Molecular Cancer Research Targeting the PI3-Kinase Pathway in Cancer Tumor Immunology: New Perspectives
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Molecular Cancer Research 1:541-550 (2003)
© 2003 American Association for Cancer Research

Signaling and Regulation

Protein Tyrosine Phosphatase {varepsilon} Inhibits Signaling by Mitogen-Activated Protein Kinases1

Hila Toledano-Katchalski1, Judith Kraut1, Tal Sines1, Shira Granot-Attas1, Galit Shohat1, Hava Gil-Henn1, Yuval Yung2 and Ari Elson1

Departments of 1 Molecular Genetics and 2 Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel

Requests for reprints: Ari Elson, Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel. Phone: 972-8-934-2331; Fax: 972-8-934-4108. E-mail: ari.elson{at}weizmann.ac.il


   Abstract
Top
 Notes
 Abstract
Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
Mitogen-activated protein kinases (MAPKs) mediate signaling from the cell membrane to the nucleus following their phosphorylation at conserved threonine and tyrosine residues within their activation loops. We show that protein tyrosine phosphatase {varepsilon} (PTP{varepsilon}) inhibits ERK1 and ERK2 kinase activity and reduces their phosphorylation; in agreement, ERK phosphorylation is increased in fibroblasts and in mammary tumor cells from mice genetically lacking PTP{varepsilon}. PTP{varepsilon} inhibits events downstream of ERKs, such as transcriptional activation mediated by Elk1 or by the serum response element. PTP{varepsilon} also inhibits transcriptional activation mediated by c-Jun and C/EBP binding protein (CHOP) but not that mediated by the unrelated NFkB, attesting that it is broadly active within the MAPK family but otherwise specific. The effect of PTP{varepsilon} on ERKs is at least in part indirect because phosphorylation of the threonine residue in the ERK activation loop is reduced in the presence of PTP{varepsilon}. Nonetheless, PTP{varepsilon} is present in a molecular complex with ERK, providing PTP{varepsilon} with opportunity to act on ERK proteins also directly. We conclude that PTP{varepsilon} is a physiological inhibitor of ERK signaling. Slow induction of PTP{varepsilon} and its lack of nuclear translocation following mitogenic stimulation suggest that PTP{varepsilon} functions to prevent inappropriate activation and to terminate prolonged, rather than acute, activation of ERK in the cytosol.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
Phosphorylation of tyrosine residues in proteins is an ubiquitous physiological process that regulates protein structure and function. Tyrosine phosphorylation is a reversible process that is controlled by the opposing activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). PTPs are a structurally diverse superfamily containing several dozens of receptor-type and non-receptor-type enzymes (1–4). Studies during the past decade have shown that PTPs are key regulators of physiological processes, but in many cases, precise details of how this is achieved are still missing.

Depending on the protein involved and on the specific site phosphorylated, phosphorylation can activate proteins in some situations and inactivate them in others. Accordingly, PTPs typically participate in both activation and down-regulation of physiological pathways. For example, while many PTPs can inhibit cellular transformation (e.g., 5, 6), a small group of PTPs has been shown to augment transformation. This group includes the receptor-type PTPs {alpha} and {varepsilon} (RPTP{alpha} and RPTP{varepsilon}), which can dephosphorylate and activate the Src tyrosine kinase. This ability allows RPTP{alpha} to transform rat embryo fibroblasts (7) and makes RPTP{varepsilon} an important factor in maintaining the transformed phenotype of mouse mammary tumor cells transformed by Neu (16). Also included in this group are CDC25 (8) and FAP-1, whose down-regulation of Fas-induced apoptosis may aid tumor cells to evade regulatory mechanisms (9).

PTP{varepsilon} exists as a family of four proteins, all products of the single PTP{varepsilon} gene. The two most prevalent forms are the receptor-type (RPTP{varepsilon}) and non-receptor-type (cyt-PTP{varepsilon}) forms of PTP{varepsilon}, each the product of a distinct PTP{varepsilon} mRNA species. The other two forms of PTP{varepsilon} are p67 and p65; these are shorter molecules that are expressed together with either RPTP{varepsilon} or cyt-PTP{varepsilon}, and the production of which is regulated at the levels of translation and post-translational processing (10–15). All forms have the same two catalytic domains but distinct N termini, which result in unique subcellular localization patterns and physiological roles (11, 13).

Depending on the precise context, PTP{varepsilon} can activate some physiological processes and down-regulate others. As indicated, RPTP{varepsilon} activates Src and plays a role in maintaining the transformed phenotype of mammary tumor cells induced by Neu in transgenic mice (16). PTP{varepsilon} is also required for ensuring proper function of macrophages in vivo (17). On the other hand, RPTP{varepsilon} can down-regulate insulin receptor signaling in BHK cells expressing the insulin receptor (18, 19), and cyt-PTP{varepsilon} can dephosphorylate and down-regulate delayed-rectifier, voltage-gated potassium (Kv) channels in Schwann cells in vivo. The latter finding correlates with transient hypomyelination of sciatic nerve axons in young mice lacking PTP{varepsilon} (20). PTP{varepsilon} can also suppress endothelial cell proliferation (21) and can inhibit JAK-STAT signaling induced by various cytokines in M1 leukemia cells (22, 23). These findings raise the question of whether PTP{varepsilon} can down-regulate key steps in mitogenic signaling other than the JAK-STAT pathway specifically examined in the above studies.

