©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of a Novel Dual Specific Phosphatase, HVH2, Which Selectively Dephosphorylates the Mitogen-activated Protein Kinase (*)

(Received for publication, November 29, 1994; and in revised form, January 9, 1995)

Kun-Liang Guan (§) Elizabeth Butch

From the Department of Biological Chemistry and the Institute of Gerontology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mitogen-activated protein kinase (MAPK) also known as extracellular signal-regulated kinase (ERK) plays a crucial role in various signal transduction pathways. ERK is activated by its upstream activator, MEK, via threonine and tyrosine phosphorylation. ERK activity in the cell is tightly regulated by phosphorylation and dephosphorylation. Here we report the cloning and characterization of a novel dual specific phosphatase, HVH2, which may function in vivo as a MAP kinase phosphatase. The deduced amino acid sequence of HVH2 shows significant identity to the VH1-related dual specific phosphatase family. In addition, the N-terminal region of HVH2 also displays sequence identity to the cell cycle regulator, Cdc25 phosphatase. Recombinant HVH2 phosphatase exhibited a high substrate specificity toward activated ERK and dephosphorylated both threonine and tyrosine residues of activated ERK1 and ERK2. Immunofluorescence studies with an epitope-tagged HVH2 showed that the enzyme was localized in cell nucleus. Transfection of HVH2 into NIH3T3 cells inhibited the v-src and MEK-induced transcriptional activation of serum-responsive element containing promoter, consistent with the notion that HVH2 promotes the inactivation of MAP kinase. HVH2 mRNA showed an expression pattern distinct from CL100 (human homologue of mouse MKP1) and PAC1, two previously identified MAP kinase phosphatases. Our data suggest a possible role of HVH2 in MAP kinase regulation.


INTRODUCTION

A group of protein serine/threonine kinases, known as mitogen-activated protein kinase (MAPK) (^1)or extracellular signal-regulated kinase (ERK), is acutely stimulated by various extracellular signals, including mitogenic growth factors such as insulin, epidermal growth factor (EGF), and phorbol esters (for review, see (1, 2, 3, 4) ). ERK activation is believed to play an essential role in mitogenic growth factor signal transduction. Evidence indicates that ERK can phosphorylate nuclear transcription factors (5, 6, 7) , protein kinases(8) , cytoskeletal proteins(9) , and proteins involved in regulation of cell growth(10, 11) , suggesting an essential role in cellular signal transduction. ERK must be phosphorylated on both threonine and tyrosine residues to exert its full enzymatic activity(12, 13) . A single protein kinase, MEK, activates ERK2 by phosphorylating threonine 183 and tyrosine 185(14, 15, 16) . Constitutive activation of MEK can cause transformation in NIH3T3 cells and differentiation in PC12 cells(17, 18) , demonstrating the importance of the MAP kinase pathway in signal transduction. In Swiss3T3 cells, MAP kinase reaches maximum activity approximately 5-10 min after EGF stimulation followed by a rapid inactivation(19) . Western blotting demonstrated that the amount of ERK protein did not change after mitogen stimulation, suggesting that ERK is inactivated by post-translational modifications(20) . Two protein phosphatases, MKP1/CL100 and PAC1, have been implicated in dephosphorylation of ERK (21, 22, 23, 24, 25) .

Protein phosphatases are generally divided into Ser/Thr and Tyr phosphatases, based on the phosphoamino acid specificity. Unlike the protein kinases, protein Ser/Thr phosphatases share no sequence identity to the tyrosine-specific phosphatases. However, a growing number of phosphatases have recently been identified to dephosphorylate both Ser/Thr and Tyr residues (for review, see (26) ). The prototype of this dual specific phosphatase is the VH1 phosphatase encoded by the vaccinia virus(27) . Cellular proteins homologous to VH1 phosphatase have been identified. These include the cell cycle regulator Cdc25 (for review, see (28) ), the nitrogen-induced yeast YVH1(29) , and the human VHR(30) . KAP and Cdi1 were two dual specific phosphatases isolated by virtue of their interaction with the cyclin-dependent kinases(31, 32) . These enzymes have been implicated to play a role in cell cycle control.

