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The Journal of Immunology, 1999, 163: 1282-1288.
Copyright © 1999 by The American Association of Immunologists

Direct Suppression of TCR-Mediated Activation of Extracellular Signal-Regulated Kinase by Leukocyte Protein Tyrosine Phosphatase, a Tyrosine-Specific Phosphatase1

Masatsugu Oh-hora*, Masato Ogata2,*, Yoshiko Mori*, Masaaki Adachi{dagger}, Kohzoh Imai{dagger}, Atsushi Kosugi{ddagger} and Toshiyuki Hamaoka*

* Biomedical Research Center, Osaka University Medical School, Osaka, Japan; {dagger} Department of Internal Medicine, Sapporo Medical School of Medicine, Sapporo, Japan; and {ddagger} School of Allied Health Science, Faculty of Medicine, Osaka University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leukocyte protein tyrosine phosphatase (LC-PTP)/hemopoietic PTP is a human cytoplasmic PTP that is predominantly expressed in the hemopoietic cells. Recently, it was reported that hemopoietic PTP inhibited TCR-mediated signal transduction. However, the precise mechanism of the inhibition was not identified. Here we report that extracellular signal-regulated kinase (ERK) is the direct target of LC-PTP. LC-PTP dephosphorylated ERK2 in vitro. Expression of wild-type LC-PTP in 293T cells suppressed the phosphorylation of ERK2 by a mutant MEK1, which was constitutively active regardless of upstream activation signals. No suppression of the phosphorylation was observed by LC-PTPCS, a catalytically inactive mutant. In Jurkat cells, LC-PTP suppressed the ERK and p38 mitogen-activated protein kinase cascades. LC-PTP and LC-PTPCS made complexes with ERK1, ERK2, and p38{alpha}, but not with the gain-of-function sevenmaker ERK2 mutant (D321N). A small deletion (aa 1–46) in the N-terminal portion of LC-PTP or Arg to Ala substitutions at aa 41 and 42 resulted in the loss of ERK binding activity. These LC-PTP mutants revealed little inhibition of the ERK cascade activated by TCR cross-linking. On the other hand, the wild-type LC-PTP did not suppress the phosphorylation of sevenmaker ERK2 mutant. Thus, the complex formation of LC-PTP with ERK is the essential mechanism for the suppression. Taken collectively, these results indicate that LC-PTP suppresses mitogen-activated protein kinase directly in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Protein tyrosyl phosphorylation plays important roles in various signal transduction pathways in lymphocytes. For example, TCR initiates signal transduction by the phosphorylation of various proteins by cytoplasmic protein tyrosine kinases (PTKs)3 (1, 2). The phosphorylation level of tyrosine is regulated by not only PTKs but also protein tyrosine phosphatases (PTPs) (3, 4, 5).

LC-PTP/HePTP (6, 7) is a human cytoplasmic PTP that is expressed preferentially in hemopoietic cells, including T lymphocytes. Very recently, Saxena and co-workers reported that overexpression of HePTP suppressed TCR-induced activation of the extracellular signal-regulated kinase (ERK) family mitogen-activated protein kinase (MAPK) as well as the transcriptional activation of a reporter gene driven by NF-AT/AP-1 elements (8). HePTP is almost identical with LC-PTP, but LC-PTP and HePTP differ in the presumptive location of the translation initiation codon. The molecular target(s) of HePTP/LC-PTP was not identified.

MAPK is activated through the phosphorylation of both tyrosine and threonine residues by MAPK kinases, dual specificity kinases. The dephosphorylation of either residue results in the complete loss of the activity. Three different classes of protein phosphatases, dual specificity phosphatases such as MKP-1/CL100 (9) and PAC-1 (10), serine/threonine phosphatases such as PP2A, and tyrosine-specific phosphatases, are implicated in the dephosphorylation and negative regulation of MAPK (reviewed in Refs. 3, 11, 12). It is well established that both dual specificity phosphatases and tyrosine-specific phosphatases are involved in the dephosphorylation and inactivation of MAPK in yeast (12). However, no mammalian tyrosine-specific phosphatase was known to dephosphorylate MAPK in hemopoietic cells.

MAPKs play important regulatory roles in lymphocytes. For example, ERK1 and ERK2 are implicated in the positive selection and lineage commitment of T cells (13, 14, 15, 16, 17, 18, 19), while p38 MAPK seems to be involved in the negative selection (19). In fact, transgenic introduction of a gain-of-function mutant of murine ERK2, ERK2sem, into developing thymocytes enhances the differentiation into CD4 lineage (18). ERK2sem has the sevenmaker Asp-to-Asn substitution at aa 319 and is analogous to the Drosophila mutant MAPK found in the sevenmaker gain-of-function mutant (20).