Previous studies have examined this issue from the opposite direction and have shown that cyt-PTP{varepsilon} expression can be regulated by mitogenic signaling. Specifically, cyt-PTP{varepsilon} mRNA is induced in a transient manner in NIH3T3 cells by short-term treatment with mitogenic factors, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), serum, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) (11). Induction of cyt-PTP{varepsilon} mRNA is dependent on protein kinase C activity, and is first detected 2 h following application of stimuli. Inhibition of protein synthesis following stimulation prevents induction of cyt-PTP{varepsilon} mRNA, indicating that cyt-PTP{varepsilon} is a delayed-early response gene in NIH3T3 cells (11). Constant mitogenic stimulation, such as when NIH3T3 cells are transformed by activated Ras, leads to stable expression of high levels of cyt-PTP{varepsilon}.2 None of these effects are noted when RPTP{varepsilon} mRNA is examined (11), highlighting functional differences between the two promoters of the PTP{varepsilon} gene that separately regulate expression of either RPTP{varepsilon} or cyt-PTP{varepsilon} (11, 14). The relatively late induction of cyt-PTP{varepsilon} and its dependence on prior protein synthesis indicate that cyt-PTP{varepsilon} is not among the first targets of mitogenic stimulation. The physiological meaning of this finding is, however, ambiguous; it is consistent with cyt-PTP{varepsilon} participating in down-regulating mitogenic signaling cascades and in shutting down the effects of mitogenic signals or conversely, with PTP{varepsilon} being a delayed participant in growth-promoting effects of mitogenic stimuli.

Among the major mitogenic signaling pathways are those that include receptor tyrosine kinases, Ras, Raf, and a mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade that includes ERK1 and ERK2 is typically triggered by mitogenic stimuli and preferentially activates the transcription factor Elk1. MAPK cascades that include JNK or p38 function primarily in stress responses and tend to activate c-Jun or C/EBP homologous protein (CHOP), respectively (24–30). MAPKs are activated following phosphorylation of both a threonine and a tyrosine in their activation loop, while dephosphorylation of either residue is sufficient to inhibit kinase activity (30–32). Accordingly, several phosphatases, which are specific either for serine/threonine or for tyrosine, can inactivate MAPKs by dephosphorylating either the threonine or the tyrosine residue of the activation loop (e.g., 31, 33, 34). These enzymes are joined by the large family of dual-specificity phosphatases, which are structurally similar to tyrosine phosphatases but which can dephosphorylate both residues of the activation loop (reviewed in 31, 35).

Induction of cyt-PTP{varepsilon} expression by stimuli that target primarily the ERK1/ERK2 cascade makes it likely that this pathway controls cyt-PTP{varepsilon} expression and might be affected by it in return. Nonetheless, cross-talk between the pathways involving ERK1/ERK2, JNK, and p38 retains the latter two cascades as reasonable targets for PTP{varepsilon} activity. A recent study has indeed shown that PTP{varepsilon} can down-regulate a MAPK-sensitive reporter in Jurkat cells in response to cross-linking the T-cell receptor, and that cyt-PTP{varepsilon} can reduce activities of ERK1 and ERK2 in 293 cells (36). In this study, we dissect the effects of various forms of PTP{varepsilon} on MAPK signaling. We show that expression of cyt-PTP{varepsilon} significantly reduces kinase activity of ERK1 and ERK2. Presence of PTP{varepsilon} also reduces phosphorylation of ERK proteins at both Thr and Tyr residues of their activating TEY motif in NIH3T3 cells and in mammary tumor cells. In agreement, ERK phosphorylation is increased in embryonic fibroblasts and in mammary tumor cells from mice genetically lacking PTP{varepsilon}. The effect of PTP{varepsilon} is at least partially indirect, because PTP{varepsilon} reduces phosphorylation of ERK2 at Thr183 following TPA stimulation of NIH3T3 cells. Expression of all known forms of PTP{varepsilon} strongly reduces activity of various downstream reporters of ERK, JNK, and p38 MAPK activities, but not of the unrelated NFkB signaling pathway. This finding indicates that PTP{varepsilon} affects the major MAPKs, but does not affect all signaling pathways indiscriminately. The effect of PTP{varepsilon} on ERK proteins is dependent on the catalytic activity or substrate-binding abilities of PTP{varepsilon}. cyt-PTP{varepsilon} and the ERK proteins are present in the same molecular complex, providing the phosphatase with physical opportunity to regulate ERK activity either directly or indirectly. In all, the data indicate that PTP{varepsilon} is a physiological inactivator of ERK proteins. Slow induction of cyt-PTP{varepsilon} and its lack of nuclear translocation following various forms of mitogenic stimulation suggest that PTP{varepsilon} functions as a "fail-safe" mechanism to prevent inappropriate activation or to terminate prolonged activation of ERK in the cytosol.


   Results and Discussion
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
Materials and Methods
 Acknowledgements
 References
 
PTP{varepsilon} Reduces Activity and Phosphorylation of ERK Proteins
To examine whether cyt-PTP{varepsilon} affects MAPK signaling, we examined ERK activity in the presence or absence of the phosphatase. For this purpose, various combinations of ERK1, ERK2, and cyt-PTP{varepsilon} proteins were expressed in 293 cells; ERK proteins were precipitated from cell lysates and their kinase activity measured using myelin basic protein (MBP) as a substrate. ERK activity in unstimulated cells was low but increased significantly following stimulation of the cells with TPA (Fig. 1A). Expression of cyt-PTP{varepsilon} reduced kinase activity of both ERK1 and ERK2 from stimulated cells by two-thirds, indicating that cyt-PTP{varepsilon} can negatively regulate ERK activity (Fig. 1A).



