Regulation of Dictyostelium Protein-tyrosine Phosphatase-3 (PTP3) through Osmotic Shock and Stress Stimulation and Identification of pp130 as a PTP3 Substrate*

Osmotic shock and growth-medium stimulation ofDictyostelium cells results in rapid cell rounding, a reduction in cell volume, and a rearrangement of the cytoskeleton that leads to resistance to osmotic shock. Osmotic shock induces the activation of guanylyl cyclase, a rise in cGMP mediating the phosphorylation of myosin II, and the tyrosine phosphorylation of actin and the ∼130-kDa protein (p130). We present data suggesting that signaling pathways leading to these different responses are, at least in part, independent. We show that a variety of stresses induce the Ser/Thr phosphorylation of the protein-tyrosine phosphatase-3 (PTP3). This modification does not alter PTP3 catalytic activity but correlates with its translocation from the cytosol to subcellular structures that co-localize to endosomal vesicles. This translocation is independent of PTP3 activity. Mutation of the catalytically essential Cys to a Ser results in inactive PTP3 that forms a stable complex with tyrosine-phosphorylated p130 (pp130) in vivo and in vitro, suggesting that PTP3 has a substrate specificity for pp130. The data suggest that stresses activate several interacting signaling pathways controlled by Ser/Thr and Tyr phosphorylation, which, along with the activation of guanylyl cyclase, mediate the ability of this organism to respond to adverse changes in the external environment.

In order to survive, cells need to adapt rapidly to environmental stresses. New environmental conditions are sensed by plasma membrane-associated proteins, activating signal transduction cascades that, in turn, regulate metabolism, cytoskeletal changes, secretion, or uptake of compounds, and gene expression (1,2). Recently, research has predominantly focused on the role of MAP 1 kinase pathways in stress response regu-lation. In mammalian cells, the MAP Jun N-terminal kinases (JNKs) or stress-activated protein kinases are activated by a diverse set of stimuli, leading to the phosphorylation and activation of transcription factors (1,3,4). UV irradiation and osmotic stress are believed to induce membrane perturbation or conformational changes in membrane proteins, which promote cell-surface receptor clustering, autophosphorylation, activation, and eventually, through a MAP kinase cascade, the activation of JNK (5). p38, another MAP kinase, is also activated by osmotic shock (6), but the signaling pathway seems to be at least partially different from the JNK pathway (3,7). In yeast Saccharomyces cerevisiae, the pathway induced by hyperosmotic condition is very well elucidated. As in Escherichia coli, in which a two-component system composed of a histidine kinase (EnvZ) and a response regulator (OmpR) is involved in osmoregulation (8), hyperosmolarity in yeast is sensed by a transmembrane histidine kinase (SLN1; Ref. 9). Under normal, low osmotic conditions, SLN1 is active and autophosphorylated on histidine. The phosphate is transferred in three steps via YPD1 to an aspartic acid residue of the response regulator SSK1 (10). Phosphorylated SSK1 prevents the activation of the HOG1 MAP kinase cascade, whereas under high osmotic conditions, SLN1 is inactive, SSK1 is not phosphorylated, and the HOG1 MAP kinase cascade is active, leading to gene expression and glycerol production (10). From the above mentioned components, only a histidine kinase (DokA, see below) has been found in Dictyostelium. Other signaling pathways, activated in Dictyostelium in response to stress stimulation, are summarized below.
In this study, we examine stress responses and osmotic shock stimulation in Dictyostelium and the potential role of a protein-tyrosine phosphatase in mediating these responses. Dictyostelium grows as single-celled amoebae, but upon starvation the cells aggregate, differentiate, and form a multicellular organism (11). Within 5-10 min after single Dictyostelium cells are exposed to high osmolarity or growth medium, the cells round up and shrink to ϳ50% of their original volume (2,(12)(13)(14). Phosphorylation of myosin II on three Thr residues, the subsequent disassembly of myosin filaments, the reduced myosin-actin interaction, and the relocalization of myosin play key roles in this process and are crucial for the cells to survive hyperosmotic stress (2). Exposure of the cells to 0.3 M glucose leads to an intracellular rise in cGMP (2,14) which is required for the phosphorylation of myosin II (2). This rise in cGMP is thought to activate a cGMP-dependent protein kinase, which in turn activates a myosin II heavy chain-specific protein kinase C (15,16). Similarly, extracellular cAMP induces guanylyl cyclase activity and myosin II phosphorylation during Dictyostelium aggregation, which mediates chemotaxis (15,17). However, the kinetics of intracellular cGMP accumulation and the signal transduction pathway leading to guanylyl cyclase stimulation are different than after osmotic shock stimulation (2,18).
Cellular stresses such as ATP depletion, as well as the exposure of cells previously starved in non-nutrient buffer to growth medium, lead to rapid cell rounding and transient tyrosine phosphorylation of certain proteins, including actin and p130 (12, 19 -22). Actin tyrosine phosphorylation, as with the activation of guanylyl cyclase, correlates with cell-shape change and a rearrangement of actin filaments and is affected by the level of the protein-tyrosine phosphatase PTP1 (12,20). The tyrosine phosphorylation of p130, however, is affected in strains overexpressing wild-type or mutant forms of proteintyrosine phosphatase PTP3 but not PTP1, suggesting it might be a substrate of PTP3 and play a different role in these response pathways (22). PTP3, determined to be a nonreceptor PTP by sequence analysis, was found to be transiently phosphorylated in response to growth medium stimulation, supporting the involvement of PTP3 in p130 regulation. PTP3 is expressed in growing cells, and its expression is induced to higher levels during multicellular development (22). Recently, a putative intracellular histidine kinase (DokA) was reported, and a dokA null strain appears to be less osmo-tolerant than wild-type cells, indicating a potential role of this enzyme in osmoregulation (13). Although it is likely that MAP kinase cascades are involved in Dictyostelium osmoregulation, no members of a stress-activated MAP kinase pathway have been identified.
