©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Mitogen-activated Protein Kinase Phosphatase
STRUCTURE, EXPRESSION, AND REGULATION (*)

Anita Misra-Press (1), Caroline S. Rim (1), Hong Yao (1) (2), Mark S. Roberson (4), Philip J. S. Stork (1) (3)(§)

From the (1)Vollum Institute, the (2)Department of Microbiology and Immunology, the (3)Department of Pathology, and the (4)Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland, Oregon 97201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mitogen-activated protein (MAP) kinase lies at the convergence of various extracellular ligand-mediated signaling pathways. It is activated by the dual-specificity kinase, MAP kinase kinase or MEK. MAP kinase inactivation is mediated by dephosphorylation via specific MAP kinase phosphatases (MKPs). One MKP (MKP-1 (also known as 3CH134, Erp, or CL100)) has been reported to be expressed in a wide range of tissues and cells. We report the identification of a second widely expressed MKP, termed MKP-2, isolated from PC12 cells. MKP-2 showed significant homology with MKP-1 (58.8% at the amino acid level) and, like MKP-1, displayed vanadate-sensitive phosphatase activity against MAP kinase in vitro. Overexpression of MKP-2 in vivo inhibited MAP kinase-dependent gene transcription in PC12 cells. MKP-2 differed from MKP-1 in its tissue distribution and in its extent of induction by growth factors and agents that induce cellular stress, suggesting that these MKPs may have distinct physiological functions.


INTRODUCTION

Mitogen-activated protein kinases (MAP()kinases) mediate multiple cellular pathways regulating growth (1) and differentiation(2, 3) . In neuronal cells, MAP kinase activity mediates the actions of growth factors like EGF that stimulate cellular proliferation as well as factors like NGF that maintain neuronal survival and differentiation(4, 5, 6) . Such ligand-activated signal transduction pathways involve activation of receptor-tyrosine kinases, which initiates a series of phosphorylation events that activate a cascade of serine/threonine kinases converging on the MAP kinase (also called extracellular signal-regulated kinase (ERK)) isoforms, ERK1 and ERK2 (7-9).

Activation of MAP kinase involves specific phosphorylations on threonine and tyrosine residues within the Thr-Glu-Tyr motif (10) by MAP kinase kinase (MAP kinase and ERK kinase or MEK)(2, 11) . Phosphorylation of both these residues is required for MAP kinase activation(11, 12) . It has been suggested that the inactivation of MAP kinase is a critical event that regulates the physiological response to MAP kinase activation(13) . This inactivation is mediated, in part, by dephosphorylation of MAP kinases by dual specificity phosphatases called MAP kinase phosphatases (MKPs) that dephosphorylate both the threonine and tyrosine residues phosphorylated by MEK (13-16). The activation of MAP kinase appears to be tightly regulated through the coordinate action of MEK and MKPs. By regulating the extent of MAP kinase activation, these MKPs may dictate the choice of differentiation or proliferation within a developing cell(17) .

The prototype dual-specificity phosphatase, VH1, was identified in vaccinia and showed similarity to cdc25, a protein that controls cell entry into mitosis(18) . VH1 homologues from human (PAC-1, CL100, and most recently B23), mouse (MKP-1 (3CH134 or Erp)), and yeast (Yop51, MSG5) have also been isolated(19, 20, 21, 22, 23, 24) . All are dual-specificity phosphatases that specifically dephosphorylate MAP kinase in vitro(25) and in vivo(13, 15, 26) . MKP-1 (also called 3CH134 or Erp) was discovered as an immediate early gene whose rapid transcription and subsequent translation are suggested to provide a feedback loop to terminate growth factor signals(13, 19, 26) . Overexpression of mouse MKP-1 was shown to dramatically inhibit fibroblast proliferation, suggesting that the inactivation of MAP kinase in vivo by MKP-1 has a profound negative effect on cellular proliferation(25, 26) .

MAP kinase activation by growth factors has been extensively studied in PC12 cells(27) . PC12 cells originate from a rat pheochromocytoma and retain many features of neural crest-derived cells, most notably the ability to undergo neuronal differentiation upon stimulation by NGF (28). Transfection with activated forms of the oncogenes ras, raf-1, and src into PC12 cells is sufficient for differentiation in the absence of NGF stimulation(6, 8, 29) . As each of these genes has been shown to converge on MAP kinase activation, this implies that components of the MAP kinase cascade are required for neuronal differentiation. More recently it has been shown that the activation of MAP kinase kinase is required and sufficient for PC12 cell differentiation(3) . Despite our understanding of MAP kinase activation in neuronal differentiation, we know relatively little about MAP kinase inactivation. Initial studies presented here characterize a family of MKPs expressed in PC12 cells.