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  		FIGURE 1. cyt-PTP{varepsilon} inhibits activities and reduces phosphorylation of ERK1 and ERK2. A. cyt-PTP{varepsilon} and HA-tagged ERK proteins were expressed in 293 cells as indicated, and activities of immunoprecipitated ERK were measured by its phosphorylation of MBP. In some cases, cells were stimulated with TPA before lysis as indicated. Top, 32P radioactivity incorporated into MBP by ERK. Middle panel shows amount of cyt-PTP{varepsilon} in cell lysates; bottom panel shows amount of ERK1 or ERK2 in the assayed precipitates. Shown is an experiment representative of three performed. B. Left, reduced dual phosphorylation (Diphosphorylation) of ERK2 on the TEY motif in TPA-stimulated NIH3T3 cells following expression of HA-ERK2 and RPTP{varepsilon} or cyt-PTP{varepsilon}. Data (means ± SD) are presented relative to ERK dual phosphorylation in TPA-stimulated cells not expressing exogenous PTP{varepsilon}. Right, representative blot showing reduced dual phosphorylation of ERK2 in cells expressing PTP{varepsilon}. NIH3T3 cells were transfected with HA-labeled ERK2, and RPTP{varepsilon} (R), cyt-PTP{varepsilon} (cyt), or empty vector (M) as indicated. Following serum starvation, cells were stimulated with TPA, lysed immediately thereafter, and immunoprecipitated with anti-HA antibodies. Shown are levels of dual phosphorylated (pThr183/pTyr185) ERK2 (top panel), amount of ERK2 in the precipitates (gen ERK, second panel), and expression of PTP{varepsilon} (third panel). Note that NIH3T3 cells express endogenous cyt-PTP{varepsilon}. C. Bar diagram as in B, showing reduced ERK2 phosphorylation at Thr183 using an antibody specific for that site.

 
ERK1 and ERK2 are activated by phosphorylation on both threonine and tyrosine residues of the activating TEY sequence (analogous to Thr183 and Tyr185 in rat ERK2). Reduced ERK activity in the presence of PTP{varepsilon} should therefore correlate with reduced phosphorylation of one or both of these residues. To examine this issue, cyt-PTP{varepsilon} or RPTP{varepsilon} were transiently expressed in NIH3T3 cells together with HA-ERK2, after which cells were starved for serum and then stimulated with TPA. Lysates prepared from these cells were blotted and analyzed with a phospho-specific antibody, which detects ERK proteins phosphorylated exclusively on both threonine and tyrosine residues of the activating TEY sequence (37). In agreement with measurements of ERK activity, TPA treatment significantly increased ERK2 phosphorylation at both sites, while expression of cyt-PTP{varepsilon} or RPTP{varepsilon} in TPA-treated cells reduced ERK phosphorylation by 63–74% (Fig. 1B). Similar experiments with antibodies that specifically detect ERK phosphorylated only on the threonine residue of the activation loop (Thr183; 37) indicated that PTP{varepsilon} activity reduced phosphorylation also at this residue in ERK2 (Fig. 1C). This finding suggests that the effect of the tyrosine-specific PTP{varepsilon} on ERK proteins is at least in part indirect. In agreement with these results, dual phosphorylation of endogenous ERK1 and ERK2 on both Tyr and Thr residues in primary embryonic fibroblasts [murine embryo fibroblasts (MEFs)] isolated from mice genetically lacking PTP{varepsilon} (Ptpre-/- mice; 20) was increased 2.1- to 2.5-fold (Fig. 2A). Dual phosphorylation of both ERK proteins was similarly affected in relative terms in this experiment, despite ERK2 being significantly more phosphorylated than ERK1 in MEFs. Experiments in which MEFs from WT or from Ptpre-/- mice were stimulated with TPA indicated that the time course and relative extent of stimulation of ERK1 and ERK2 were similar in cells from both genotypes (Fig. 2B).



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  		FIGURE 2. PTP{varepsilon} reduces dual phosphorylation of ERK in MEFs. A. Left, increased phosphorylation of ERK1 (black bars) and ERK2 (white bars) in primary MEFs from wild-type (WT) and PTP{varepsilon}-deficient (KO) mice, as determined by blotting with anti-diphospho-ERK antibodies. Results are based on three experiments. *, P <= 0.028; **, P <= 0.0007 by Student's t test. Right, representative blot showing increased ERK phosphorylation in primary MEFs from wild-type (WT) or PTP{varepsilon}-deficient (KO) mice. Shown are levels of dual phosphorylated endogenous ERK1 and ERK2 (pERK1, pERK2; top two panels) and of total ERK1 and ERK2 (bottom panel). Middle panel is an overexposed version of the top panel to allow examination of ERK1, the phosphorylation levels of which are significantly lower than pERK2. B. Time course stimulation of ERK1 and ERK2 from WT and Ptpre-/- MEFs by TPA. Serum-starved MEFs were stimulated with TPA for the times indicated, after which dual phosphorylation of ERK1 and of ERK2 was determined by protein blotting. Shown are averages and SEs of two experiments. Differences between WT (W) and Ptpre-/- (K) values at each time point were not statistically significant.