In this report, we further investigate the role of PTP3. We find that PTP3 becomes phosphorylated on Ser and Thr residues after osmotic shock or other stress stimulations, which also lead to the tyrosine phosphorylation of actin and p130. However, by using different concentrations of osmotically active substances, we find that the signaling pathways mediating actin and p130 tyrosine phosphorylation, as well as guanylyl cyclase activation, seem to be distinct. We demonstrate that PTP3 specifically interacts with pp130 in vivo and in vitro, suggesting that pp130 is a PTP3 substrate. Another tyrosinephosphorylated protein (pp60) was found to interact with PTP3, but the interaction seems to be different than with pp130. In addition, we show that PTP3 phosphorylation does not alter PTP3 activity but correlates with a translocation of PTP3 from the cytoplasm to subcellular structures. Our results indicate that osmotic shock and other stresses result in the activation of multiple, interactive response pathways, including Tyr and Ser/Thr phosphorylation of multiple components in the pathway that permit Dictyostelium cells to respond to environmental changes.

EXPERIMENTAL PROCEDURES
Plasmid Constructions and Culturing of Dictyostelium Strains-Most plasmids have been described previously (22). In all PTP3 overexpression constructs, the PTP3 promoter is localized upstream of the wild-type or mutated PTP3 gene, and overexpression is achieved by multiple integrations of these plasmids into the chromosome. For the fusion of the FLAG tag (DYKDDDDK) to the C terminus of PTP3, an oligonucleotide was designed that contained the antisense sequence encoding the last 8 amino acids of PTP3 and the FLAG amino acids followed by an Asp718 restriction site (5Ј-GTT TGG TAC CTT TTT TTT  ACT TGT CAT CGT CAT CTT TGT AAT CAA AAC ATT TAA TTG GTG  TAA CTC T-3Ј). This oligonucleotide and an outside T7 primer were used for polymerase chain reaction amplification of the last ϳ500 base pairs of the PTP3 gene, and after the confirmation of the correct sequence, the BglII-Asp718 fragments of the PTP3(C649S) and the PTP3⌬1(C649S) overexpression constructs were replaced by the BglII-Asp718 fragment containing the FLAG tag (for the restriction sites see Fig. 5A). Similar to the FLAG-tagged construct, a C-terminal Myc tag (EQKLISEEDLN) fusion was made (Myc oligonucleotide, 5Ј-GTT TGG  TAC CTT TTT TTT AAT TTA AAT CTT CTT CTG AAA TTA ATT TTT  GTT CAA AAC ATT TAA TTG GTG TAA CTC T-3Ј), and the BglII-Asp718 fragment of the wild-type PTP3 overexpression construct was replaced by the BglII-Asp718 fragment containing the Myc tag. The GST-PTP3(C649S) construct pMG35 is essentially the same as the previously described pMG24 except for a single base pair change that converts the catalytic cysteine to a Ser (Fig. 5A).
In most of our studies, the wild-type strain KAx-3 was used, and if not specifically indicated overexpression plasmids were transformed into this strain. The partial ptp3 null strain, lacking one of the PTP3 genes, has been described (22). For mitochondrial localization studies, PTP3 overexpression constructs were transformed into the cluA null mutant (23). Transformation and clonal selection was carried out as described earlier (22). The growth medium was HL5 supplemented with 56 mM glucose as described by Franke and Kessin (24).
Growth Medium or Osmotic Shock Stimulation, Harvesting of Total Protein Samples-Prior to growth or osmotic shock stimulation, Dictyostelium cultures were grown for 2-3 days in shaking cultures and the cells were washed, in either 12 mM sodium/potassium phosphate buffer (pH 6.1) or phosphate-free MES-PDF buffer (25). The cells were resuspended in the same buffers at 1.0 ϫ 10 7 cells/ml and shaken for 2 to 4 h at room temperature at 150 rpm. Growth medium, osmotic shock, or other stress stimulations were performed as indicated in the figure legends. At different time points, total protein samples of 5.0 ϫ 10 6 cells were taken and boiled in 80 l of SDS sample buffer. Usually, 2-3 l were loaded per lane on an 8% SDS gel.
FITC-dextran was used to label endosomal compartments as described (29). Briefly, starved cells were placed on coverslips, placed in dishes, and flooded with either sodium/potassium phosphate buffer or HL5 growth medium containing FITC-dextran (2 mg/ml; Sigma) for 30 min.
Phosphoamino Acid Analysis-KAx-3 cells overexpressing PTP3⌬1-(C649S) were starved for 2-4 h in MES-PDF buffer in the presence of 1 mCi/ml [ 32 P]orthophosphate (35 mCi/70 l; ICN Biomedicals, Costa Mesa, CA). After starvation 2.0 ϫ 10 7 cells were withdrawn for the first IP, and sorbitol was added to the remaining cells at a final concentration of 200 mM. After an additional 15 min of shaking, 2.0 ϫ 10 7 cells were taken for the second IP. The anti-PTP3 IPs were boiled in SDS sample buffer and loaded on a preparative SDS gel (10% polyacrylamide gel; 14 ϫ 16 cm 2 ). After the gel run, the proteins were transferred to an Immobilon-P membrane (Millipore) for 1 h as described previously (22). The membrane was exposed to a Kodak XAR film to detect the 32 P incorporation and subjected to Western blot analysis by using the anti-PTP3 antibody. The hot spots were cut out, and after two washing steps in 100% methanol and H 2 O, the pieces of membrane were submerged in "constant boiling HCl" and incubated at 110°C for 1 h. Afterward, the hydrolysates were lyophilized and dissolved in H 2 O containing markers for Ser(P), Thr(P), and Tyr(P). The phosphoamino acids were separated by two-dimensional electrophoresis (pH 1.9 and pH 3.5) as described previously (30).
