From the Department of Biology, Center for Molecular Genetics,
University of California, San Diego, La Jolla, California 92093-0634 and ¶ Molecular Biology and Virology Laboratory, The Salk
Institute for Biological Studies, San Diego,
California 92186-5800
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ABSTRACT |
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Osmotic shock and growth-medium stimulation of
Dictyostelium 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
MAP1 kinase pathways in
stress response regulation. 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 ~50% of their original volume (2, 12-14).
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 protein-tyrosine
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 tyrosine-phosphorylated 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.
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 (5'-GTT TGG TAC CTT TTT TTT ACT TGT CAT CGT CAT CTT
TGT AAT CAA AAC ATT TAA TTG GTG TAA CTC T-3'). This oligonucleotide and
an outside T7 primer were used for polymerase chain reaction
amplification of the last ~500 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
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 × 107 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 × 106 cells were taken and boiled in 80 µl of SDS sample
buffer. Usually, 2-3 µl were loaded per lane on an 8% SDS gel.
Antibodies, Western Blot, Immunoprecipitation, PP2A Assay, and
Immunostainings--
The polyclonal, affinity purified anti-PTP3
antibody (22), the monoclonal anti-Tyr(P) antibody PY72 (26), and the
monoclonal anti-FLAG antibody M2 (IBI/Kodak, New Haven, CT) were used
for Western blot analysis and/or immunoprecipitation (IP). Western blots, IPs, and the PP2A assay were done as described previously (22),
except that for the PP2A assay, the PP2A holoenzyme was used. For the
anti-FLAG IPs, 2 µg of the M2 antibody were used per 2.0 × 107 lysed cells. For the immunostainings, the following
antibodies at the indicated dilutions were used: polyclonal anti-PTP3
antibody (1:20) (22), anti-Myc (1:1000) (Invitrogen, La Jolla, CA), and anti-F1B antibody (27). Immunostainings were done as
described by Araki et al. (28).
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 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 × 106 cells (usually 100 µl) were withdrawn (Fig. 4).
The aliquots were diluted in 100 µl (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 µl of
50% saturated KHCO3 and incubated for another 60 min on
ice with occasional vigorous shaking. After a final spin for 10 min at
4 °C, 100 µl of the supernatant was analyzed with the cyclic GMP
3H 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 (1× PBS (pH 7.4), 50 mM NaF, 1%
Nonidet P-40, 2 mM EDTA (pH 7.2), 1 mM sodium
pyrophosphate, 1.6 µg/ml leupeptin, 4 µg/ml aprotinin). Sodium
orthovanadate (Na3VO4) 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 × 107 cells in
1.1 ml of lysis buffer was added to ~80 µl 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 1× 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 After Osmotic Shock Stimulation PTP3 Is Phosphorylated on Ser and
Thr--
When PTP3
To determine the amino acids that were phosphorylated on PTP3, cells
overexpressing PTP3 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 (Fig.
3A). Because growth medium stimulation and osmotic shock led
to phosphorylation of PTP3, we tested whether osmotic conditions
induced changes in protein tyrosine phosphorylation. Surprisingly, the
results varied with the stimulant. Lower concentrations of sorbitol
(0.10 or 0.15 M) resulted in a strong p130 phosphorylation
(Fig. 3B; Table I), whereas
higher sorbitol concentrations ( 0.30 M Glucose or Sorbitol, but Not Growth Medium,
Leads to a Strong Transient Accumulation of cGMP--
The activation
of guanylyl cyclase and the tyrosine phosphorylation of actin were
maximal 5-25 min after osmotic stress induction (Figs. 3A,
and 4A; 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 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 ~100-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 ~230-amino 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 Cys-to-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-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
starvation 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
tyrosine-phosphorylated 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 ~110 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 ~60 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
strongly tyrosine-phosphorylated proteins, among them actin and a
protein of ~200 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 p130cas (36).