 
Alterations in ERK phosphorylation were observed also in mammary epithelial tumor cells, which were isolated from tumors induced in wild-type or in Ptpre-/- mice by mammary gland-specific expression of an activated Neu transgene (38,16). Dual phosphorylation of ERK in these cells was readily detectable, most likely due to strong constitutive activation of the Ras-MAPK pathway by the Neu protein. In agreement with results obtained in MEFs, dual phosphorylation of the two ERK proteins was increased 2.0- to 2.3-fold in cells from Ptpre-/- mice as compared with cells from wild-type mice (Fig. 3A). Additional experiments revealed that expression of either cyt-PTP{varepsilon} or RPTP{varepsilon} in tumor cells from Ptpre-/- mice reduced dual phosphorylation of ERK by 51–67% (Fig. 3B). Together, these results indicate that PTP{varepsilon} reduces the level of phosphorylated ERK proteins, in agreement with phosphorylation and kinase activity measurements presented above. As results presented above were obtained using a diverse set of cell types, the effect of PTP{varepsilon} on ERK appears to be widespread. Furthermore, the endogenous form of PTP{varepsilon} expressed in MEFs is cyt-PTP{varepsilon}, while mammary tumor cells express RPTP{varepsilon}; the effect of PTP{varepsilon} on ERK is therefore not limited to a particular isoform of PTP{varepsilon}.



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  		FIGURE 3. PTP{varepsilon} reduces dual phosphorylation of endogenous ERK proteins in Neu-induced mouse mammary tumor cells. A. Diagram depicting dual phosphorylation of ERK1 (black bars) and ERK2 (white bars) in tumor cells expressing endogenous RPTP{varepsilon} from WT (WT) or from Ptpre-/- mice (KO). B. Top, similar diagram depicting ERK phosphorylation in Neu-induced mammary tumor cells derived from Ptpre-/- mice and reconstituted with cyt-PTP{varepsilon} or RPTP{varepsilon}. Data (means ± SD) are from eight experiments. **, t <= 0.0072 by Student's t test. Bottom, Neu-induced mammary tumor cells genetically lacking PTP{varepsilon} were infected with retroviral expression constructs, which were empty (M) or which contained cDNAs for cyt-PTP{varepsilon} (cyt) or for RPTP{varepsilon} (R). Top, levels of doubly phosphorylated ERK. Middle, same blot re-probed with non phospho-specific anti-ERK antibodies. Bottom, expression of PTP{varepsilon} in the various cells. Note significantly higher phosphorylation of ERK proteins from mock-infected cells (top) despite lower amount of ERK protein present in this lane (bottom). *, non-specific band.

 
PTP{varepsilon} Inhibits Transcriptional Activation Mediated by Elk1 and by the Serum Response Element
Reduced phosphorylation and activity of ERK proteins and possibly of other MAPKs should reduce activation of molecules located further downstream in signaling cascades. To examine the effect of PTP{varepsilon} on downstream molecules, NIH3T3 cells were transiently transfected with PTP{varepsilon}, and Elk1-mediated transcription in these cells in response to various stimuli was examined. Elk1 activity was determined using a reporter system, which included a hybrid protein consisting of the activation domain of Elk1 fused to the Gal4 DNA binding domain (Elk1/Gal4-DBD). On phosphorylation of the Elk1 domains of this molecule by MAPK molecules, the hybrid molecule binds the Gal4 upstream activating sequence (UAS) located 5' to a luciferase reporter gene (Gal4-UAS/luciferase), ultimately resulting in luciferase activity proportional to the extent of MAPK activity.

Luciferase activity was detected following transient transfection of the Elk1/Gal4-DBD and Gal4-UAS/luciferase plasmids into NIH3T3 cells (Fig. 4A). Deletion of the Elk1 domain from Elk1/Gal4-DBD virtually eliminated luciferase activity, verifying that luciferase activity depended on the Elk1 domain. Activity measured in these experiments most likely reflects basal signaling levels present in the cells. On transient expression of RPTP{varepsilon} or cyt-PTP{varepsilon}, luciferase activity was reduced by 87% and 92%, respectively (Fig. 4A). In separate experiments, stimulation of serum-starved NIH3T3 cells with bFGF resulted in a 27.7-fold increase in luciferase activity; transient expression of RPTP{varepsilon} reduced 87% of this increase, while cyt-PTP{varepsilon} reduced 90% (Fig. 4B). Stimulation of NIH3T3 cells with TPA enhanced Elk1-mediated transcription by approximately 13.2-fold, while transient expression of constitutively active EJ-Ras caused an increase of approximately 8-fold. In both cases, expression of cyt-PTP{varepsilon} or RPTP{varepsilon} reduced luciferase activity by 74–86% (Fig. 4B). We conclude that PTP{varepsilon} can effectively down-regulate Elk1-mediated transcription in mitogen-stimulated or non-stimulated NIH3T3 cells. Protein blotting experiments revealed modest expression levels of transfected PTP{varepsilon} in NIH3T3 cells which, bringing into account transfection efficiencies, were estimated as two to three times that of endogenous cyt-PTP{varepsilon} (Fig. 1B and results not shown).