Guanylyl Cyclase Assays-Wild-type cells were starved in 12 mM sodium/potassium phosphate buffer as described above. After the addition of the osmotic active solution, the cells were kept in shaking culture. At the indicated time points, aliquots of 2.0 ϫ 10 6 cells (usually 100 l) were withdrawn (Fig. 4). The aliquots were diluted in 100 l (1 volume) of 3.5% perchloric acid and incubated on ice for 30 -60 min with periodic vigorous shaking. The solution was neutralized by the addition of 45 l of 50% saturated KHCO 3 and incubated for another 60 min on ice with occasional vigorous shaking. After a final spin for 10 min at 4°C, 100 l of the supernatant was analyzed with the cyclic GMP 3 H assay system (Amersham Pharmacia Biotech).
GST Fusion Protein Isolation and Adsorption of Cell Lysates-The isolation of GST fusion proteins from E. coli strain BL21(DE3) was done as previously reported (22) except that the proteins were not eluted from the glutathione-Sepharose beads after the washing steps. The in vitro adsorption of Dictyostelium proteins was performed as follows.
After starvation and 15 min of growth medium incubation, wild-type cells were lysed in lysis buffer (1ϫ PBS (pH 7.4), 50 mM NaF, 1% Nonidet P-40, 2 mM EDTA (pH 7.2), 1 mM sodium pyrophosphate, 1.6 g/ml leupeptin, 4 g/ml aprotinin). Sodium orthovanadate (Na 3 VO 4 ) was only added when indicated. After a cell lysis on ice for 5 min and a centrifugation at 4°C for 10 min, the lysate of 2.0 ϫ 10 7 cells in 1.1 ml of lysis buffer was added to ϳ80 l of glutathione-Sepharose beads carrying the GST fusion proteins. Following an incubation at 4°C with gentle rocking for 1 h, the beads were washed in lysis buffer and 1ϫ PBS (pH 7.4) and, only if indicated, in 0.5 M NaCl. The proteins were finally eluted from the beads by boiling in SDS sample buffer.

PTP3 Is Phosphorylated in Response to
Stress-When Dictyostelium cells were starved for 4 h in non-nutrient buffer and resuspended in growth medium, PTP3 became transiently phosphorylated. This modification was evident by anti-PTP3 Western blot analysis since it led to a slower migrating form of PTP3 on an SDS gel (22). We were interested in examining other conditions that might induce PTP3 phosphorylation. For this purpose, cells overexpressing an inactive form of PTP3 with an internal deletion of 116 amino acids (PTP3⌬1(C649S) (22)) were used. The truncated version of PTP3 was used instead of the full-length protein of 989 amino acids because it gave a higher level of expression in Dictyostelium and a greater mobility shift on SDS gels. Thus, PTP3⌬1(C649S) presumably contains the critical phosphorylation site(s) for the shift. Osmotically active small molecules, such as 0.3 M glucose, 0.2 M sorbitol, or 0.4 M sodium chloride, induced a mobility shift of PTP3⌬1(C649S) on an SDS gel (Fig. 1A). Within the resolution of this assay, the shift was identical for the three active compounds at these concentrations and is similar to observations when starved cells are shifted to growth medium. In addition, stresses such as ATP depletion, heavy metal ions, or heat shock induce tyrosine phosphorylation of actin and p130 (20), as previously shown for growth medium addition to starved cells (12). Since pp130 is a potential PTP3 substrate (22), we tested whether the exposure of cells to 1 mM sodium azide (to deplete ATP), 100 M cadmium chloride, or a heat shock at 33°C induced a mobility shift of PTP3 on an SDS gel. All of the stresses led to PTP3⌬1(C649S) phosphorylation (Fig. 1B). We observed mobility shifts to those shown in Fig. 1B when cells were taken from growth medium and exposed to the stresses mentioned above (data not shown). Our data suggest a possible general role for PTP3 in osmo-and stress regulation.
To determine the amino acids that were phosphorylated on PTP3, cells overexpressing PTP3⌬1(C649S) were labeled in vivo with [ 32 P]orthophosphate. In vivo 32 PO 4 -labeled cell lysates were made from cells starved for 4 h and stimulated or not stimulated with 0.2 M sorbitol. PTP3⌬1(C649S) was immunoprecipitated with anti-PTP3 antibodies, and the IPs were separated on an SDS gel and blotted onto a membrane. The membrane was exposed to a film and also subjected to Western blot analysis using anti-PTP3 antibodies (Fig. 2B). The Western blot confirmed that equal amounts of PTP3⌬1(C649S) were immunoprecipitated in the samples and that the PTP3⌬1-(C649S) exhibited a mobility shift after sorbitol stimulation. The autoradiogram indicated that PTP3⌬1(C649S) was phosphorylated before and after sorbitol treatment and the mobility shift correlated with an increase in the level of phosphorylation. Interestingly, both bands of the PTP3⌬1(C649S) doublet were labeled in starved cells and the sorbitol stimulation led to a very broad, fuzzy series of bands. The labeled PTP3⌬1(C649S) proteins were excised from the membrane and examined by phosphoamino acid analysis (30). In starved, unstimulated cells, only Ser(P) was detected, whereas after sorbitol induction the amount of label in the Ser(P) increased, and some Thr(P) was also detected (Fig. 2C). Since the 116-amino acid region that was deleted in PTP3⌬1(C649S) does not contain any tyrosines and since neither anti-Tyr(P) Western analysis nor in vivo 32 PO 4 -labeling detected any PTP3 tyrosine phosphorylation (22), we conclude that PTP3 is phosphorylated exclusively on serines and threonines.