Specific Interaction of Tyrosine-phosphorylated pp130 with
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
(DYKDDDDK) was fused in-frame at the C terminus to full-length
PTP3(C649S) or the truncated version PTP3 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 (1× PBS
(pH 7.4), 50 mM NaF, 1% Nonidet P-40, 2 mM
EDTA (pH 7.2), 1 mM sodium pyrophosphate, 1.6 µg/ml
leupeptin, 4 µg/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 (~5-fold for the PTP3(WT) overexpressor strain; ~2.5-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 Myc-tagged 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 dot-like structures
are mitochondria by transforming the Myc-tagged 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 F1B, 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 FITC-labeled 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).
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
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 ~0.16 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 13-acetate 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 tyrosine-phosphorylated p130cas 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 (As3+) are toxic and highly
carcinogenic. As3+ 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. 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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, 5'-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-3'), 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).
1(C649S) were starved for 2-4 h in MES-PDF buffer in the
presence of 1 mCi/ml [32P]orthophosphate (35 mCi/70 µl;
ICN Biomedicals, Costa Mesa, CA). After starvation 2.0 × 107 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 × 107 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 × 16 cm2). 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 32P 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 H2O, 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
H2O 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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 µM
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.
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Fig. 1.
Modification of PTP3 in response to osmotic
shock and other cellular stresses. KAx-3 cells overexpressing
PTP3 1(C649S) were starved in sodium/potassium phosphate or MES-PDF
buffer. Total protein was lysed in SDS sample buffer before
(unstimul.) or 15 min after the indicated stimulations. The
PTP3 shifts were detected by anti-PTP3 Western blot. A,
response to different concentrations of osmotic active substances.
B, response to other stresses, such as anoxia, heavy metal
ions, and heat shock (33 °C). The arrows point to
non-stimulated and modified PTP3. sorb., sorbitol;
gluc., glucose.
1(C649S) was immunoprecipitated from lysates of
sorbitol-stimulated cells and treated with protein serine-threonine phosphatase PP2A, the mobility shift of PTP3
1(C649S) was reversed. The PTP3
1(C649S) migrated more rapidly on an SDS gel with the same
mobility as PTP3
1(C649S) from unstimulated cells (Fig.
2A), as we previously
demonstrated for PTP3
1(C649S) from growth medium-stimulated cells
(22). In control experiments, incubation of phosphorylated PTP3
1(C649S) with PP2A in the presence of microcystin LR, a potent inhibitor of PP2A, or with reaction buffer alone did not affect PTP3
1(C649S) migration. Thus, both growth medium and osmotic shock
stimulation resulted in a similar PTP3
1(C649S) phosphorylation.
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Fig. 2.
Phosphorylation of PTP3 on Ser
and Thr after osmotic shock stimulation. For these experiments,
KAx-3 cells overexpressing PTP3 1(C649S) were starved
(starv.) in MES-PDF buffer for 2-4 h and incubated in 0.2 M sorbitol (sorb.) for an additional 15 min.
A, PTP3 stimulated by 0.2 M sorbitol is
dephosphorylated in vitro by PP2A. The following protein
samples were analyzed by an anti-PTP3 Western blot: total protein
lysates after starvation and after 15 min of subsequent incubation in
0.2 M sorbitol, anti-PTP3 IPs after starvation and after
sorbitol addition. Anti-PTP3 IPs isolated after sorbitol stimulation
were treated with either PP2A (1.1 milliunits), PP2A in presence of the
PP2A-inhibitor microcystin LR (10 µM), or PP2A buffer
alone. inhib., inhibitor. B, in vivo
32P phosphorylation of PTP3. 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
(32PO4). 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.
1(C649S) were labeled in vivo with [32P]orthophosphate. In vivo
32PO4-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
32PO4-labeling detected any PTP3 tyrosine
phosphorylation (22), we conclude that PTP3 is phosphorylated
exclusively on serines and threonines.
0.20 M) resulted in weak phosphorylation (Fig. 3C; Table I). By a GST-PTP3(C649S)
interaction (pull-down) assay (Fig. 5B), we verified that
the faint Tyr(P) bands at 130 kDa visible after 0.20 and 0.30 M sorbitol stimulation represent tyrosine-phosphorylated
p130 (data not shown). In some experiments, a strong Tyr(P) signal at
130 kDa in unstimulated 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.
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Fig. 3.