To understand the function of LC-PTP/He PTP in lymphocytes, it is very important to identify its target molecule(s). Here we report the direct suppression of ERK family and p38 family MAPKs by LC-PTP. Furthermore, the failure of an ERK binding-deficient mutant of LC-PTP to suppress TCR-mediated activation of ERK strongly suggests that the direct inactivation of ERK is the major mechanism of the suppression. To our knowledge, LC-PTP is the first mammalian tyrosine-specific phosphatase that directly suppresses MAPK in lymphocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfection

293T cells were cultured in DMEM containing 10% FCS. The cDNAs were subcloned in the pEF-BOS expression vector (21) and used for the transfection experiments. 293T cells (1 x 106) in a 6-cm dish were transfected with a plasmid encoding LC-PTP or its mutants and a plasmid encoding Flag-tagged ERK1 or ERK2 by the calcium phosphate coprecipitation method. Total amounts of DNA were kept constant (10 µg) by the addition of empty pEF-BOS vector. Jurkat cells were cultured in RPMI 1640 supplemented with 10% FCS, 50 µM 2-ME, 2 mM L-glutamine, and gentamicin. Jurkat cells were transfected using Superfect transfection reagent (Qiagen, Chatsworth, CA).

Immunoprecipitation, precipitation with GST fusion proteins, and immunoblotting analysis

Cells were lysed in 1% (v/v) Triton X-100, 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM PMSF, 5 mM EDTA, 1 mM Na3VO4, 50 mM NaF, and 5 mM sodium pyrophosphate. The lysates were precleared and used for the immunoprecipitation with protein G-Sepharose beads preloaded with the appropriate Abs. For precipitation with GST fusion proteins, 293T cell lysate or Jurkat cell lysate (5 x 107 cell equivalent in 0.5 ml lysis buffer) was precleared with glutathione-Sepharose beads and incubated at 4°C for 1 h with the beads preloaded with 4 µg of various GST fusion proteins. The precipitated proteins were visualized by immunoblotting using the enhanced chemiluminescence system (Amersham, Arlington Heights, IL).

Reporter assay

Jurkat cells (2.5 x 105) in 24-well tissue culture plates were transfected with pFA-Elk1 and 200 ng of pFR-Luc (firefly luciferase reporter) with or without LC-PTP plasmids. Total amounts of DNA were kept constant (1 µg) by the addition of empty pEF-BOS vector. To activate Elk-1/Gal4, 25 ng of pFC-MEK plasmid was used. Alternatively, 2 days after transfection, cells were stimulated by the incubation in the well precoated with OKT3 mAb (1 µg/ml), OKT3 mAb plus PMA (10 ng/ml), or ionomycin (1 µM) plus PMA for 7 h. The reporter firefly luciferase activities were measured and normalized by the transfection efficiencies estimated by the activities of Renilla luciferase constitutively expressed from cotransfected pRL-TK (50 ng).

The NF-AT-luciferase reporter construct (NF-AT-luc) (22) was a gift from G. Crabtree (Stanford University, Palo Alto, CA). Five hundred nanograms of NF-AT-luc was used for the assay.

Phosphatase assay

LC-PTP and LC-PTP{Delta}N transiently expressed in 293T cells were immunoprecipitated, and the catalytic activity was measured using p-nitrophenyl phosphate (p-NPP) as a substrate (23). The beads capturing LC-PTP and LC-PTP{Delta}N were suspended in 50 µl of assay buffer (20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM DTT, and 1 mM EDTA), and the reaction was initiated by adding 50 µl of assay buffer containing 24 mM p-NPP. After incubation at 37°C, the reaction was stopped by adding 17 µl of 2.5 N NaOH. The amount of p-nitrophenol released was determined from absorbance at 405 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the OD without the immunoprecipitates (beads alone).

GST-LC-PTP is a recombinant fusion protein in which LC-PTP was fused C-terminally to GST. To produce GST-LC-PTP, a cDNA fragment encoding aa 2–360 of LC-PTP was subcloned in the pGEX-3X plasmid. GST-LC-PTP was expressed and purified as previously described (23). Dephosphorylation of ERK2 by GST-LC-PTP in vitro was conducted as follows. ERK2-Flag protein was phosphorylated by an active MEK1 mutant in 293T cells. After immunoprecipitation with an anti-Flag Ab (M2), phosphorylated ERK2-Flag was incubated in 50 mM Tris-HCl (pH 7.4), 10 mM EDTA, 10 mM DTT, 1 mg/ml of BSA, and 0.1 mM PMSF with 8 µM GST-LC-PTP or GST for 60 min at 37°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dephosphorylation of ERK by LC-PTP in vitro

First, we examined whether LC-PTP could dephosphorylate ERK in vitro. LC-PTP was expressed in Escherichia coli as a GST fusion protein (GST-LC-PTP) and purified. ERK2 phosphorylated in vivo was immunoprecipitated and used as a substrate. The tyrosine dephosphorylation of ERK2 was induced by GST-LC-PTP (Fig. 1GoA, lane 2) but not by GST.