We report the cloning of a novel MKP, MKP-2, that is highly expressed in PC12 cells. MKP-2 mRNA is expressed at moderate levels in nearly all tissues and cells and encodes a phosphatase that inactivates MAP kinase in vitro and MAP kinase-dependent gene transcription in vivo. Its distribution in the central nervous system and regulation in the hippocampus suggest a potential role for this phosphatase in neuronal signaling pathways.


EXPERIMENTAL PROCEDURES

Materials

Restriction and modification enzymes were purchased from New England Biolabs (Beverly, MA), Boehringer Mannheim, and Promega. Superscript reverse transcriptase was from Life Technologies, Inc.; Taq DNA polymerase from Perkin-Elmer and Sequenase from U. S. Biochemical Corp. All enzymes were used according to the instructions from the manufacturer. [-P]dATP (3000 Ci/mmol), S-dATP, S-UTP (1500 Ci/mmol), [-P]ATP (800 Ci/mmol), [S]cysteine (1075 Ci/mmol), and [-P]UTP (800 Ci/mmol at 40 mCi/ml) were purchased from Dupont NEN. Oligonucleotides were synthesized by a core facility at Oregon Health Sciences University. Antisera to MKP-1 was purchased from Santa Cruz Biotechnology Inc. An anitibody to phosphotyrosine (clone 4G10) was kindly provided by Brian Druker (OHSU, Portland, OR)(30) .

Reverse Transcriptase PCR Amplification

One µg of total RNA from PC12 cells was used to generate first strand cDNA after an initial annealing reaction to 0.1 µg of random hexamers at 70 °C for 10 min. Following equilibration to ambient temperatures, a buffer containing 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl, 10 mM dithiothreitol, 500 µM of each of four dNTPs, and 200 units/µg of Superscript reverse transcriptase was added, and the mix was incubated at 37 °C for 1 h. The reaction was terminated by placing the tubes on ice, and the cDNA was recovered by ethanol precipitation. The pellet was washed with 70% ethanol and resuspended in 100 µl of 5 mM Tris, 0.5 mM EDTA mix. Five µl of this cDNA was used as a template for PCR amplification. Initially, two degenerate oligonucleotides were synthesized that generated a 204-bp cDNA fragment. The 5` primer corresponded to the conserved WFNEAI sequence present in MKP-1 (26) and PAC-1 (23) (5`-TGG-TT(T/C)-AA(T/C)-GA(G/A)-GC(G/A/T/C)-AT-3`), while the 3` primer corresponded to the conserved NFSFMG sequence present in MKP-1 (26) and PAC-1 (23) (5`-C-CAT-(A/G)AA-(G/A/T/C)(G/C)(A/T)-(A/G)AA-(A/G)TT-3`) (see Fig. 2). Two additional oligonucleotides were made to confirm the novelty of MKP-2. The 5` primer was a degenerate primer corresponding to the conserved YDQGGP sequence (5`-TA(T/C)-GA(T/C)-CA(A/G)-GG(G/A/T/C)-GG(G/A/T/C)-CC-3`), while the 3` primer was specific for MKP-2 (5`-ATGAAGAAACGGGTGCGG-3`) corresponding to MKKRVR sequence (Fig. 1B and 2). This set of primers generated a 336-bp cDNA fragment corresponding to nucleotides 628-963 (Fig. 1B). The PCR reaction consisted of 50 mM KCl, 1.5 mM MgCl, 0.2 mM dNTPs, 15 mM Tris-HCl, pH 8.4, and 0.5 µg of each primer pair. PCR was allowed to proceed for 30 cycles. Each cycle consisted of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C in a thermocycler (Perkin-Elmer Cetus). The PCR products were purified and subcloned into pBluescript (SK-) (Stratagene) using restriction enzymes engineered at the ends of each of the primers.


Figure 2: Amino acid homology between MKP-2, MKP-1, PAC-1, and B23. The amino acid sequences of rat MKP-2, mouse MKP-1, mouse PAC-1, and human B23 are aligned with each other, and the areas of homology are shown as shaded boxes. The catalytic domain is boxed. Dots represent spaces put in for alignment. The dark gray boxes represent the two CH2 domains present in all MKPs. Arrows correspond to the primers used in the reverse transcriptase PCR screening strategy for cloning MKPs.