Different concentrations of osmotic active
substances regulate the tyrosine phosphorylation of actin and
pp130. Anti-Tyr(P) Western blots are shown. A and
B, KAx-3 wild-type cells were starved in 12 mM
sodium/potassium phosphate buffer for 2-4 h, and a first protein
sample was withdrawn (0'). The cells were resuspended in
growth medium (A) or 0.15 M sorbitol
(B), 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 ~130-kDa band
migrates as two distinct bands as seen in B. B,
the ~130-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.
Comparison of actin and p130 tyrosine phosphorylation and cGMP
accumulation after different stimulation
0.30 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 ~5-10 min (data not shown).
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Fig. 4.
Activation of guanylyl cyclase in response to
different osmotic shocks. Wild-type cells were starved for
2.5 h in sodium/potassium phosphate buffer and stimulated with
hypertonic solutions as indicated. At different time points, samples
were withdrawn and subjected to a guanylyl cyclase assay. The
curves presented are representative of repeated experiments.
A, the cell culture was split after starvation and
stimulated with different osmotic active solutions. B, these
results were obtained on a different day. In contrast to this slow rise
in cGMP, cAMP-stimulated induction of guanylyl cyclase, which is
required for chemotaxis, is much more rapid and cGMP levels peak at
~10 s (18).
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Fig. 5.
In vitro interaction of PTP3 with
pp130. A, maps of PTP3 genomic DNA and the
GST-PTP3(C649S) fusion plasmid pMG35. The location of the catalytic PTP
domain in respect to the open reading frame is shown as well as the PTP
signature motif with the conserved Cys or the Ser mutation.
Abbreviations for the restriction sites are as follows:
EcoRV (E), NsiI (N),
SspI (S), HaeIII (H),
PstI (P), BglII (B),
XbaI (X), 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(C649S), or GST
alone. To eliminate the unbound proteins, the beads were washed in
lysis buffer and 1× 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).
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 FLAG-tagged 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.
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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 full-length 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.
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Fig. 7.
Intracellular localization of PTP3 in starved
cells and cells stimulated with growth medium. A,
anti-Myc staining of KAx-3 cells overexpressing PTP3-Myc(WT) after
starvation and (B) growth medium addition. Ca,
anti-Myc staining of cluA null cells overexpressing
PTP3-Myc(WT) after starvation and growth medium addition.
Cb, anti-F1B staining (mitochondrial protein) of
the same cells. Da, anti-Myc staining of KAx-3 cells
overexpressing PTP3-Myc(WT) after starvation and growth medium
addition. Db, FITC-dextran staining of endosomal
compartments in the same cell.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
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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 <5 min (fast responses) or 10 min
(slow responses). See "Discussion" for details.
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ACKNOWLEDGEMENTS |
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We thank Jill Meisenhelder for expert technical assistance and members of the Firtel and Hunter laboratories for helpful suggestions. We thank Stephan Schuster for sending the dokA null strain; Margaret Clarke for sending the cluA null strain; Gernot Walter for providing PP2A; and Michael Yaffe for giving us the anti-F1B antibody.
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FOOTNOTES |
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* This work was supported by U. S. Public Health Service grants (to T. H. and R. A. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Laboratorium für Organische Chemie, ETH Zentrum, CHN D39, Universitätstrasse 16, 8092 Zürich, Switzerland.
Supported in part by an American Cancer Society Postdoctoral
Fellowship Grant PF-3983. Present address: Ligand Pharmaceuticals, 9393 Towne Centre Dr., San Diego, CA 92121.
** Frank and Else Schilling American Cancer Society Research Professor.
To whom correspondence should be addressed: Center for
Molecular Genetics, Rm. 225, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0634. Tel.: 619-534-2788; Fax: 619-534-7073; E-mail: rafirtel{at}ucsd.edu.
2 M. Gamper and R. A. Firtel, unpublished observations.
3 H. Ma and R. A. Firtel, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: MAP, mitogen-activated protein; JNKs, Jun N-terminal kinases; PTP3, protein-tyrosine phosphatase-3; PBS, phosphate-buffered saline; GST, glutathione S-transferase; WT, wild type; IP, immunoprecipitation; FITC, fluorescein isothiocyanate.
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