An immediate-early gene, 3CH134, induced by serum and growth factors in mouse fibroblasts, was isolated and shown to have significant amino acid sequence identity to VH1(33) . The human homolog of 3CH134, CL100, was cloned by Keyse and Emslie (34) as an immediate-early gene in response to oxidative stress and heat shock. Another VH1-related immediate-early gene, PAC1, was isolated from mitogen activated T-cells (35) . CL100, 3CH134, and PAC1 have been demonstrated to specifically dephosphorylate threonine and tyrosine residues of ERKs(21, 22, 23, 24, 25) . A possible function of these mitogen-induced phosphatases may be to down-regulate ERK. Therefore, Sun et al.(23) have suggested the name of MKP1 (map kinase phosphatase) for 3CH134 as an indication of its biological function. Genetic studies in yeast Saccharomyces cerevisiae identified a dual specific phosphatase, MSG5, which inactivated the FUS3 and KSS1 kinases, two MAP kinase homologs in the yeast mating pathway(36) .

In this report, a novel dual specific phosphatase, HVH2 (for human VH1 homologous phosphatase 2), was isolated and characterized. The deduced HVH2 protein shares 62 and 55% sequence identity to CL100 and PAC1, respectively. Purified recombinant HVH2 specifically hydrolyzed the phosphothreonine and phosphotyrosine residues of the activated ERK1 and ERK2. HVH2 was found to be a nuclear protein and capable of blocking activation of a MAP kinase-regulated reporter gene expression.


EXPERIMENTAL PROCEDURES

Cell Culture

NIH3T3 and Swiss3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Hela cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfection was performed using Lipofectin (Life Technologies, Inc.) as described previously (37) . Hep G2 cells were cultured in Eagle's minimal essential medium supplemented with 10% fetal calf serum. For RNA induction, Hep G2 cells were starved in serum free medium for 24 h and then stimulated with one of the following reagents: insulin-like growth factor 1 (160 ng/ml), EGF (160 ng/ml), phorbol 12-myristate 13-acetate (500 nM), H(2)O(2) (100 µM) or 3-isobutyl-1-methylxanthine (IBMX, 500 µM)/forskolin (25 µM). Cells were stimulated for 30 min, 1 and 3 h. RNA isolation and Northern hybridization were performed following standard procedures(38) .

Cloning and Sequence Analysis

HVH2 cDNA was isolated by screening a human placenta cDNA library (Stratagene) using CL100 cDNA (39) as a probe at moderate stringency. Hybridization was performed under conditions with 40% formamide, 5 times SSPE, 5 times Denharnt's solution, 0.1% SDS at 42 °C(38) . The filters were washed in 1 times SSC at 60 °C. Plaques showing weak hybridization signals were isolated and purified by secondary and tertiary screening. Purified clones were converted into phagemid following manufacturer's instructions (Stratagene). Synthetic oligonucleotides were used to obtain the complete nucleotide sequence. Sequence alignment was performed using the Wisconsin Genetic Computation Group software.

Expression and Purification

The full-length HVH2 clone contained an insert of 2.3 kb. The cDNA was digested with NcoI, located at the initiation ATG, and SacI, located within the 3`-noncoding region. The 1.3-kb NcoI-SacI fragment containing the entire coding sequence was subcloned into pGEX-KG (40) digested with NcoI and SacI to produce pGEX-HVH2. This plasmid was then introduced into Escherichia coli strain TG-1 to express GST-HVH2 fusion protein. The GST-HVH2 was induced with 10 µM isopropylthio-beta-galactoside at room temperature for 8-12 h. GST-HVH2 was purified as described (40) and stored at -80 °C in 10% glycerol. Rat ERK2 was expressed with a histidine tag and purified by NAT-Ni affinity chromatography (Qiagen)(41) . ERK1, GST-MEK2, and GST-CL100 were expressed and purified as described(25) . Protein concentrations were determined by densitometric scanning of SDS-PAGE using bovine serum albumin as a standard.