Tyrosine Phosphorylation of Actin and p130 Is Induced at Different Concentrations of Osmotic Active Substances-When Dictyostelium cells were starved for 2-4 h in non-nutrient buffer and then incubated with growth medium, we observed several distinct changes in the tyrosine phosphorylation pattern of certain proteins (Fig. 3A) (12,22). p130 was fully phosphorylated within 5 min, whereas actin phosphorylation was first detected at 10 min and was maximal at 25 min after stimulation. When the cells were shifted back to low osmotic phosphate buffer, both proteins became dephosphorylated ( stimulated cells (Fig. 3C) is visible. Since this Tyr(P) protein never showed any interaction with PTP3 (in GST-PTP3(C649S) interaction assays (Fig. 5B) or co-immunoprecipitation assays with PTP3(C649S) (Fig. 6A), data not shown), it presumably is a protein other than pp130, or it is pp130 phosphorylated on another tyrosine that is not recognized by PTP3 (see Fig. 3B). The tyrosine phosphorylation of actin was regulated differently than that of p130; 0.10 M sorbitol produced only a low level of actin tyrosine phosphorylation (data not shown); intermediate osmotic concentrations (0.15 M (Fig. 3B) and 0.20 M) led to strong actin phosphorylation, and high osmolarity (0.30 M and above) had only a minor effect (Table I; Fig. 3C). Analysis of osmotically active substances showed that ionic and non-ionic molecules had equal responses with respect to differential p130 and actin tyrosine phosphorylation and were dependent on the osmotic concentration (Table I). As the osmolarity response curves of actin and p130 tyrosine phosphorylation are different, we suggest the responses may be regulated, at least in part, by different signaling pathways.  Table I; see Refs. 2, 12, and 14). However, despite these similar slow activation kinetics, actin phosphorylation was maximal at osmolarities between 0.15 and 0.20 M (Table I). For guanylyl cyclase activation, maximal stimulation was observed at osmolarities of Ն0.30 M (Fig. 4A; Table I; see Ref. 14). Stimulation with 0.20 M glucose or growth medium produced only a small increase in cGMP, whereas stimulation with 0.20 M sorbitol had little effect (Fig. 4, A and B). These data suggest that a distinct signaling pathway is responsible for the strong guanylyl cyclase activation. Overexpression of PTP3(WT) or the deletion of one of the two chromosomal PTP3 genes in Dictyostelium did not affect guanylyl cyclase activation (data not shown). 0.20 and 0.15 M sorbitol stimulation led to cell rounding, with kinetics similar to growth medium stimulation. 0.10 M sorbitol produced cell rounding, but the initiation of the rounding was delayed by ϳ5-10 min (data not shown).
Specific Interaction of Tyrosine-phosphorylated p130 with a Catalytically Inactive Form of PTP3 in Vitro-Since our preliminary data suggested that pp130 might be a PTP3 substrate (22), we further investigated the potential interaction between the two proteins. For this purpose, two nearly identical ϳ100-kDa fusion proteins were designed in which the N-terminal 242 amino acids of PTP3 were replaced by GST. One protein had an active catalytic site (GST-PTP3(WT), pMG24; see Ref. 22), whereas in the other protein, a Ser was substituted for the Cys essential for catalysis (GST-PTP3(C649S), pMG35; Fig. 5A; see Ref. 31), resulting in a catalytically inactive enzyme. The catalytic Cys is localized in a highly conserved region of the ϳ230amino acid catalytic domain within the signature motif characteristic for PTPs, HCXXGXXRS(T) (31,32). These conserved amino acids bind the tyrosine phosphate, and in the initial step of the catalysis, the cysteine thiolate acts as a nucleophile yielding a covalent thiol phosphate intermediate (33). The Cysto-Ser mutation still allows substrate recognition and binding, but the inability to hydrolyze the phosphate is reported to give a prolonged and more stable interaction with the substrate (34).
The two GST-PTP3 fusion proteins and the GST protein alone were expressed in E. coli and isolated using glutathione- Anti-PTP3 IPs of in vivo labeled cell lysates taken before and after 0.2 M sorbitol addition were separated on an SDS gel. The proteins were transferred to a membrane and exposed to a film ( 32 PO 4 ). To verify the amounts of immunoprecipitated PTP3, the same membrane was subjected to anti-PTP3 Western blot hybridization (Western blot). The two arrows point to the PTP3 doublet visible before stimulation. C, phosphoamino acid analysis of PTP3 after starvation and after sorbitol stimulation. The origins where the samples were applied to the thin layer plate are indicated. The first electrophoresis was done at pH 1.9 (upward) and the second at pH 3.5 (to the left). The markers were visualized by ninhydrin staining, and the labeled phosphoamino acids were identified after exposure to a film. part. hydrol., partial hydrolysis.