Figure 1: Restriction map and sequence of MKP-2 cDNA. A, the 4.8-kb MKP-2 cDNA was digested with various restriction enzymes, and a schematic representation of some of these sites is shown. The following abbreviations are used: RI, EcoRI; Pst, PstI; A, ApaI; H, HindIII; RV, EcoRV; Sma, SmaI; Bam, BamHI; 5` UT, 5`-untranslated region; 3` UT, 3`-untranslated region; ATG, translation start site; TAG, translation stop site. The coding region of MKP-2 is shown as a rectangularbox with two different domains highlighted. The stippled box represents CH2 domains (42) while the hatched box represents the catalytic domain. B, nucleotide sequence and the encoded amino acid sequence of rat MKP-2 cDNA is shown. The translation start site is denoted as +1. The consensus catalytic site, the AU-sequence motifs in the 3` untranslated region, and the putative polyadenylation signal are underlined. The 5`- and 3`-untranslated regions are depicted in lower case letters.



Screening of the PC12 cDNA Library and Isolation of the Full-length Clone

The 336-bp PCR fragment generated by using the specific MKP-2 primer (described above) was labeled by random primed synthesis and used to screen a PC12 oligo(dT)-primed cDNA library in gt10 with 5 10 individual recombinants that had been size-selected prior to ligation for clones greater than 2 kb. The library was plated onto 20 LB plates and allowed to grow at 42 °C to a concentration of 50,000 recombinants/plate. The plaques were then transferred onto nitrocellulose filters in duplicate. The filters were soaked in prehybridization/hybridization buffer (6 SSC, 5 Denhardt's solution, 1% SDS, 0.01 M EDTA, 50% formamide, 100 µg/ml denatured salmon sperm DNA) at 42 °C for 1-2 h with gentle agitation. Random primed probe was made as described in the Boehringer Mannheim kit. One to two million cpm/ml of boiled MKP-2 specific probe was added directly to the prehybridization/hybridization mix, and hybridization was allowed to proceed at 42 °C for 24 h. The filters were washed in 2 SSC and 1% SDS for 2 h at 65 °C with frequent changes in the wash solution. The final wash was in 1 SSC and 1% SDS after which the filters were air dried and put on film. After the tertiary screen, three positive plaques were obtained. Phage DNA was isolated by standard methods and digested with EcoRI to release the insert. The inserts obtained were cloned into pBluescript (SK-) and subjected to restriction enzyme mapping and sequencing.

Sequencing

MKP-2 cDNA inserts obtained were sequenced on both strands by the method of Sanger (31) using Sequenase reagents (U.S. Biochemical Corp.) according to the protocol suggested by the manufacturer. Multiple internal primers were made to allow sequencing of the complete 800-bp insert. The 4-kb insert was also sequenced using multiple internal primers on both strands through the termination codon and partially into the 3`-untranslated region. Sequences proximal to the polyadenylation signal were also obtained as shown (Fig. 1B).

Cell Culture and Drug Treatments

PC12-GR5 cells (courtesy of Rae Nishi, OHSU, Portland, OR) were grown at 5% CO in Dulbecco's modified Eagle's medium containing 5% fetal calf serum, 10% horse serum, and L-glutamine. Prior to drug treatments, the cells were serum-starved for 24 h with Dulbecco's modified Eagle's medium containing no serum and treated with either 100 ng/ml NGF or 20 ng/ml EGF for the indicated times.

RNA Isolation

Total cellular RNA was isolated using RNAzol B (Biotecx Lab, Inc.) according to the manufacturer's protocol. Briefly, cells were grown to 30-50% confluency in a 100-mm plate, rinsed with cold phosphate-buffered saline, and scraped into 1 ml of RNAzol B. After vortexing, 0.1 ml of chloroform was added and incubated on ice for 15 min. The suspension was centrifuged, and the RNA was precipitated from the upper aqueous layer with an equal volume of isopropyl alcohol. After pelleting, the RNA was resuspended in water, quantitated at A, and used directly for reverse transcriptase PCR or Northern analysis.

Northern Blot Analysis

Ten µg of total RNA was electrophoresed through a 1.2% agarose formaldehyde gel and transferred onto Magna NT filter (MSI, Westboro, MA) using standard methodology in 6 SSC. Filters were prehybridized in 3 ml of hybridization buffer (5% SDS, 400 mM NaPO pH 7.2, 1 mM EDTA, 1 mg/ml bovine serum albumin, 50% formamide) at 65 °C for 4 h in a rotating hybridization oven. 2-5 10 cpm/ml of the antisense riboprobe was then directly added to the hybridization buffer, and hybridization was allowed to proceed for 24 h. The next day, filters were initially washed in 1 SSC at room temperature for 15 min and then washed in (0.05 SSC, 0.1% SDS, 5 mM EDTA, pH 8) at 70 °C for 3-4 h. Filters were autoradiographed at -70 °C on Kodac XAR-5 film using Dupont intensifying screens. Quantitations were performed using a Molecular Dynamics PhosphorImager 445 SF, and all signals were normalized to the 18 S and cyclophilin signals respectively.