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  		FIGURE 1. Dephosphorylation and suppression of ERK2 by LC-PTP. A, Dephosphorylation of ERK2 in vitro. ERK2-Flag protein phosphorylated by an active MEK1 mutant in 293T cells was immunoprecipitated and incubated with 8 µM GST (lane 1) or GST-LC-PTP (containing aa 2–360 of LC-PTP) (lane 2) for 60 min at 37°C. The phosphorylation levels and the amounts of ERK2 were determined by the immunoblotting using an anti-phosphotyrosine (PY20) mAb (upper panel) and an anti-Flag mAb (M2; lower panel), respectively. B, Suppression of MEK1-mediated ERK2 phosphorylation in 293T cells. 293T cells were transfected with the plasmid (2 µg) coding for ERK2-Flag without (lanes 1 and 4) or with the plasmid (4 µg) for HA-tagged LC-PTP (lanes 2 and 5) or LC-PTPCS (lanes 3 and 6). pFC-MEK plasmid (200 ng) coding for an active MEK1 mutant was cotransfected to induce the phosphorylation of ERK2 (lanes 4–6). The total amounts of DNA were kept constant (10 µg) by the addition of empty pEF-BOS vector. ERK2-Flag was immunoprecipitated by M2 mAb, and the phosphorylation levels and the amounts of ERK2 were determined as described above.

 
LC-PTP suppressed the phosphorylation and activation of ERK in vivo

It was reported that the overexpression of HePTP suppressed the TCR-induced phosphorylation and activation of ERK in Jurkat cells. It was not clear, however, whether HePTP suppressed ERK directly or through other signaling molecules upstream of ERK. Thus, we tested the ability of LC-PTP to suppress the phosphorylation of ERK induced by a constitutively active MEK1 mutant (24, 25) in vivo. MEK1 is a MAPK kinase for ERK, and this mutant is active regardless of the activation state of the upstream signaling molecules. LC-PTP should suppress the phosphorylation of ERK if ERK would be the direct target. 293T human embryonic kidney cells were transfected with a plasmid encoding Flag-tagged ERK2, with or without expression plasmids encoding HA-tagged LC-PTP and the active MEK1 mutant. The expression of wild-type LC-PTP strongly suppressed the phosphorylation of ERK2 (Fig. 1GoB, lane 5). In contrast, a catalytically inactive mutant, LC-PTPCS, revealed no suppression (Fig. 1GoB, lane 6). LC-PTPCS was made by altering the conserved Cys291 to Ser as previously described (8).

We further confirmed the suppression of ERK cascade by LC-PTP in Jurkat cells. The activation level of ERK cascade was measured by the reporter assay using an Elk-1/Gal4 chimeric trans-activator. Elk-1 is a transcription factor that is phosphorylated and activated by ERK. The TCR cross-linking by the OKT3 mAb increased the reporter activities, and they were suppressed by the overexpression of LC-PTP (Fig. 2GoA), consistent with the previous observation that the overexpression of HePTP suppressed the TCR-induced activation and phosphorylation of ERK in Jurkat cells (8). In addition, like HePTP (8), LC-PTP suppressed the NF-AT reporter activity (Fig. 2GoB).