Sepharose beads. As expected, GST-PTP3(WT) dephosphorylated p-nitrophenyl phosphate and a tyrosine-phosphorylated peptide; GST-PTP3(C649S) had no detectable activity toward these substrates (see Ref. 22; data not shown). To identify tyrosine-phosphorylated Dictyostelium proteins that interact with PTP3, wild-type Dictyostelium cells were lysed after star-vation in non-nutrient buffer or after a subsequent stimulation with growth medium, and the lysates were incubated with the GST fusion proteins coupled to glutathione-Sepharose beads. After washing the resin, the retained proteins were eluted with SDS sample buffer, separated by polyacrylamide gel electrophoresis, and blotted onto a membrane. Anti-Tyr(P) Western blot analysis revealed that one tyrosine-phosphorylated 130-kDa protein bound very specifically to GST-PTP3(C649S). Because this protein had the same mobility as pp130 and was only detectable after growth medium stimulation (Fig. 5B), it is very likely that the protein is pp130. The active GST-PTP3(WT) did not bind stably to pp130, presumably because it dephosphorylated and released this substrate. From these results, we can conclude that GST-PTP3(C649S) interacts with the tyrosinephosphorylated p130 specifically through the PTP3 catalytic domain. This interaction was quite strong, since the treatment of the adsorbed beads with 0.5 M NaCl did not decrease pp130 binding (Fig. 5C). In addition to pp130, two other bands were detected in the anti-Tyr(P) Western blots. The band at ϳ110 kDa corresponded to the very abundant GST-PTP3 protein that was bound to the glutathione-Sepharose (Fig. 5C) and results from a very weak binding of the antibody to this highly abundant protein on the blot. The band at ϳ60 kDa (pp60) is another tyrosine-phosphorylated protein that was present in lysates before and after medium stimulation and was not dephosphorylated by GST-PTP3(WT) (Fig. 5B). Since glutathione-Sepharose beads carrying GST alone did not bind pp60 (Fig. 5B), the interaction of pp60 with PTP3 is specific but most likely not mediated through the catalytic active site. Other , and subsequently, every 5 min, another total protein sample was taken. After 25 min, the cells were washed and resuspended in sodium/potassium phosphate buffer, and again, samples were taken every 5 min for 25 min. C, essentially the same experiments were performed as in A and B, but only two protein samples were taken, one after starvation (unstimul.) and the other 35 min after the different osmotic stimulations (as indicated). Note that 35 min after stimulation with growth medium or 0.15 M sorbitol, high levels of pp130 and actin phosphorylation were found (data not shown). The results shown in C were confirmed with full time courses as presented in A and B. In some gels, the ϳ130-kDa band migrates as two distinct bands as seen in B. B, the ϳ130-kDa phosphotyrosine band is seen as two bands, a faster mobility, lighter band observed in unstimulated cells that disappears with a stronger, slower mobility band (pp130) appearing within 5 min. After removal of the sorbitol, the slower mobility band disappears and the faster mobility band reappears. Ϫ Ϫ ND a Cell lysates taken 25 min after stimulation were analyzed by anti-Tyr(P) Western blot and Tyr(P)-levels of actin and p130 were compared. b cGMP levels of cell lysates taken 10 min after stimulation were compared. Ϫ not visible; (ϩ) very weak response; ϩ weak response; ϩϩ strong response; ϩϩϩ very strong response; ND, not determined.
c The calculated osmolarity of HL5 is ϳ0.10 M.
strongly tyrosine-phosphorylated proteins, among them actin and a protein of ϳ200 kDa, did not interact with the GST-PTP3 fusion proteins. Recent structural data for Yersinia PTP Yop51 indicates that sodium orthovanadate inhibits PTPs through a covalent bond between vanadate and the active site Cys (35). One mM vanadate did not inhibit the interaction between GST-PTP3(C649S) and pp130, presumably because the active site cysteine thiolate was absent. In fact, 1 mM vanadate increased the amount of bound pp130 to the GST-PTP3(C649S) resin, possibly because it inhibited endogenous PTP activities present in the cell lysate. A higher concentration (10 mM) of vanadate did prevent the interaction GST-PTP3(C649S) with pp130 (data not shown), as was also observed for the interaction of PTP-PEST(C231S) with its substrate p130 cas (36).
Specific Interaction of Tyrosine-phosphorylated pp130 with  Asp718 (A). B and C, specific binding of pp130 to inactive GST-PTP3(C649S). Lysates of wild-type cells harvested after starvation (unstimul.) and subsequent growth medium addition (stimul.) were incubated with glutathione-Sepharose beads carrying active GST-PTP3(WT), inactive GST-PTP3(C-649S), or GST alone. To eliminate the unbound proteins, the beads were washed in lysis buffer and 1ϫ PBS and, only when indicated, in 0.5 M NaCl (C). The adsorbed tyrosine-phosphorylated proteins were detected by anti-Tyr(P) Western blots. As a control, the GST-PTP3(C649S) protein was also incubated in lysis buffer alone, without any Dictyostelium cell lysate (C, no extract).

