patterns of pTyr proteins in cells lacking or overexpressing PTP1 and PTP2. Examination of pTyr proteins in PTP2-overexpressing strains and null strains shows specific changes in the pTyr pattern from the wild-type cells. The observation that not all pTyr proteins are equally affected in the null and overexpressing strains suggests that the changes we see are specific and may represent either direct PTP2 substrates or proteins whose tyrosine phosphorylation is influenced by PTP2. In contrast to PTP1 overexpression, PTP2 overexpression has little if any effect on the tyrosine phosphorylation of actin during the growth shift. Thus, a phosphatase other than PTP2 must catalyze the actin dephosphorylation in ptpl null strains. Our data on PTP1 (19) and PTP2 suggest that important aspects of D. discoideum growth and development are regulated by reversible protein-tyrosine phosphorylation. ACKNOWLEDGMENTS We thank members of the Firtel and Hunter laboratories for helpful suggestions. We thank Christina Allen for carrying out the PCR and screening of potential PTPs and isolation of genomic clone 1-G. We thank Youhang Jiang for initial screening of the 12- to 16-h XZAP library and Gavin Schnitzler for the use of that library. We thank Joseph Dynes for the Sau3A partial digest genomic library. P.K.H. was supported, in part, by an American Cancer Society postdoctoral fellowship (grant PF-3983). M.G. was supported, in part, by a postdoctoral fellowship from the Swiss National Science Foundation. T.H. is an American Cancer Society Research Professor. This work was supported by USPHS grants to R.A.F. and T.H. REFERENCES 1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. 2. Ceccarelli, A., H. Mahbubani, and J. G. Williams. 1991. Positively and negatively acting signals regulating stalk cell and anterior-like cell differentiation in Dictyostelium. Cell 65:983-989. 3. Charbonneau, H., and N. K. Tonks. 1992. 1002 protein phosphatases? Annu. Rev. Cell Biol. 8:463-493. 4. Cool, D. E., N. K. Tonks, H. Charbonneau, K. A. Walsh, E. H. Fischer, and E. G. Krebs. 1989. cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family. Proc. Natl. Acad. Sci. USA 86:5257-5261. 5. Devine, K., and W. F. Loomis. 1985. Molecular characterization of anterior-like cells in Dictyostelium discoideum. Dev. Biol. 107:364372. 6. Dingermann, T., N. Reindl, H. Werner, M. Hildebrandt, W. Nellen, A. Harwood, J. Williams, and K. Nerke. 1989. Optimization and in situ detection of Escherichia coli beta-galactosidase gene expression in Dictyostelium discoideum. Gene 85:353-362. 7. Dunphy, W. G., and A. Kumagai. 1991. The cdc25 protein contains an intrinsic phosphatase activity. Cell 67:189-196. 8. Dynes, J. L., and R. A. Firtel. 1989. Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library. Proc. Natl. Acad. Sci. USA 86:7966-7970. 9. Esch, R. K., and R. A. Firtel. 1991. cAMP and cell sorting control the spatial expression of a developmentally essential cell-typespecific ras gene in Dictyostelium. Genes Dev. 5:9-21. 10. Esch, R. K., P. K. Howard, and R. A. Firtel. 1992. Regulation of the Dictyostelium cAMP-induced, prestalk-specific DdrasD gene: identification of cis-acting elements. Nucleic Acids Res. 20:13251332. 11. Firtel, R. A., and A. L. Chapman. 1990. A role for cAMPdependent protein kinase A in early Dictyostelium development. Genes Dev. 4:18-28.

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