Phosphatase Assay

The p-nitrophenyl phosphate (pNPP) hydrolysis activity of GST-HVH2 was assayed in 200 µl of buffer A (50 mM HEPES, pH 7.5, 0.1% 2-mercaptoethanol) containing 20 mM pNPP at 37 °C for 30 min. One unit of phosphatase activity was defined as the amount of enzyme required to hydrolyze 1 µmol of pNPP at 37 °C in 1 min. Purified GST-HVH2 had a specific activity of 0.25 units/mg compared to 0.102 units/mg of GST-CL100(25) .

Activated ERK1 and ERK2 were used as substrates for GST-HVH2. ERK (21.6 µg) was activated by GST-MEK2 (2.75 µg) in buffer B (18 mM HEPES, pH 7.5, 50 µM ATP, 10 mM magnesium acetate) at 30 °C for 20 min. The GST-MEK2 was depleted by absorption to glutathione-agarose (Sigma). Under these conditions, ERK was usually activated by more than 100-fold. The activated ERK was then used for HVH2 inactivation assay in 10 µl of buffer A at 30 °C for 10 min. Activity of HVH2-treated ERK was directly determined by the MBP kinase assay.

P-Labeled ERK1, ERK2, and GST-MEK2 were prepared by autophosphorylation in the presence of [-P]ATP in buffer B. Casein was phosphorylated by either the catalytic subunit of protein kinase A or P43 kinase as described(27) . ERK1 and ERK2 were also phosphorylated by GST-MEK2 in the presence of [-P]ATP. Dephosphorylation of P-labeled proteins was performed in 20 µl of buffer A at 30 °C for 10 min using 3.8 microunits of GST-HVH2 or 1,000 microunits of PTP1. Samples were analyzed by SDS-PAGE and visualized by autoradiography. Phosphoamino acid analysis was performed as described (42) .

The coding sequence of human ERK1 was subcloned into plasmid pALTER-1 (Promega) for site-directed mutagenesis. The catalytic essential lysine residue 71 of human ERK1 was mutated to arginine by oligonucleotide-directed mutagenesis (Promega) to produce a kinase-deficient ERK1*. The threonine 202 and tyrosine 204 were independently mutated to alanine and phenylalanine, respectively, in the kinase-deficient ERK1*. Mutations were confirmed by DNA sequencing and subcloned into pGEX-2T (43) for expression and purification. ERK1*, ERK1*T202A, and ERK1*Y204F were phosphorylated by GST-MEK2 as described above. Dephosphorylation of these mutant ERKs was performed as described for wild type ERK1. Dephosphorylation reactions were terminated by addition of SDS sample treatment buffer and resolved by SDS-PAGE. The samples were then transferred to nitrocellulose and quantitated by phosphoimaging or scintillation counting.

Activated ERK1 or ERK2 (0.4 µg) was inactivated by 5.9 microunits of GST-HVH2 in 30 µl of buffer A at 30 °C for 10 min. Sodium vanadate was added to inhibit HVH2. Half of the sample was directly used for MBP kinase assays. The other half was subjected to reactivation by 0.32 µg of GST-MEK2 in 20 µl of buffer B.

Kinase Assay

ERK activity was determined as described(44) . In order to visualize the phosphorylated MBP, reactions were also directly analyzed by 15% SDS-PAGE and followed by autoradiography.

Immunofluorescence

The myc epitope (45) was incorporated into the C terminus of HVH2 by polymerase chain reaction. The epitope-tagged HVH2 cDNA was subcloned into pCMV4 vector (46) to produce pCMV-HVH2 myc. This plasmid was transfected into NIH3T3 or Hela cells by the Lipofectin method. Immunofluorescence of transfected cells with anti-myc antibody was performed following published methods(37) . Synthetic peptide, EQKLISEEDL, corresponding to the myc epitope was used for competition.