PTP3(C649S) in Vivo-
To determine whether the observed in vitro interaction of PTP3 with pp130 is biologically relevant, we tried to co-immunoprecipitate these two proteins from Dictyostelium cell lysates. For this purpose, the FLAG tag (DYKD-DDDK) was fused in-frame at the C terminus to full-length PTP3(C649S) or the truncated version PTP3⌬1(C649S). Dictyostelium cells expressing the FLAG-tagged proteins were lysed before and after medium stimulation, and the lysates were precipitated with an anti-FLAG antibody. The IPs were first analyzed by an anti-Tyr(P) Western blot (Fig. 6A), and the filter was stripped and probed with an anti-PTP3 antibody (Fig. 6B). After medium stimulation, the full-length and truncated forms of PTP3(C649S) co-immunoprecipitated pp130 (Fig. 6A). No pp130 was immunoprecipitated in the wild-type control strain in which no FLAG-tagged protein was expressed (Fig. 6A). Since full-length PTP3 and pp130 migrated similarly on this SDS gel and since the tyrosine in the sequence of the FLAG tag could potentially be phosphorylated, we tested whether the tyrosine-phosphorylated band was not the FLAGtagged PTP3. As seen in Fig. 6A, the truncated PTP3⌬1(C649S), which migrates more rapidly than full-length PTP3 and pp130 (Fig. 6B), was not tyrosine-phosphorylated. As suggested previously (22), the internal deletion of 116 amino acids that contains the sequence between the first NsiI site and the SspI site (Fig. 5A) did not affect substrate interaction in vivo. No other tyrosine-phosphorylated proteins were visible. These data provide further evidence for the specificity of the PTP3 interaction with pp130.
Phosphorylation of PTP3 Correlates with an Intracellular Translocation-To examine the possible physiological significance of PTP3 phosphorylation and how this might affect its interaction with pp130, we performed two series of experiments. First, the PTP activity of anti-PTP3 IPs was determined before and after growth medium stimulation. IPs of wild-type cells and wild-type cells overexpressing full-length PTP3(WT) were analyzed for enzymatic activity before and after growth medium stimulation against a Tyr(P)-containing Cdc2 peptide (37,38). Samples were taken from the reaction mixture, and the free phosphate was measured by scintillation counting (38). In the presence of 1 mM dithiothreitol in the IP buffer (1ϫ PBS (pH 7.4), 50 mM NaF, 1% Nonidet P-40, 2 mM EDTA (pH 7.2), 1 mM sodium pyrophosphate, 1.6 g/ml leupeptin, 4 g/ml aprotinin) to keep the catalytic Cys of PTP3 reduced and active (39), similar PTP3 activities were found before and after stimulation (data not shown). In the absence of dithiothreitol, the PTP3 activity after starvation was significantly higher (ϳ5-fold for the PTP3(WT) overexpressor strain; ϳ2.5-fold for the wild-type strain) than after subsequent growth medium addition (data not shown). These results suggest that Ser/Thr phosphorylation does not affect PTP3 activity but possibly results in a conformational change of PTP3 that makes the active center more accessible to oxidation during protein isolation. In vivo, this conformational change could lead to altered substrate interaction or subcellular localization.
Second, we examined the intracellular localization of PTP3 before and after growth medium stimulation of wild-type cells overexpressing PTP3(WT) and PTP3(C649S). For these immunostaining experiments, two antibodies were used, the monoclonal anti-Myc antibody, directed against a C-terminal Myctagged PTP3(WT), and the polyclonal anti-PTP3 antibody, directed against PTP3(WT) and PTP3(C649S). After starvation, staining was visible throughout the cell for both forms, and cytoplasmic membranes remained unstained (Fig. 7A). In some experiments, nuclei whose localizations were determined by DNA (Hoechst dye) staining appeared as dark spots in the immunofluorescence experiments using the anti-PTP3 or Myc antibodies (data not shown). After growth medium addition, we observed a dramatic change in the PTP3-staining pattern. With both antibodies and the PTP3(WT) and PTP3(C649S) overexpressor strains, we found a scattered, dot-like staining throughout the cell after 15 min of stimulation (Fig. 7B, data for PTP3(C649S)). After a more extended period, PTP3 accumulates in larger domains (Fig. 7, Ca and Da).
The staining pattern suggested that PTP3 may be associated with an organelle. We excluded the possibility that these dotlike structures are mitochondria by transforming the Myctagged PTP3(WT) into the cluA null strain (23). In this strain, all mitochondria are clustered near the cell center (23). After 30 min stimulation with growth medium, the mitochondria, as visualized by immunostaining the mitochondrial protein F 1 B, were found localized near the center of the cell (Fig. 7Cb), whereas PTP3 accumulated in domains that excluded the mitochondria (Fig. 7Ca). We examined whether the PTP3 may associate with an endosomal compartment. Cells were starved for 4 h and stimulated with growth medium containing FITClabeled dextran to label endosomal compartments. As shown in Fig. 7D, there was a direct correlation between the distribution of dextran-containing compartments and PTP3 staining after stimulation. Non-stimulated cells show a random distribution of dextran (data not shown).
FIG. 6. Co-immunoprecipitation of pp130 with PTP3(C649S). Lysates from wild-type cells or wild-type cells overexpressing the FLAG-tagged full-length PTP3(C649S) or the truncated PTP3⌬1-(C649S) were immunoprecipitated with the anti-FLAG antibody. A, co-immunoprecipitated tyrosine-phosphorylated proteins were detected by an anti-Tyr(P) Western blot. B, the presence of fulllength or truncated PTP3 was verified by an anti-PTP3 Western blot of the same membrane. As discussed previously for Fig. 3, B and C (see "Results"), the strong Tyr(P) signal at 130 kDa in unstimulated cells presumably belongs to a protein other than pp130. starv., starved; med., medium.