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  		FIGURE 4. A. PTP{varepsilon} suppresses Elk1-mediated transcriptional activation in unstimulated NIH3T3 cells. Cells were transiently transfected with pcDNA3 vector, RPTP{varepsilon}, or cyt-PTP{varepsilon} as indicated, as well as with constituents of the Elk1 reporter system and a CMV-ß-galactosidase plasmid as indicated in "Materials and Methods." Shown is luciferase activity detected in the cells, normalized to relative transfection efficiency determined by ß-galactosidase activity, in one experiment representative of five performed for a total of 10 determinations per data point. B. PTP{varepsilon} suppresses Elk1-mediated transcription in stimulated NIH3T3 cells. Transfected cells were serum starved for 12 h, after which they were stimulated with 10 ng/ml bFGF or 100 ng/ml TPA for 10 min. Medium was then replaced with fresh low-serum medium for 12 additional hours, followed by assay of reporter activity as in panel A. Some cells were transfected with EJ-Ras in regular growth medium. Shown is one experiment representative of three performed, for six determinations per data point. C. PTP{varepsilon} can suppress serum response element (SRE)-mediated transcription in stimulated NIH3T3 cells. Cells were transiently transfected with an SRE-luciferase reporter plasmid and with a cytomegalovirus (CMV)-ß-galactosidase plasmid and were then starved and stimulated with bFGF or TPA as described in panel B. Results from one experiment, representative of three performed, are shown for a total of six determinations per data point. In all panels, values shown are means ± SD.

 
We next examined the effect of PTP{varepsilon} on transcription mediated by the serum response element (SRE), a sequence motif present in promoters of many immediate-early genes (39, 40). SRE is triggered by endogenous Elk1 and related proteins, allowing us to monitor the effect of PTP{varepsilon} on mitogen-induced transcription at a step further downstream and in a manner independent of the Elk1 reporter system. NIH3T3 cells were transfected with an SRE-luciferase reporter construct and, following serum starvation, stimulated with either bFGF or TPA. In agreement with results presented above, expression of cyt-PTP{varepsilon} or RPTP{varepsilon} inhibited both bFGF- and TPA-mediated activation by 66–73% (Fig. 4C).

PTP{varepsilon} Inhibits Transcriptional Activation Mediated by ERKs, JNK, and p38, but not by NFkB
Elk1 can be activated also by JNK and p38 (30), raising the issue of whether PTP{varepsilon} can also affect signaling by these MAPKs. We therefore introduced into NIH3T3 cells reporter systems in which downstream transcription was mediated by c-Jun or CHOP, the major targets of JNK or of the p38 MAPKs, respectively. We also examined a reporter activated by NFkB, which is part of an unrelated signaling mechanism. As seen in Fig. 5, A–C, transcription mediated by Elk1, CHOP, or c-Jun was reduced by 70–92% following expression of either RPTP{varepsilon} or cyt-PTP{varepsilon}. In contrast, RPTP{varepsilon} and cyt-PTP{varepsilon} had significantly weaker effects on NFkB-mediated transcriptional activation (Fig. 5D). Similarly, weak inhibition was obtained in NIH3T3 cells following stimulation of NFkB signaling by TNF{alpha} (not shown). We conclude that both forms of PTP{varepsilon} can inhibit transcriptional activation by the three MAPK pathways. In this respect, PTP{varepsilon} resembles several of the dual-specificity MAPK phosphatases (MKPs) as well as several PTPs and serine/threonine phosphatases, which do not exhibit strict specificity toward a particular MAPK (31). Nonetheless, PTP{varepsilon} is not promiscuous and can differentiate between the three MAPK pathways and the unrelated NFkB pathway.



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  		FIGURE 5. PTP{varepsilon} suppresses transcriptional activation mediated by Elk1, c-Jun, and CHOP, but not by NFkB. NIH3T3 cells were transiently transfected with reporter plasmids for the above transcription factors, a CMV-ß-galactosidase plasmid, and pcDNA3 or PTP{varepsilon} in pcDNA3 as indicated. Luciferase activity, normalized to ß-galactosidase activity, was determined 24 h post-transfection. Results are presented relative to luciferase activity in cells transfected with empty pcDNA3 in each experiment. Shown is one experiment (means ± SD), representative of three performed, for a total of six determinations per data point.

 
Membrane or Nuclear Localization of PTP{varepsilon} Are Not Required for Inhibition of Elk1-Mediated Transcription
Data presented thus far indicate that both RPTP{varepsilon}, which is an integral membrane protein, and cyt-PTP{varepsilon}, which is not, can inhibit Elk1-mediated transcriptional activation. Nonetheless, some cyt-PTP{varepsilon} molecules are found at the cell membrane (11, 13), leading us to examine whether membrane localization is required for PTP{varepsilon} to exert its effect. We therefore examined the ability of two naturally occurring, exclusively cytosolic isoforms of PTP{varepsilon}, p67 PTP{varepsilon} and p65 PTP{varepsilon}, to affect Elk1-mediated transcription (13). Expression of p65 or of p65 together with p67, in the absence of full-length RPTP{varepsilon} or cyt-PTP{varepsilon}, inhibited Elk1-mediated transcription as efficiently as full-length cyt-PTP{varepsilon} or RPTP{varepsilon} (Fig. 6A), leading us to conclude that membrane localization is not required for PTP{varepsilon} to exert its effect in our experimental system. In this respect, PTP{varepsilon} is somewhat similar to PTP-SL, the transmembrane and cytosolic isoforms of which are able to dephosphorylate ERK2 (41). A fraction of cyt-PTP{varepsilon} molecules are present in the cell nucleus (42). The ability of the cytosolic p67 and p65 PTP{varepsilon} molecules to affect ERK therefore also indicates that nuclear localization is not required for cyt-PTP{varepsilon} to down-regulate ERK.