Luciferase Assay

The HVH2 cDNA was subcloned into pCMV4 (46) to construct pCMV-HVH2. Human MEK1 cDNA (47) was subcloned into the BamHI site of pCMV4 to produce pCMV-MEK. pJH2 (containing v-src) was a generous gift of Dr. Taparowsky (Purdue University). Plasmid SRE-luc containing the luciferase under the control of minimum promoter of thymidine kinase and serum-responsive element of c-fos was a generous gift of Dr. Pessin (University of Iowa)(48) . pCMV-luc containing the luciferase under the control of the CMV promoter was a gift of Dr. Cui (University of Michigan). Plasmid SRE-luc (0.2 µg) was transfected into NIH3T3 cells (60-mm plates) together with a different combination of plasmid pJH2 (1.0 µg) or pCMV-MEK (1.0 µg) and varying amounts of pCMV-HVH2 or pCMV-HVH2 myc in the presence of pCMV-SEAP (0.4 µg, for expressing alkaline phosphatase as an internal control)(49) . Two days after transfection, cells were washed twice with ice-cold phosphate-buffered saline and harvested in 350 µl of cell lysis buffer(50) . Cell lysates (50-100 µl) were directly used for luciferase assays following standard protocol(50) . Cultured media from transfected cells were heated at 65 °C for 5 min and directly used for alkaline phosphatase assays(49) .


RESULTS

Cloning of HVH2 cDNA

To test the possibility that new ERK-specific phosphatases may exist, low stringency hybridization was performed to isolate new members of the dual specific phosphatases. Using CL100 as a probe(39) , 10 moderate hybridizing clones were isolated from a human placenta cDNA library. Restriction digestion with EcoRI followed by Southern hybridization revealed that three of the 10 clones contained an internal EcoRI site which is absent in CL100 cDNA. DNA sequencing analysis demonstrated that all three cDNAs encoded the same protein. The longest clone of 2.3 kb, designated as HVH2, encoded an open reading frame of 394 amino acid residues (Fig. 1).


Figure 1: Sequence alignment of HVH2 with CL100 and PAC1. The deduced amino acid sequence of HVH2 was aligned with CL100 (34) and PAC1 (35) by the BESTFIT program of Wisconsin Genetics Computation Group. Conserved residues were highlighted. Gaps (as spaces) were introduced for the maximum alignment. The catalytically essential cysteine in all PTPs and VH1-related phosphatases is indicated by an asterisk (*).



The deduced amino acid sequence of HVH2 displays 62 and 55% overall sequence identity to the complete sequences of CL100 and PAC1, respectively (Fig. 1)(34, 35) . The highest sequence conservation occurs in the C-terminal half of the molecules, where the catalytically essential cysteine (Cys for HVH2) found in all protein tyrosine phosphatases is located. This C-terminal domain also shares significant sequence identity to the active site region of dual specific phosphatases such as VH1, YVH1, KAP, and Cdi1. In contrast, the N-terminal 181 residues of HVH2 share only 33 and 25% sequence identity to the corresponding regions of CL100 and PAC1, respectively (Fig. 1). Interestingly, the N-terminal region of HVH2 showed significant sequence identity with the cell cycle regulator Cdc25 phosphatases. This sequence similarity has been observed in CL100 and MKP1(39, 51) . It is worth noting that the catalytically essential cysteine in Cdc25 is absent in the N-terminal regions of both CL100 and HVH2, suggesting that the catalytic domain of HVH2 phosphatase resides in the C-terminal and not the N-terminal region of the polypeptide.