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  		FIGURE 2. The effects of LC-PTP on the ERK and NF-AT cascades in Jurkat cells. The activation levels of ERK and NF-AT cascades were measured by the reporter assay. Only the representative results from at least three independent experiments are shown. The luciferase activities were measured 2 days after the transfection. The reporter firefly luciferase activity was normalized by the control Renilla luciferase activity. The basal activity (the activity without stimulations) was expressed as 1. A, The effects of LC-PTP on the TCR-induced Elk-1/Gal4 reporter activity. Jurkat cells were transiently transfected with (arrowheads) or without the plasmid for HA-tagged LC-PTP (500 ng) with plasmids for Elk-1/Gal4 (pFA-Elk1, 60 ng), Gal4-driven firefly luciferase reporter (pFR-Luc; 200 ng), and Renilla luciferase (pRL-TK; 50 ng). The total amounts of DNA were kept constant (1 µg) by the addition of empty pEF-BOS vector. After 2 days, cells were stimulated by OKT3 mAb, OKT3 mAb plus PMA (10 ng/ml), or ionomycin (1 µM) plus PMA for 7 h. B, The effects of LC-PTP on TCR-induced NF-AT reporter activity. Jurkat cells were transiently transfected with (arrowheads) or without the plasmid for LC-PTP (500 ng) with NF-AT-luc (500 ng) and pRL-TK (50 ng). C, Inhibition of the mutant MEK1-induced Elk-1/Gal4 reporter activity by LC-PTP. Jurkat cells were transfected with the reporter plasmids described above and the various amounts (0–100 ng) of the plasmid coding for LC-PTP (closed circle) or LC-PTPCS (open circle). Ten nanograms of pFA-Elk1 was used. To activate the ERK cascade, 25 ng of pFC-MEK plasmid encoding an active MEK1 mutant was used.

 
LC-PTP also suppressed the ERK cascade activated by PMA and ionomycin, which should bypass the requirement for the early activation signals (Fig. 2GoA). Furthermore, as in 293T cells, transfection with various amounts of the LC-PTP plasmid suppressed the ERK cascade activated by the active MEK1 mutant in a dose-dependent manner (Fig. 2GoC). These observations are in a good accordance with the idea that ERK is the direct target of LC-PTP in Jurkat cells. More than 50% suppression was observed with as little as 20 ng of the LC-PTP expression plasmid. In contrast, LC-PTPCS revealed the only marginal inhibition, if any. In Fig. 2GoC, the luciferase activity was somewhat increased in the presence of 20 ng of the LC-PTPCS plasmid, but this stimulatory effect was not very reproducible.

Complex formation of LC-PTP with ERK and p38 MAPK

Next, we tested whether LC-PTP had the potential to make complexes with ERK. HA-tagged LC-PTP and Flag-tagged ERK2 were coexpressed in 293T cells. After the immunoprecipitation of Flag-tagged ERK2, LC-PTP coprecipitated was visualized by the immunoblotting analysis. LC-PTP was coprecipitated with ERK2 (Fig. 3GoA, lane 2) and ERK1 (data not shown). Similar results were obtained using LC-PTPCS, a catalytically inactive mutant (Fig. 3GoA, lane 6). In this experiment about 5–10% of LC-PTP and LC-PTPCS were estimated to make complexes with ERK2.



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  		FIGURE 3. In vivo complex formation of LC-PTP and LC-PTPCS with ERK2 and p38 MAPK. A, Coprecipitation of LC-PTP and LC-PTPCS with ERK2. 293T cells were transfected with the plasmid (4 µg) coding for HA-tagged LC-PTP (lanes 1–4) or LC-PTPCS (lanes 5–8) with 4 µg of the empty vector plasmid, (lanes 1 and 5), the plasmid for ERK2-Flag (lanes 2 and 6), p38{alpha}-Flag (lanes 3 and 7), or JNK2-Flag (lanes 4 and 8). After the immunoprecipitation of ERK2, p38{alpha}, and JNK2 by anti-Flag mAb (M2), coprecipitated LC-PTP and LC-PTPCS were visualized by immunoblotting using a biotinylated anti-HA mAb, 12CA5 (upper panel). LC-PTP and LC-PTPCS in the cell lysates were detected by 12CA5 mAb (middle panel). To detect ERK2, p38{alpha}, and JNK2, the Abs were removed, and the membrane was reprobed using M2 mAb (lower panel). The open arrowhead shows the location of the signal caused by the incomplete removal of 12CA5 mAb. B, Coprecipitation of the kinase activity with LC-PTP. Jurkat cells (3 x 107) were activated by the incubation with 10 ng/ml PMA and 100 nM ionomycin at 37°C for 5 min, lysed, and used for the immunoprecipitation with 3 µl of control rabbit serum (lane 1) or anti-LC-PTP serum (lane 2) (37 ). In vitro kinase reaction was performed as previously described (30 ), and after SDS-PAGE, phosphoproteins were visualized by an imaging plate scanner BAS2000. C, Schematic drawings of LC-PTP and its mutants. LC-PTP{Delta}N is a deletion mutant lacking a small N-terminal portion (aa 1–46) of LC-PTP. LC-PTPA41A42 has Arg-to-Ala substitutions at aa 41 and 42. The closed box indicates the region conserved among LC-PTP/HePTP, PTPBR7, and STEP. PTP, protein tyrosine phosphatase domain. D, Coprecipitation of LC-PTP, but not LC-PTP{Delta}N, with ERK2. HA-tagged LC-PTP (lanes 1 and 2) and LC-PTP{Delta}N (lanes 3 and 4) were transiently expressed without (lanes 1 and 3) or with (lanes 2 and 4) ERK2-Flag in 293T cells. E, Undetectable binding of LC-PTPA41A42 to ERK2. HA-tagged LC-PTP (lanes 1 and 2) and LC-PTPA41A42 (lanes 3 and 4) were transiently expressed without (lanes 1 and 3) or with (lanes 2 and 4) ERK2-Flag in 293T cells.