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  		FIGURE 6. A. The D2 domain of PTP{varepsilon} and membrane localization are not required for inhibition of Elk1-mediated transcription. NIH3T3 cells were transfected, starved, stimulated with TPA, and processed as detailed in Fig. 2B. Transfections included, in addition to reporter plasmids and CMV-ß-galactosidase, pcDNA3-based plasmids containing full-length RPTP{varepsilon} (R), cyt-PTP{varepsilon} (cyt), their D1 domains (R-D1 and cyt-D1), the D2 domain (D2), or PTP{varepsilon} cDNAs expressing both p67 and p65 or only p65. B. Inactive, non-binding mutants of PTP{varepsilon} do not inhibit Elk1-mediated transcription. NIH3T3 cells were transfected, starved, and stimulated with TPA, and processed as above. Cells were transfected with wild-type RPTP{varepsilon} or cyt-PTP{varepsilon}, or with their D-to-A or R-to-M mutants, as indicated. Both figures show ß-galactosidase-normalized luciferase activities, and are presented relative to activity in non-stimulated cells transfected with empty pcDNA3. Results (means ± SD) from one experiment, representative of two or three performed, are shown, for a total of four to six determinations per data point.

 
Catalytic Activity or Substrate-Binding Ability Is Required for PTP{varepsilon} to Inhibit Elk1-Mediated Transcription
We next examined the ability of each of the two catalytic domains of PTP{varepsilon} to affect Elk1-mediated transcription. Of these, only the D1 proximal domain is active (43). D2 distal PTP domains are typically inactive and have been suggested to play regulatory roles by promoting protein-protein interactions (44–47). The PTP{varepsilon} D2 domain could therefore in principle interfere with Elk1-mediated transcriptional activation by interacting with a protein necessary for activating Elk1. To examine this possibility, we studied the effect of the D1 domains of RPTP{varepsilon} and cyt-PTP{varepsilon} and of the D2 domain, which is common to both PTP{varepsilon} forms, on Elk1-mediated transcription. As seen in Fig. 6A, RPTP{varepsilon} or cyt-PTP{varepsilon} molecules lacking the D2 domain reduced Elk1-mediated transcription as efficiently as the full-length PTPs. In contrast, the isolated D2 domain had virtually no effect. As before, the relative effect of the D1 domains was similar in freely growing and in TPA-stimulated NIH3T3 cells (Fig. 6A and not shown). We conclude that the D1 domain of PTP{varepsilon} is sufficient to mediate the full effect of PTP{varepsilon}, and that the D2 domain has little if any direct effect in the experimental system examined here.

We next examined the ability of two types of catalytically inactive mutants of PTP{varepsilon} to affect Elk1-mediated transcriptional activation. Experiments included the substrate-trapping mutants D302A RPTP{varepsilon} and D245A cyt-PTP{varepsilon} (20), as well as R340M RPTP{varepsilon} and R283M cyt-PTP{varepsilon}. Both classes of mutants are virtually inactive; however, D-to-A mutants retain the ability to bind phosphorylated substrates via their catalytic cleft, while R-to-M mutants do so much less efficiently (48). As shown in Fig. 6B, the R-to-M mutants of cyt-PTP{varepsilon} and RPTP{varepsilon} did not significantly inhibit Elk1-mediated transcription, indicating that PTP{varepsilon} catalytic activity is required for this effect. In contrast, the D-to-A mutants inhibited Elk1 in a manner similar to wild-type PTP{varepsilon}. As D-to-A mutants can bind physiological substrates at tyrosine residues that the wild-type enzyme would dephosphorylate, binding could sequester or otherwise incapacitate substrates, thereby mimicking inactivating dephosphorylation. Of note, a similar phenomenon was noted previously, as the catalytically inactive D245A cyt-PTP{varepsilon} was able to bind and partially reduce activity of the Kv2.1 voltage-gated potassium channel in transfected cells and in microinjected Xenopus oocytes (20).

cyt-PTP{varepsilon} and ERK Proteins Are Present in the Same Molecular Complex
Results presented above raise the possibility that the ERK proteins interact with or might be substrates of PTP{varepsilon}. We therefore performed a series of studies in which the ability of ERK1 or ERK2 to coprecipitate with cyt-PTP{varepsilon} was examined. As seen in Fig. 7, both ERK proteins clearly bound cyt-PTP{varepsilon} at levels significantly above background, indicating that cyt-PTP{varepsilon} and ERKs are found in the same molecular complex. Yet, attempts to determine whether ERK proteins bound the D245A substrate-trapping mutant of cyt-PTP{varepsilon} stronger than wild-type cyt-PTP{varepsilon} yielded inconclusive results (not shown). Detection of enzyme-substrate binding using substrate trapping mutants of phosphatases is occasionally problematic and not straightforward. The results presented here therefore do not strictly rule out the possibility that ERK proteins are substrates of PTP{varepsilon}, although reduced phosphorylation of ERK2 at Thr183 in the presence of PTP{varepsilon} argues that at least part of the effects of PTP{varepsilon} on ERKs are indirect. Involvement of a third protein, possibly a scaffold, in the ERK-PTP{varepsilon} complex is possible because PTP{varepsilon} lacks an obvious kinase interaction motif (KIM; 32, 41, 49) of the type found in tyrosine phosphatases thought to directly bind and dephosphorylate MAPKs. In all, existence of a molecular complex comprised of PTP{varepsilon} and ERK offers PTP{varepsilon} physical opportunity to regulate ERK activity, either directly or indirectly.