Dephosphorylation and Inactivation of ERKs

To determine the substrate specificity of HVH2, several phosphoproteins were tested, including protein kinase A-phosphorylated casein (on serine), v-abl-phosphorylated casein (on tyrosine), autophosphorylated ERK1 (on tyrosine and serine), autophosphorylated GST-MEK2 (on serine and threonine), MBP (on threonine), and activated ERK1 (on serine, threonine, and tyrosine). Under the same assay conditions, GST-HVH2 dephosphorylated the activated ERK1 while no significant dephosphorylation was observed with all the other phosphoproteins tested (data not shown). These results indicated a high substrate specificity of HVH2 toward ERK. The efficiency of GST-HVH2 to inactivate ERK1 and ERK2 were determined and compared with that of PTP1 (52) . GST-HVH2 effectively inactivated both ERK1 and ERK2 while PTP1 did not (Fig. 2A). GST-HVH2 also displayed a specific activity three times higher than GST-CL100 in an ERK1 inactivation assay. As expected, vanadate (2 mM) completely inhibited HVH2 activity while okadaic acid (5 µm) had no effect (not shown).


Figure 2: A, inactivation of ERK1 and ERK2 by GST-HVH2. The activated ERK1 was inactivated by GST-HVH2 (closed circles) or PTP1 (open circles). The activated ERK2 was inactivated by GST-HVH2 (open triangles). ERK activity was determined by the MBP kinase assay. B, tyrosine and threonine phosphatase activity of HVH2. ERK1 was phosphorylated by GST-MEK2 before dephosphorylation (lane 1), or dephosphorylated by GST-HVH2 (3.8 µU, lane 2) or PTP1 (1 milliunit, lane 3). Positions of free phosphate, phosphoserine, phosphothreonine, phosphotyrosine, and origin are denoted by P, pS, pT, pY, and O, respectively.



If HVH2 dephosphorylated ERK on the same threonine and tyrosine residues which were recognized by MEK, the HVH2-dependent ERK inactivation should be reversible. To demonstrate this point, the HVH2-inactivated ERK2 was subjected to reactivation by MEK2 in the presence of 2 mM sodium vanadate, which inhibited the HVH2 activity. We observed that the HVH2-inactivated ERK2 could be quantitatively reactivated by MEK (not shown), indicating that HVH2 and MEK recognized the same residues on ERK.

Dual Specific Phosphatase Activity

The MEK2-activated ERKs contained phosphoserine, threonine, and tyrosine (Fig. 2B). Phosphoamino acid analysis revealed that GST-HVH2 dephosphorylated the threonine and tyrosine residues, which were phosphorylated by MEK, but not the autophosphorylated serine residue of the activated ERK1, while PTP1 dephosphorylated tyrosine only (Fig. 2B). GST-HVH2 could not completely dephosphorylate ERK1 because the autophosphorylated serine in ERK1 was resistant to the HVH2. Similar observations were obtained with the activated ERK2 (not shown).

An intriguing difference between the activated ERK and other phosphoproteins was that the activated ERK contained adjacent phosphothreonine and phosphotyrosine. It is possible that the two neighboring phosphorylated residues serve as a recognition determinant for HVH2. To test this hypothesis, ERK1 phosphorylated on either threonine (ERK1*Y204F) or tyrosine alone (ERK1*T202A) was utilized as a substrate for HVH2. Threonine 202 and tyrosine 204 in ERK1 (53) correspond to threonine 183 and tyrosine 185 in ERK2 which are the activation-phosphorylation sites by MEK(14, 15, 16) . ERK1*, a kinase-deficient mutant, was phosphorylated on both threonine and tyrosine by MEK2 (Fig. 3B). ERK1*T202A, having threonine 202 substituted by an alanine, was phosphorylated only on tyrosine while ERK1*Y204F, having tyrosine 204 substituted by a phenylalanine, was phosphorylated only on threonine (Fig. 3B). GST-HVH2 dephosphorylated all three ERK1* mutants (Fig. 3A), suggesting that double phosphorylations of adjacent threonine and tyrosine were not a prerequisite for HVH2 recognition. However, HVH2 dephosphorylated ERK1* and ERK1*T202A more efficiently than ERK1*Y204F (Fig. 3A), indicating that HVH2 preferred phosphotyrosine over phosphothreonine. Interestingly, MEK also phosphorylated tyrosine residues more efficiently than threonine residues of ERK(54) . (^2)