Multiple, Discrete Pathways Are Activated in Response to
Stress-In this study, we analyzed stress responses in Dictyostelium in general and the regulation and role of PTP3 in these pathways in particular. We have shown that different osmolarities lead to different intracellular responses, suggesting that subtle regulatory mechanisms exist for the adaptation of cells to small changes in the extracellular environment. Considering the changes in the natural environment that Dictyostelium cells may experience, such mechanisms guarantee the ability of the cells to respond appropriately and to survive. Since p130 phosphorylation, actin phosphorylation, and the maximum activation of guanylyl cyclase are induced by different osmotic conditions, we suggest that the pathways leading to these events are, at least in part, different. A knock-out of the histidine kinase DokA or a mutation that reduces guanylyl cyclase activity leads to an osmosensitive phenotype (2,13). However, cGMP accumulation is not affected in dokA null strains, indicating that DokA acts downstream of guanylyl cyclase or in another pathway (13). We have not observed an altered osmosensitivity for any PTP3 mutant, including the partial ptp3 null strain lacking one copy of PTP3 or the wild-type strain overexpressing active or inactive PTP3. Moreover, PTP3⌬1-(C649S) expressed in the dokA null background was phosphorylated in response to stress. 2 This most likely excludes the possibility that DokA lies upstream of PTP3 in a signaling cascade. A MAP kinase kinase (DdMEK1; see Ref. 18) is hyperphosphorylated in response to stress, and interestingly, this occurs with kinetics similar to those of PTP3 and p130 phosphorylation. 3 DdMEK1 does not appear to be upstream of PTP3 because overexpressed PTP3⌬1(C649S) is hyperphosphorylated in the ddmek1 null background as well. 2 One possibility is that both DdMEK1 and PTP3 are phosphorylated by a common, stress-activated kinase.
Previously, we and others (12,21,22) investigated responses of starved cells to growth medium stimulation. The data presented here cannot exclude the possibility that the observed cellular events were, fully or partially, a consequence of the osmolarity of the growth medium. Stimulation with 0.15 M sorbitol mimics the protein tyrosine phosphorylation pattern induced by growth medium, which has a calculated osmolarity of ϳ0.16 M. The results of cells stimulated with HL5 lacking the 0.056 M glucose support this possibility, as the tyrosine phosphorylation is similar to that of 0.10 M sorbitol induction (Table I).
Stress-induced Phosphorylation of PTP3 Correlates with a Translocation of PTP3-In response to high osmolarity, we found PTP3 to be hyperphosphorylated on Ser and Thr. PTP3 is a large protein (989 amino acids) with 153 (15.4%) Ser and 64 (6.5%) Thr residues. The broad fuzzy band that is observed after sorbitol stimulation (Fig. 2B) can be explained by differential Ser/Thr phosphorylation at multiple sites. Analysis of the PTP3 sequence by eye or by the ppsearch program (EMBL Data Library) identifies the following potential PTP3 phosphorylation sites for known protein kinases: MAP kinase, 14 minimal proline-directed recognition sites (Ser/Thr-Pro; see Ref. 40); protein kinase A and cGMP-dependent protein kinase, 1 recognition site (Lys-Arg-Arg-Ser); protein kinase C, 16 recognition sites (Ser/Thr-Xaa(hydrophobic)-Arg/Lys); and casein kinase II, 12 recognition sites (Ser/Thr-Xaa-Xaa-Asp/Glu).
The Ser/Thr phosphorylation of PTP3 correlated with a translocation of PTP3 from the cytoplasm to subcellular structures, but it did not affect PTP3 activity toward a phosphopeptide substrate. Since both wild-type PTP3 and the catalytically inactive PTP3(C649S) translocated in response to osmotic stress, the translocation is independent of PTP3 activity. We suggest that PTP3 translocation is regulated through Ser/Thr phosphorylation. Our data suggest that PTP3 translocates to an endosomal compartment, although our analysis cannot distinguish between the compartments. As the response is transient when cells are placed in growth medium and can also be readily reversed by placing the cells in starvation medium, 2 we suggest that the association with endosomal vesicles is probably on the outside of the structures. The functional reason for this translocation is not known, although we note that PTP3 is more resistant to oxidation under these conditions. Whereas this property is observed upon cell lysis and may not be an in vivo property of PTP3 in osmotically stressed cells, it is an indication of a change in the property of PTP3 that is associated with its phosphorylation and/or subcellular localization and thus suggests some change in the in vivo properties of PTP3. There are other examples of intracellular translocation of PTPs upon stimulation as follows: phorbol 12-myristate 13acetate induces the differentiation of human HL-60 cells to macrophages. In this process, the activity and expression level of PTP1C increase 2-3 times; PTP1C is Ser-phosphorylated and translocates from the cytoplasm to the plasma membrane (41). In thrombin-activated platelets, SH-PTP1 translocates to the cytoskeleton (42).
pp130 Is a Substrate of PTP3-The catalytically inactive PTP3(C649S) binds tyrosine-phosphorylated pp130 in vivo and in vitro. These results show that PTP3 per se has a substrate specificity for pp130. Because pp130 did not associate with active PTP3(WT) in the in vitro binding experiments and because high vanadate concentrations inhibited PTP3(C649S) association with pp130 in vitro, the interaction between PTP3 and pp130 is presumably mediated through the catalytic site of PTP3 and the Tyr(P) and surrounding residues of pp130. Similarly, inactive PTP-PEST(C231S) selectively binds tyrosinephosphorylated p130 cas in vitro and in vivo, whereas inactive PTP1B has no substrate specificity in in vitro binding assays and binds practically any tyrosine-phosphorylated protein present in the cell lysate (36).