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  		FIGURE 7. cyt-PTP{varepsilon} binds ERK1 and ERK2 proteins. FLAG-tagged cyt-PTP{varepsilon} and HA-tagged ERK1 or ERK2 proteins were expressed in 293 cells as indicated, followed by immunoprecipitation of cyt-PTP{varepsilon}. Top, levels of coprecipitated ERK proteins. Middle, levels of cyt-PTP{varepsilon} protein in precipitate. Bottom, expression levels of ERK proteins in cell lysates. *, non-specific bands.

 
Mitogenic stimulation of NIH3T3 cells induces cyt-PTP{varepsilon} only after several hours (11). Although some cyt-PTP{varepsilon} is present in the nucleus (13, 42), these amounts are relatively small and do not seem to be required to mediate down-regulation of ERK by PTP{varepsilon}. Together, these data indicate that cyt-PTP{varepsilon} is not among the earlier proteins induced by MAPK signaling, and that cyt-PTP{varepsilon} affects MAPKs mainly in the cytosol. PTP{varepsilon} might therefore function either to down-regulate the activity of MAPKs toward cytosolic substrates following cellular stimulation, or to prevent premature activation of MAPKs in the cytosol. PTP{varepsilon}, in particular, cyt-PTP{varepsilon}, which is highly inducible, may then be part of a "fail-safe" mechanism which acts to counter effects of dysregulated mitogenic stimulation by shutting down MAPK signaling in cases of prolonged, as opposed to acute, activation of MAPK signaling. The latter possibility is consistent with induction of cyt-PTP{varepsilon} also by non-mitogenic stimuli, such as in macrophage differentiation and activation (11, 14). For example, TPA-induced differentiation of the human promyelocytic leukemia HL60 cell line along the monocyte-macrophage pathway is accompanied by massive up-regulation of cyt-PTP{varepsilon}, which correlates with cessation of cell division and induction of differentiation (11). PTP{varepsilon} then appears to be somewhat similar to the PTP subfamily containing STEP, PTP-SL, and PTPBR7/PC12-PTP1, and that containing HePTP and LC-PTP. These phosphatases preferentially target ERK1/2 and p38 (reviewed in 31) but are not found in the cell nucleus, leading to suggestions that they function in the cytosol to prevent improper activation of MAPKs.

The wide range of cell types examined in this study, as well as the previous documentation of PTP{varepsilon} inhibition of a MAPK reporter in Jurkat T cells in response to T-cell receptor activation (36), suggests that the effect of PTP{varepsilon} on MAPK proteins is most likely widespread. It should be noted, although, that while results presented here implicate all four PTP{varepsilon} forms in inactivation of ERK proteins, it is conceivable that they are not physiologically equivalent. This suggestion is based on the different expression patterns of the PTP{varepsilon} proteins among cells and tissue types and within cells. For example, RPTP{varepsilon} and cyt-PTP{varepsilon} have virtually non-overlapping patterns of expression in mouse tissues (11); furthermore, the stably expressed RPTP{varepsilon} is an integral membrane protein, whereas the highly inducible cyt-PTP{varepsilon} is primarily cytosolic (11). Divergent expression patterns typically result in distinct molecular environments for each PTP{varepsilon} protein, possibly modulating their abilities to affect ERK phosphorylation and activity.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
Acknowledgements
 References
 
Reagents
The following cDNAs were cloned into the pcDNA3 eukaryotic expression vector (Invitrogen, Carlsbad, CA) and used in transfection experiments: mouse RPTP{varepsilon} (50); cyt-PTP{varepsilon} (11); p67 PTP{varepsilon} and p65 PTP{varepsilon} (13); D245A cyt-PTP{varepsilon} (20); and HA-tagged ERK1 and ERK2 cDNAs (51). The following mutants were constructed by PCR or by site-directed mutagenesis: D302A RPTP{varepsilon}; R340M RPTP{varepsilon}; R283M cyt-PTP{varepsilon}; D1 domain of RPTP{varepsilon} (amino acid residues 1–451 of RPTP{varepsilon}, numbering as in Genbank sequence accession no. U35368); D1 domain of cyt-PTP{varepsilon} (residues 1–394, as in sequence U36758); and the D2 domain (D2: residues 417–699 of RPTP{varepsilon}, identical to residues 360–642 of cyt-PTP{varepsilon}). Following sequence verification, all were cloned into pcDNA3. Antibodies used include rabbit polyclonal anti-PTP{varepsilon} (50), monoclonal anti-dual phosphorylated (pThr/pTyr) ERK (37; Sigma, St. Louis, MO), monoclonal anti-monophosphorylated pThr ERK (37; Sigma), polyclonal anti-general (non-phospho-specific) ERK (Sigma), polyclonal anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal anti-HA (clone HA11, BABCO), and anti-FLAG M2 affinity beads (Sigma).