 
Then we tested the possibility of complex formation of endogenous LC-PTP with ERK. Cell lysates were prepared from Jurkat cells and incubated with beads preloaded with GST-LC-PTP or GST-ERK2. Endogenous ERK2 and LC-PTP were coprecipitated with GST-LC-PTP and GST-ERK2, respectively (Fig. 4GoC, lanes 6 and 8). About 5% of ERK2 and 12% of LC-PTP in the lysate were coprecipitated. Then, LC-PTP was immunoprecipitated from the lysate of activated Jurkat cells, and the coprecipitation of the kinase activity was detected by the immune complex kinase assay. As shown in Fig. 3GoB, LC-PTP was phosphorylated in vitro by the coprecipitated kinase. We also confirmed that purified active ERK2 directly phosphorylated purified LC-PTP in vitro (data not shown).



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  		FIGURE 4. In vitro complex formation of LC-PTP and ERK2. A, Schematic drawings of GST fusion proteins containing various LC-PTP mutants. The closed box indicates the region conserved among LC-PTP/HePTP, PTPBR7, and STEP. PTP, protein tyrosine phosphatase domain. B, GST fusion proteins used in the coprecipitation experiments. Two micrograms of GST fusion proteins were separated by 12% SDS-PAGE and stained with Coomassie blue. C, Coprecipitation of LC-PTP and ERK2 with various GST fusion proteins. Cell lysates from 293T cells overexpressing ERK2-Flag (lanes 1–5) or Jurkat cells (5 x 107 cell equivalent in 0.5 ml of lysis buffer; lanes 6–8) were incubated with glutathione-Sepharose beads preloaded with 4 µg of GST (lanes 1 and 7), GST-LC-PTP (lanes 2 and 6), GST-LC-N (lane 3), GST-LC-{Delta}N (lane 4), GST-LC-A41A42 (lane 5), and GST-ERK2 (lane 8). Coprecipitated ERK and LC-PTP were visualized by the immunoblotting using anti-ERK (E17120, Transduction Laboratories, Lexington, KY) or anti-LC-PTP (37 ) Abs. Note that the size of the endogenous ERK2 in Jurkat cell (lane 6) is smaller than the Flag-tagged ERK2 (lanes 2 and 3).

 
LC-PTP and LC-PTPCS were coprecipitated with p38{alpha} MAPK too (Fig. 3GoA, lanes 3 and 7). Therefore, we investigated whether LC-PTP suppresses p38 MAPK. MKK6 is a MAPK kinase for p38 MAPK. A constitutively active MKK6 mutant (S207E/T211E MKK6) was reported (26), and it was used to activate the p38 MAPK cascade in Jurkat cells. The activation level of p38 MAPK cascade was measured by the reporter assay using the ATF2/Gal4 chimeric trans-activator. As shown in Fig. 5Go, transfection with various amounts of LC-PTP plasmid suppressed the MKK6-mediated activation of p38 MAPK in a dose-dependent manner.



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  		FIGURE 5. The effect of LC-PTP on the MKK6-mediated p38 MAPK activation in Jurkat cells. The ERK (closed circle) or p38 (closed square) cascade was activated by the transfection of Jurkat cells with 10 ng of plasmids for the constitutively active MEK1 or MKK6, respectively. The activation levels of the ERK cascade were measured by the reporter assay as described in Fig. 2Go. The activation levels of the p38 cascade were measured using pFA-ATF2 plasmid (10 ng) coding for an ATF2/Gal4 chimeric trans-activator. Various amounts (0–500 ng) of the plasmid coding for LC-PTP were cotransfected, and the luciferase activity without LC-PTP was expressed as 100%. The basal activities of ERK and p38 cascades (the activities without MEK1 and MKK6) were 4.2 ± 0.2 and 4.2 ± 1.5%, respectively.

 
The coprecipitation of LC-PTP with JNK2 was only marginal (Fig. 3GoA, lanes 4 and 8), consistent with the previous observation that HePTP did not suppress JNK (8).