Figure 3: A, dephosphorylation of ERK1* mutants. The MEK2-phosphorylated ERK1* mutants were dephosphorylated by HVH2 for various times (xaxis). Equal amounts (3,000 counts/min for each assay) of phosphorylated substrates were used for ERK1* (closed circles), ERK1*T202A (open triangles), and ERK1*Y204F (open circles). B, phosphoamino acid analysis of ERK1*, ERK1*T202A, and ERK1*Y204F. Kinase-deficient ERK1* mutants were phosphorylated by GST-MEK2 and subjected to phosphoamino acid analysis.



Different Expression Patterns of HVH2 and CL100

Biochemical characterizations of CL100 and HVH2 indicated that they shared many common properties(21, 22, 23, 24, 25) . A multiple human tissue mRNA blot was probed with CL100 or HVH2 under high stringency to determine the tissue distribution of these two enzymes. Two mRNA species (2.5 and 6 kb) were detected by the HVH2 probe (Fig. 4B). The size of the 2.5-kb mRNA was consistent with the cloned cDNA (2.3 kb). At present, we have no evidence to distinguish whether the 6-kb mRNA represents an alternative splicing form of HVH2 or a closely related gene transcript. Interestingly, CL100 (33, 34, 39) and HVH2 showed a very different tissue distribution pattern (Fig. 4, A and B). We also noticed that HVH2 mRNA was expressed at a level significantly lower than CL100. HVH2 mRNA was not detectable in Swiss3T3 cells. The HVH2 mRNA was induced by phorbol ester (approximately 6-fold) and IBMX-forskolin (4-fold) in Hep G2 cells, which also showed a low basal HVH2 mRNA. No significant induction was observed by either EGF or H(2)O(2), which are known to induce the CL100 mRNA(33, 34, 39) . These observations suggest that HVH2 and CL100 may be important for ERK inactivation in response to different stimuli in distinct cell types.


Figure 4: A, tissue distribution of CL100 mRNA. A multiple human tissue RNA blot was probed with CL100 cDNA for comparison to HVH2. Lanes: 1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; and 8, pancreas. B, tissue distribution of HVH2 mRNA. The RNA blot is the same as stated in panel A and was probed with HVH2 cDNA. Lanes correspond to those in panel A. C, induction of HVH2 mRNA by phorbol 12-myristate 13-acetate but not by EGF. The arrow indicates the HVH2 mRNA (2.5 kb). Cyclophilin mRNA (the lower band) was detected as an internal control. Hep G2 cells were treated for 1 h by insulin-like growth factor (lane 1); EGF (lane 2); IBMX-forskolin (lane 3); phorbol 12-myristate 13-acetate (lane 4); hydrogen peroxide (lane 5); and control (lane 6). Hybridization signals were quantitated by phosphoimaging and normalized.



Nuclear Localization

Analysis of the HVH2 sequence showed two putative nuclear localization signal sequences(55) . We determined the subcellular localization of HVH2 by epitope tagging and immunofluorescence which have been successfully used to study subcellular localization of many proteins. The myc-tagged HVH2 was exclusively found in the nuclei of cells transfected with pCMV-HVH2 myc (Fig. 5). Similar results were obtained in pCMV-HVH2 myc-transfected NIH3T3 cells (not shown). No nuclear staining could be detected if the antibody was preincubated with competing peptide or cells transfected with pCMV-HVH2 (not shown).


Figure 5: Nuclear localization of HVH2. Myc-tagged HVH2 was transfected into Hela cells and followed by immunofluorescence using a monoclonal anti-myc antibody. Panels are labeled as follows: A, immunofluorescence; B, nuclear staining by 4,6-diamidino-2-phenylindole; and C, phase contrast.