Cell Culture and Reporter Assays
NIH3T3 and 293 cells were grown in DMEM (Invitrogen), supplemented with 10% (v/v) FCS (Invitrogen), 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. Cells were transfected using the LipofectAMINE reagent (Invitrogen) or by the calcium phosphate method (52). For reporter assays, monolayers of non-confluent NIH3T3 cells in six-well plates were transiently transfected with 1 µg PTP{varepsilon} cDNAs cloned in pcDNA3 or with empty pCDNA3 vector. Transfections also included 0.5 µg of a CMV-ß-galactosidase plasmid as an internal control for transfection efficiency, 50 ng of the Gal4-UAS/luciferase plasmid, and 50 ng of either the Elk1/Gal4-DBD or Gal4-DBD plasmids (PathDetect trans-reporter system, Stratagene, La Jolla, CA). In some experiments, the Elk1/Gal4-DBD plasmid was replaced by analogous plasmids for reporting of c-Jun, CHOP, or NFkB activities. SRE reporter activity was monitored using an SRE-luciferase plasmid, kindly provided by Drs. Rony Seger and Batya Cohen. All transfections contained the same total amount of DNA. Twenty-four hours post-transfection, cells were transferred to a medium containing 0.5% serum for 24 h, lysed, and analyzed for luciferase and ß-galactosidase activities. Experiments involving mitogenic stimulation were performed as above, except following 12 h in 0.5% serum, cells were stimulated with 10 ng/ml bFGF or 100 ng/ml TPA for 10 min, after which medium was replaced with fresh low-serum medium for 12 additional hours.

MEF cells were prepared from E14 embryos and were grown in DMEM supplemented with 60 µM ß-mercaptoethanol, and with FCS, glutamine, and antibiotics as above. MEFs were used at passage 4. Mammary tumor cells were isolated from Neu-induced tumors that arose in PTP{varepsilon}-knockout mice (20), following their matings with mice transgenic for activated Neu under the direction of the mouse mammary tumor virus (MMTV) promoter/enhancer (38). Tumor cells were grown in DMEM medium containing 10% iron-supplemented calf serum (Hyclone Laboratories, Logan, UT), glutamine, and antibiotics as above. RPTP{varepsilon} and cyt-PTP{varepsilon} were expressed in these cells by retroviral infection, using the relevant cDNAs cloned in pBABE (53). For TPA stimulation, cells were grown in medium containing 0.5% FCS for 24 h, after which 100 ng/ml TPA were added for the times indicated in Fig. 2B. ERK dual phosphorylation was analyzed by blotting as described below.

Protein Blotting and Immunoprecipitation
Cells were lysed in buffer A [50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 1% NP40], supplemented with 0.5 mM sodium pervanadate, 25 mM ß-glycerophosphate, 1 mM NaF, and protease inhibitors (1 mM AEBSF, 40 µM bestatin, 15 µM E-64, 20 µM leupeptin, 15 µM pepstatin; Sigma). Five to 20 µg total protein were analyzed on 7% or 10% SDS-polyacrylamide gels, followed by transfer to nitrocellulose membranes (Protran, Schleicher & Schuell, Dassel, Germany), and hybridization to antibodies. Complete protein transfer following blotting was verified routinely by noting transfer of pre-stained molecular size marker proteins of the proper size range; absence of lane-to-lane variations in blotting was verified by staining the blotted membranes with Ponceau S (Sigma). When required, band intensity was determined using a scanning densitometer. For immunoprecipitations, 1 mg of total cell protein was incubated with anti-HA antibodies coupled to protein A-Sepharose beads (Amersham Biosciences, Piscataway, NJ) or with M2 anti-FLAG affinity beads for 3–4 h, followed by three extensive washes in buffer A.

ERK Activity Assay
293 cells were transfected with HA-labeled ERK1 or ERK2 cDNAs and with FLAG-tagged cyt-PTP{varepsilon} cDNA. Cells were grown in medium containing 0.1% FCS for 24 h, after which 100 ng/ml TPA were added for 10 min. Cells were then lysed in buffer A supplemented with protease inhibitors as above and with 1 mM NaF, 80 mM ß-glycerophosphate, and 0.5 mM sodium pervanadate. The latter phosphatase inhibitors were present throughout all subsequent precipitations and washes. Following pre-clearing with protein G beads, ERK proteins were precipitated from 1 mg total cell lysate protein using monoclonal anti-HA antibodies. Precipitated material was washed extensively twice in radioimmunoprecipitation assay (RIPA) buffer, followed by 0.5 M KCl in buffer A, 0.5 M LiCl in buffer A, and twice in kinase buffer [50 mM HEPES, 20 mM MgCl2 (pH 7.6)]. Kinase activity of each precipitate was assayed in kinase buffer containing 0.1 mg/ml bovine serum albumin, 50 µM ATP, 0.5 µl (= 5 µCi) of {gamma}-32P-ATP (3000 Ci/mmol, Amersham Biosciences), 5 µg MBP, 3.3 mM EGTA, 25 mM ß-glycerophosphate, 0.5 mM sodium pervanadate, 1 mM NaF, and 0.7 mM DTT. Reactions were incubated in a water bath at 30°C for 30 min and were stopped by addition of SDS-PAGE sample buffer and boiling. Samples were subject to SDS-PAGE and blotting; following exposure to film, amount of precipitated ERK protein in each reaction was determined by probing the blot with anti-HA antibodies.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Drs. Rony Seger and Batya Cohen of the Weizmann Institute, for helpful comments and for reagents, and members of the Elson lab for helpful discussions. A.E. is incumbent of the Adolfo and Evelyn Blum Career Development Chair in Cancer Research at the Weizmann Institute.


   Notes
Top
 Notes
Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 The Israel Science Foundation, founded by The Israel Academy of Sciences and Humanities, and by a research grant from Mr. and Mrs. Lon Morton. Back

2 J. Kraut, H. Toledano-Katchalski, and A. Elson, unpublished observations. Back

Received September 20, 2002; revised February 10, 2003; accepted March 11, 2003.


   References
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 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgements
 References
 

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