LC-PTP{Delta}N (Fig. 3GoC), a deletion mutant lacking a small N-terminal portion (aa 1–46), revealed no detectable binding capacity to ERK2 (Fig. 3GoD, lane 4). To study the interaction site of LC-PTP further, GST fusion proteins containing the entire LC-PTP (GST-LC-PTP) and its mutants (Fig. 4GoA) were incubated with cell lysates from 293T cells overexpressing ERK2. As expected, ERK2 was coprecipitated with GST-LC-PTP, but not with GST-LC-{Delta}N lacking aa 1–46 (Fig. 4GoC, lanes 2 and 4). In these experiments about 4% of ERK2 was coprecipitated with GST-LC-PTP. The association was observed using GST-LC-N containing only the N-terminal portion (aa 1–99) of LC-PTP. Thus, the portion outside the PTP domain of LC-PTP is crucial for binding to ERK. Furthermore, Arg41 and/or Arg42 of LC-PTP are essential for the binding, because Arg-to-Ala substitutions (LC-PTPA41A42 and GST-LC-A41A42) result in the complete loss of the ERK binding capacity in vivo (Fig. 3GoE, lane 4) and in vitro (Fig. 4GoC, lane 5).

The sevenmaker mutation is a gain-of-function mutation of Drosophila MAPK in which asparagine is substituted for aspartic acid 334. The analogous sevenmaker mutants of human ERK2 (D321N ERK2sem) and murine p38{alpha} MAPK (D315S/D316N p38{alpha}sem) were tagged with the Flag epitope and coexpressed with HA-tagged LC-PTP. LC-PTP was not coprecipitated with ERK2sem (Fig. 6GoA, lane 3) and p38{alpha}sem (Fig. 6GoA, lane 6).



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  		FIGURE 6. Failure of LC-PTP to bind to and suppress the sevenmaker MAPK mutants. A, Undetectable binding of LC-PTP to ERK2sem and p38{alpha}sem. HA-tagged LC-PTP were transiently expressed alone (lanes 1 and 4) or with ERK2-Flag (lane 2), ERK2sem-Flag (lane 3), p38{alpha}-Flag (lane 5), or p38{alpha}sem-Flag (lane 6) in 293T cells. The coprecipitation assay was performed as described in Fig. 3GoA. B, Little suppression of MEK-mediated phosphorylation of ERKsem by LC-PTP. 293T cells in six-well plates were transfected with the plasmid (500 ng) coding for ERK2-Flag (lanes 1–5) or ERK2sem-Flag (lanes 6–10) without (lanes 1, 2, 6, and 7) or with 2.5 µg (lanes 3 and 8), 1 µg (lanes 4 and 9), or 0.2 µg (lanes 5 and 10) of the plasmid for HA-tagged LC-PTP. pFC-MEK plasmid (100 ng) coding for an active MEK1 mutant was cotransfected to induce the phosphorylation of ERK2 (lanes 2–5 and 7–10). The total amounts of DNA were kept constant (5 µg) by the addition of empty pEF-BOS vector. ERK2-Flag was immunoprecipitated by M2 mAb, and the phosphorylation levels and the amounts of ERK2 were determined as described in Fig. 1GoA.

 
ERK binding activity of LC-PTP is crucial for the suppression of TCR-mediated activation of ERK

If ERK is the direct target of LC-PTP, ERK binding activity might be crucial for the suppression. Thus, we tested the effect of LC-PTP{Delta}N, a mutant lacking ERK binding activity, on the ERK cascade. This mutant revealed little inhibition of the ERK cascade activated by either TCR cross-linking (Fig. 7GoA) or the active MEK1 mutant (data not shown). The expression levels (Fig. 7GoA, upper panel) and the catalytic activities (Fig. 7GoB) of LC-PTP and LC-PTP{Delta}N were comparable. LC-PTPA41A42, another mutant lacking ERK binding capacity, also did not suppress the ERK cascade (data not shown).