Expression of HVH2 Blocks Transcriptional Activation of a MAP Kinase-regulated Reporter Gene

Transcription activation of the SRE-containing promoter requires serum response factor and p62 (ternary complex factor). The p62 has been demonstrated to be a physiological substrate of MAP kinase(5) . Phosphorylation by MAP kinase activated the transcription activity of p62. The PAC1 phosphatase has been shown to inhibit transcription of a SRE-containing promoter(24) . To test the effect of HVH2 expression on SRE promoter activity, we measured the luciferase activity driven by a SRE (from c-fos promoter)-containing promoter (48) in a transient transfection. Luciferase expression was greatly enhanced by cotransfection of v-src (approximately 80-fold) in NIH3T3 cells. This v-src-induced luciferase activity was quantitatively suppressed by cotransfection of pCMV-HVH2 or pCMV-HVH2 myc in a dose-dependent manner (Fig. 6A) while the luciferase activity was not affected by cotransfection with control pCMV vector. In fact, pCMV-HVH2 and pCMV-HVH2 myc showed identical effects on luciferase expression, suggesting that the HVH2 function was not altered by the myc tagging. Cotransfection of HVH2 had little effect on a control, a CMV promoter-driven luciferase expression. Cotransfection of pCMV-MEK with pSRE-luc increased luciferase expression (approximately 8-fold) although the magnitude was much less than v-src. The stimulating effect of MEK on SRE-controlled promoter activity was also inhibited by HVH2 cotransfection (Fig. 6B). Our data suggest that overexpression of HVH2 promotes the inactivation of MAP kinase, thus leading to inhibition of SRE-dependent transcription.


Figure 6: Inhibition of SRE dependent promoter activity by co-transfection of HVH2. A, v-src-induced transcriptional activation of a SRE-containing promoter was blocked by HVH2. The luciferase reporter plasmid was cotransfected with v-src and various amounts of pCMV-HVH2 myc. Luciferase activity was measured and normalized (yaxis). Results were representative of three independent experiments. B, inhibition of MEK-stimulated SRE promoter activity by HVH2. Cotransfection of HVH2 blocked MEK-induced luciferase expression. Luciferase activity in the absence (open bars) or presence (hatched bars) of 1 µg of pCMV-HVH2 myc. v-src (bars 1 and 2) and MEK (bars 3 and 4) used in the transfection are indicated.




DISCUSSION

Accumulating evidence supports that the mitogen-induced dual specific phosphatases play an important role in MAP kinase modulation (56) . The mouse MKP1 and human CL100 may be responsible for down-regulation of MAP kinase after growth factor stimulation(21, 22, 23, 25) , while the lymphocyte-specific PAC1 phosphatase may function in down-regulating MAP kinase in T-cells and B-cells(24, 35) . It is likely that different activators (MEKs) and inactivators (phosphatases) are required for the complex regulation of ERK. This is plausible for a number of reasons. First, ERK is known to be activated by numerous extracellular stimuli in a wide variety of cells. Second, ERK is a growing multi-enzyme family. One such example is the recently identified c-jun N-terminal kinase (JNK) which is related to ERK and activated by Thr/Tyr phosphorylation(57) . Different ERKs may be regulated by different activators (kinases) as well as inactivators (phosphatases). Furthermore, numerous different signal transduction pathways may use kinase cascades similar to the ERK pathway. For example, at least three distinct ERK-related signal transduction pathways have been identified in yeast, including the mating pheromone response, osmolarity regulation, and cell wall construction(58) . Existence of different MEKs and ERK-specific phosphatases provides a means by which the MAP kinase pathway could be differentially regulated.