It is possible that PTP3 substrates in addition to pp130 exist. Such substrates could be present only in low amounts or they may not be efficiently recognized by our anti-Tyr(P) antibody. Since PTP3 is also expressed during Dictyostelium multicellular development with a maximal expression at 8 h (22) as well as during growth, it is probable that during the multicellular stages, PTP3 interacts with proteins other than pp130 and functions in different pathways. At the moment, the molecular identity of p130 is unknown. Preliminary data from pp130 adsorbed in vitro to GST-PTP3(C649S) did not reveal any obvious autokinase activity under the conditions used. 2 Possible Association of PTP3 with Stress-response Pathways-We have no direct proof that p130 or PTP3 plays a regulatory role in stress response, but from the data presented in this paper it is intriguing to speculate that they do. We observed a correlation between the phosphorylation of PTP3 and an intracellular translocation of PTP3 after stress stimulation, as well as an interaction of PTP3 with pp130. PTP3 isolated from growing cells migrated with a mobility on SDS gels that was similar to its migration in starved cells before stimulation. Similarly, PTP3 staining in growing cells looked like PTP3 staining in starved cells (data not shown). Assuming that pp130 is also cytoplasmically localized, our accumulated data could lead to the following hypothetical model (Fig. 8A). Under normal, non-hyperosmotic conditions during growth and development, PTP3 is in the cytoplasm and acts to keep pp130 in the unphosphorylated state. Stress induction stimulates PTP3 phosphorylation and may directly stimulate pp130 tyrosine phosphorylation. We propose that PTP3 phosphorylation leads to a conformational change exposing a site for endosomal docking and a subsequent translocation from the cytoplasm, which allows tyrosine-phosphorylated pp130 to accumulate in the cytoplasm. Although p130 could be a structural protein, it is intriguing to speculate that tyrosine phosphorylation of p130 has a positive or activating effect on stress-induced signal transduction pathways, and PTP3 plays a negative role in modulating these pathways. The co-immunoprecipitation experiments (Fig. 6) do not necessarily contradict this model. Because of the high overexpression of PTP3(C649S) it is likely that, although the translocation from the cytoplasm is apparent (Fig. 7), some PTP3(C649S) remains in the cytoplasm and associates with pp130. The model in Fig. 8B summarizes the known pathways outlining Dictyostelium stress regulation. Fast stress responses are observed within minutes after stimulation and include the phosphorylation of PTP3, p130, and DdMEK1. Slow responses are detected 10 -20 min after the stress signal in wild-type cells and result in the phosphorylation of actin and myosin, the rearrangement of the cytoskeleton, and cell rounding.
Other PTPs are known to negatively regulate pathways induced by hyperosmolarity or other stresses. In S. cerevisiae, a defect in the osmosensor SLN1 histidine kinase resulted in a non-phosphorylated downstream SSK1 response regulator, which is responsible for the lethal, constitutive activation of the HOG1 MAP kinase cascade. Overexpression of PTP2 rescued this lethal phenotype, and it was proposed that PTP2 directly dephosphorylates and inactivates HOG1 (9). In fission yeast Schizosaccharomyces pombe, the Spc1 MAP kinase pathway is activated by various cytotoxic stresses such as high osmolarity, oxidative stress, and high temperature. spc1 null cells are unable to grow in high osmolarity medium (43,44). Spc1 is also required for the initiation of mitosis, meiosis, and mating (44 -46). Two PTPs, PYP1 and PYP2, negatively regulate this pathway by dephosphorylating Spc1 (43,44). Furthermore, PYP2 is a target gene of the Spc1-stimulated transcription factor Atf1, indicating a negative feedback mechanism (45,46). In mammalian cells, arsenite ions (As 3ϩ ) are toxic and highly carcinogenic. As 3ϩ is thought to directly inhibit a phosphatase containing an essential Cys. In the absence of cellular stresses, this phosphatase activity is believed to maintain low JNK and p38 MAP kinase activities (47). Recently, PTP1B has been reported to be phosphorylated on Ser in response to stress and osmotic shock, but neither the function of the phosphorylation nor the upstream kinase have been identified (48). Because no members of a stress-regulated MAP kinase pathway have been identified in Dictyostelium, we cannot test whether PTP3 is phosphorylated by such a pathway or acts as a negative regulator of a MAP kinase as discussed in the examples above. FIG. 8. Stress regulation in Dictyostelium. A, model of PTP3 and pp130 interaction in response to stress. Stress induces the Ser and Thr phosphorylation of PTP3 and, simultaneously, the translocation of PTP3 from the cytoplasm. Our data strongly suggest that pp130 is a substrate for PTP3. We speculate the following regulatory mechanism. The tyrosine phosphorylation of pp130 could be induced directly by the stress signal, but assuming a cytosolic localization of p130, it is facilitated by the translocation of PTP3 to another compartment. Cytosolic PTP3 keeps pp130 in the non-phosphorylated, possibly inactive form. Supposing that a phosphorylated active pp130 is required in stress response signaling pathways, the function of PTP3 in normal, non-stress-stimulated cells could be to negatively regulate a stress-stimulated signaling cascade through the inhibition of p130 activation. B, summary of stress response pathways in Dictyostelium. Upon stimulation of cells, signaling pathways are induced in Ͻ5 min (fast responses) or 10 min (slow responses). See "Discussion" for details.
Purification and sequence analysis of p130 are likely to provide the data necessary to define its function and the function of PTP3 in regulating stress response pathways.
In Dictyostelium, osmotic and stress response regulation appears to be complex. The data presented here indicate different pathways control different aspects of the overall response. The identification of pp130 as a specific PTP3 substrate characterizes PTP3 as a highly selective PTP. The concomitant PTP3 phosphorylation and translocation in response to stress suggest that PTP3, perhaps through its inhibition of p130 activation, may function to negatively regulates stress response pathways.