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  		FIGURE 7. The effects of LC-PTP{Delta}N on the ERK cascade stimulated by TCR cross-linking. A, Little suppression of TCR-mediated activation of ERK cascade by LC-PTP{Delta}N. Jurkat cells were transfected with various reporter plasmids described in Fig. 2GoA, and the plasmid for Flag-LC-PTP (500 or 100 ng) or Flag-LC-PTP{Delta}N (500 ng). Activation of the ERK cascade was induced by OKT3 mAb. OKT3-induced luciferase activity without PTPs was expressed as 100%. The basal activity (the activity without OKT3 stimulation) was 3.4 ± 0.1%. The amounts of LC-PTP and LC-PTP{Delta}N expressed in the representative samples were analyzed by the immunoblotting using M2 mAb (upper panel). B, The catalytic activity of LC-PTP and LC-PTP{Delta}N. Flag-LC-PTP and Flag-LC-PTP{Delta}N transiently expressed in 293T cells were immunoprecipitated, and the phosphatase activities were measured using p-NPP as a substrate. The amounts of LC-PTP and LC-PTP{Delta}N precipitated were analyzed as described above (upper panel).

 
These results strongly suggest that ERK is the direct target of LC-PTP, and the binding of LC-PTP to ERK is the key mechanism of the suppression. However, it is still possible that LC-PTP{Delta}N and LC-PTPA41A42 are not suppressive because they cannot bind to the unidentified target molecule other than ERK. To rule out this possibility, we tested the effect of wild-type LC-PTP on ERK2sem that could not bind to LC-PTP. The phosphorylation of ERK2 and ERK2sem was induced by the cotransfection of an active MEK1 mutant. LC-PTP suppressed the phosphorylation of ERK2 in the dose-dependent manner (Fig. 6GoB, lanes 3–5), but little suppression of ERK2sem was observed (Fig. 6GoB, lanes 8–10). Therefore, we concluded that the complex formation of LC-PTP with ERK is the essential mechanism for the suppression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, the suppression of TCR-mediated ERK activation by HePTP was reported (8). However, the precise mechanism of the suppression was not clear. To understand the mechanism, it is very important to identify the target molecule(s) of the PTP.

In this study we demonstrated that MAPK is the direct target of LC-PTP, and the binding of LC-PTP to MAPK is the key mechanism of the suppression. Furthermore, we identified that Arg41 and/or Arg42 of LC-PTP and Asp321 of ERK2 are essential for the complex formation and the suppression. Our conclusion that ERK is the direct target of LC-PTP is based on the following observations. 1) LC-PTP dephosphorylates ERK2 in vitro. 2) LC-PTP suppresses the activation of the ERK cascade by a MEK1 mutant that is constitutively active regardless of the state of upstream signaling elements. 3) LC-PTP makes complexes with ERK family MAPKs. 4) Both the phosphatase activity and the ERK binding activity of LC-PTP are crucial for the suppression. 5) LC-PTP does not suppress the phosphorylation of ERK2sem, which does not bind to LC-PTP.

We demonstrated the in vivo complex formation of LC-PTP and ERK using 293T cells overexpressing these molecules. This raised a question of whether the endogenous molecules could make complexes at a physiological concentration. Thus, we tested the complex formation in vitro using the cell lysate from Jurkat cells (Fig. 4Go). Substantial amounts of endogenous ERK (~5%) and LC-PTP (>10%) in the Jurkat cell lysate make complexes with GST-LC-PTP and GST-ERK2, suggesting that endogenous LC-PTP and ERK have the potential to make complexes. Furthermore, LC-PTP is coprecipitated with an ERK kinase activity. These observations strongly suggest the complex formation of LC-PTP and ERK in Jurkat cells. However, further study is necessary to demonstrate the complex formation more conclusively, especially in the primary T cells. The physiological significance of the phosphorylation of LC-PTP by ERK also remains to be studied.

Previously, we and others cloned a PTP, PTPBR7/PTP-SL/PCPTP1/PC12-PTP, that revealed a structural similarity to LC-PTP/HePTP (23, 27, 28, 29). Despite the structural similarity, the tissue distribution is totally different. LC-PTP/HePTP is found only in hemopoietic cells, while PTPBR7/PTP-SL/PCPTP1/PC12-PTP is expressed almost exclusively in neuronal cells. Very recently, we and others have found that PTPBR7/PTP-SL/PCPTP1/PC12-PTP makes complexes with ERK and suppresses the ERK activated by NGF (30) and epidermal growth factor (31). The suppression of ERK by a PTP related to LC-PTP/HePTP further supports the possibility that ERK is the direct target of LC-PTP/HePTP in the TCR signal transduction pathway.