In this report, we described the isolation of a novel dual specific phosphatase, HVH2, which showed significant sequence identity to CL100/MKP1 and PAC1 (Fig. 1). Several lines of evidence support that HVH2 is an ERK-specific phosphatase. First, HVH2 shows a high substrate selectivity toward MAP kinase and did not dephosphorylate any of the phosphoproteins tested except for the activated ERK1 and ERK2. The high substrate selectivity of HVH2 is analogous to CL100/MKP1 and PAC1 (21, 22, 23, 24, 25) . Second, HVH2 selectively dephosphorylated the threonine and tyrosine residues which were phosphorylated by MEK but not the autophosphorylated serine residue of ERK. Third, the nuclear localization is consistent with a role of HVH2 in MAP kinase regulation since the activated MAP kinase is known to be translocated into the nucleus(59) . Interestingly, PAC1 was also found in the cell nucleus (35) . Furthermore, ectopic expression of HVH2 blocked the activity of a SRE-containing promoter, consistent with the inactivation of MAP kinase. Caution should be taken to interpret the HVH2 overexpression data because nonspecific effects could occur. Nevertheless, the above observations strongly support that HVH2 phosphatase is likely to be an important component of the ERK/MEK signal transduction pathways, although the precise role of HVH2 in ERK inactivation remains to be elucidated.

PAC1 was maximally expressed in hematopoietic tissues and induced by T-cell activation(35) . MKP1/CL100 was induced by growth factors such as EGF, serum, and oxidative stress(22, 34, 39) . The MKP1/CL100 mRNA was highest in lung and also high in placenta and pancreas (Fig. 4A)(22, 39) . In contrast, HVH2 mRNA was induced by phorbol 12-myristate 13-acetate but not by EGF (Fig. 4B) and showed a tissue distribution pattern different from CL100. The HVH2 mRNA was highest in placenta followed by pancreas but was virtually undetectable in lung (Fig. 4B). A possible role of HVH2 in pancreas is consistent with the observation that cholecystokinin transiently activated MAP kinases in rat pancreatic acini(60) . Collectively, data from this report and previous studies demonstrate that different members of the ERK phosphatases, which are highly specific toward ERK, are expressed in different cells and tissues. The HVH2 described in this report represents one candidate of such phosphatases. These phosphatases are differentially regulated when cells exposed to wide variety of extracellular stimuli, providing additional mechanisms of MAP kinase modulation. Additional work on the regulation of these dual specific phosphatases will generate vital information to further elucidate signal transduction pathways utilizing MAP kinase.


FOOTNOTES

*
This work was supported in part by Grant BE-171 from the American Cancer Society, Public Health Service Grant GM51586 from the National Institute of General Medical Science, University of Michigan Geriatrics Center Grant 5P30A609908, a grant from the University of Michigan Gastrointestinal Peptide Research Center (to K. L. G.), and National Institute of Aging Training Grant 5T32AG00114 (to E. B.). Computer analysis of sequence was supported by General Clinic Research Center Grant M01RR00042. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 313-763-3030; Fax: 313-763-4581.

(^1)
The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK or ERK kinase; HVH2, human VH1 homologous phosphatase 2; MKP1, MAPK phosphatase 1; GST, glutathione S-transferase; SRE, serum-response element; MBP, myelin basic protein; EGF, epidermal growth factor; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; IBMX, 3-isobutyl-1-methylxanthine.

(^2)
E. Butch and K. L. Guan, unpublished observation.


ACKNOWLEDGEMENTS

We thank Drs. D. Brautigan and J. Chen (Brown University) for PP2A, Dr. J. Corbin (Vanderbilt University) for protein kinase A, Drs. S. L. Pelech (University of British Columbia), M. H. Cobb (University of Texas, Southwestern Medical Center), J. E. Pessin (University of Iowa), J. Cui (University of Michigan), and E. Taparowsky (Purdue University) for plasmids, Drs. S. Kwak and K. Martell for CL100 Northern blot and RNA preparation, Drs. J. E. Dixon and L. Mathews for advice and critical reading of the manuscript, X. Wu for technical assistance, and Dr. Y. Wang for immunofluorescence.


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