Although LC-PTP/HePTP and PTPBR7/PTP-SL/PCPTP1/PC12-PTP have the capacity to inactivate ERK directly, this does not necessarily mean that the direct inactivation is the major mechanism of the suppression. It is possible that the PTP inactivates both ERK and other molecules in the upstream signal transduction pathway at the same time. For example, >50% suppression of the epidermal growth factor-mediated ERK activation was induced by an ERK binding-deficient mutant of PTP-SL (31). This might suggest the presence of a target molecule other than ERK in the upstream signaling cascade. The relative importance of the direct inactivation of ERK as a mechanism of the suppression might vary depending on the signaling cascades. The failure of LC-PTP{Delta}N, an ERK binding-deficient mutant, to suppress the TCR-mediated activation of ERK strongly suggests that the direct inactivation of ERK is the major mechanism of the suppression. In line with this idea, LC-PTP suppressed the ERK cascade even if the early activation events of the TCR stimulation were bypassed by PMA and ionomycin.

In the previous report (8), Saxena and coworkers used HePTP; we used LC-PTP in this report. HePTP is almost identical with LC-PTP, but LC-PTP and HePTP differ in the presumptive location of the translation initiation codon (6, 7). This results in the difference in the predicted amino acid sequence in the most N-terminal portion. The translation initiation codon of rat HePTP is the same as that of human LC-PTP (32). Despite this difference, LC-PTP and HePTP revealed similar suppressive effects on ERK phosphorylation and NF-AT/AP-1 reporter activity. The study of the various mutants of LC-PTP shows that the ERK binding site is outside the PTP domain (between aa 1–99) and in the region conserved among LC-PTP and HePTP (this study and unpublished observations). Especially, Arg41 and/or Arg42 are essential for the ERK binding capacity, because the substitutions of these residues to Ala result in the totally loss of ERK binding activity. These residues are also conserved among related PTPs, such as PTPBR7/PTP-SL/PCPTP1/PC12-PTP (23, 27, 28, 29) and STEP (33, 34). Substitutions of the conserved Arg residues in PTPBR7 resulted in the loss of ERK binding capacity (M. Oh-hora and M. Ogata, unpublished observation).

LC-PTPCS, a catalytically inactive mutant, revealed no suppressive effect on the phosphorylation of ERK. In contrast, this mutant revealed weak, but reproducible, suppressive effects on the ERK cascade measured by the reporter assay in both Jurkat cells (Fig. 2C) and 293T cells (data not shown). This apparent discrepancy is not surprising, because a similar observation has been reported for Ptp2p, a tyrosine-specific phosphatase in S. cerevisiae (35). It was explained that the binding of the catalytically inactive PTP might interfere with the MAPK to interact with downstream signaling molecules.

The biological roles of LC-PTP/HePTP remain to be elucidated. It was reported that the expression of HePTP-C270S, a catalytically inactive mutant, revealed a stimulatory effect on ERK phosphorylation, probably due to the competition with the endogenous PTP (8). This dominant negative effect of HePTP-C270S strongly suggests the involvement of LC-PTP/HePTP in the physiological regulation of ERK kinases. In T lymphocytes, ERK is activated through the stimulation of TCR and cytokine receptors such as IL-2R. The suppression of ERK cascade by LC-PTP/HePTP suggests its involvement in the negative feedback regulation of the T cell responses. In line with this idea, the expression level of HePTP/LC-PTP is increased by the TCR cross-linking (7) and IL-2 stimulation (36, 37).

Drosophila sevenmaker mutant is a gain-of-function mutant that is caused by a single amino acid substitution (D334N) of the Drosophila MAPK (20). Although hyperactive in vivo, the sevenmaker MAPK reveals little increase in the enzymatic activity when the activity of the recombinant MAPK is tested in vitro (38). It was reported that the expression of the sevenmaker ERK2sem mutant in the developing thymocytes enhances T cell development into CD4 lineage (18). In this report we observed that LC-PTP failed to make complexes with ERK2sem. Because LC-PTP is expressed predominantly in the thymus, and the ERK binding capacity is crucial for the suppression, the less effective inactivation of ERK2sem by LC-PTP may underlie the phenotype in T cell development. If this were the case, LC-PTP might play an important role in the regulation of T cell development. It should be noted, however, that ERK2sem is also resistant to the dual specificity MAPK phosphatases, such as MAPK phosphatase-1, -2, and -3 and PAC1 (38, 39, 40).


   Acknowledgments
 
We thank Dr. Gerald R. Crabtree for providing NF-AT-luc plasmid.


   Footnotes
 
1 This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture, Japan, and the Osaka Foundation for Promotion of Clinical Immunology. Back

2 Address correspondence and reprint requests to Dr. Masato Ogata, Biomedical Research Center, Osaka University Medical School C6, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail address: Back

3 Abbreviations used in this paper: PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; HePTP, hemopoietic PTP; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; p-NPP, p-nitrophenyl phosphate; HA, hemagglutinin. Back

Received for publication February 19, 1999. Accepted for publication May 20, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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