Riboprobe Synthesis

The 336-bp cDNA fragment generated by using the specific MKP-2 primer (described under ``Reverse Transcriptase PCR Amplification'') was subcloned in pBluescript (SK-) and was used to synthesize antisense riboprobes by linearizing the plasmid with SalI that was engineered into the 5` primer. Full-length MKP-1 cDNA (kindly provided by Nicholas Tonks, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) was subcloned into pBluescript (SK-) and then linearized with BamHI to generate a 1.9-kb MKP-1 riboprobe. Rat cyclophilin (pSP65 1B15) and 18 S ribosomal RNA (18SpSP65) plasmids (kindly provided by James Douglass, Vollum Institute, Portland, OR) were linearized using PstI and HindIII respectively to generate linear antisense riboprobes. Antisense riboprobes were synthesized as described previously(32) . Briefly, 1 µg of template DNA was incubated in transcription reaction mix (40 mM Tris-HCl, pH 7.5, 6 mM MgCl, 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol, 20 units of RNasin, 0.5 mM each of rATP, GTP, and CTP, 12 µM rUTP, 50 µM [-P]UTP (800 Ci/mmol), and 15 units of the appropriate RNA polymerase) at 37 °C for 60 min. The reaction was stopped by the addition of 2 units of RNase-free DNase and incubated at 37 °C for 15 min. 25 mM EDTA was then added, samples were phenol-chloroform extracted and ethanol-precipitated. Antisense riboprobes were resuspended in water at a concentration of 1-2 10 cpm/µl.

In Situ Hybridization

Male Sprague-Dawley rats (200-300 g) were anesthetized and perfused with 1000 ml of 4% paraformaldehyde in borate buffer, pH 9.5, at 4 °C (fixation buffer). Brains were dissected and incubated in fixation buffer for 8 h and then incubated overnight in fixation buffer with 10% sucrose. Brains were sectioned serially into five series of 30-µm slices with a sliding microtome. Sections were prepared and hybridized as described previously(33) . The 336-bp MKP-2 specific cDNA fragment was used to synthesize antisense and sense riboprobes. Sections were hybridized with S-labeled riboprobes (10 cpm/ml) in 66% formamide, 0.26 M NaCl, 1.32 Denhardt's solution, 13.2 mM Tris, pH 8.0, 1.32 mM EDTA, 13.2% dextran sulfate, pH 8.0, at 65 °C for 24 h. Slides were washed in 4 SSC, digested with RNase A (20 µg/ml for 30 min at 37 °C), and then rinsed in a stringent wash containing 0.1 SSC at 65 °C for 30 min. Sections were dehydrated, dipped in NTB-2 emulsion (Kodak), and developed after 21 days. Light- and darkfield photomicrographs were taken with a Dialux 22 EB at 32 magnification.

In Vitro Transcription and Translation Reactions

Full-length MKP-1 and MKP-2 cDNAs were used in a coupled in vitro transcription and translation reaction using TNT coupled reticulocute lysate system (Promega) as per the manufacturer's instructions. Briefly, 1 µg of circular DNA was incubated with 27.5 µl of TNT rabbit reticulocyte lysate, 2 µl of TNT reaction buffer, 1 µl of T7 RNA polymerase, 1 µl of 1 mM amino acid mix minus cysteine, 2 µl of [S]cysteine (1075 Ci/mmol at 11 mCi/ml), and 1 µl of RNAsin at 40 units/µl in a final volume of 50 µl. The reaction was incubated at 30 °C for 1-2 h. The synthesized proteins were separated by SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography.

PC12 Transfection Assays

PC12 cells were grown to approximately 60% confluence prior to transfection. For transient transfection experiments, 3 µg of 5 Gal4-E1B luciferase and 3 µg of cytomegalovirus (CMV) Gal4-Elk1 transactivation domain()were used in combination with either 6 µg of Rous sarcoma virus promoter coupled to globin (control) or constitutively active form of Raf kinase (Raf BxB) (34) and 30 µg of either pCDNA3 (Invitrogen, Inc.) containing the CMV promoter, CMV MKP-1, or CMV MKP-2. Cells were transfected by calcium phosphate-mediated DNA transfer as described previously(35) . Cell lysates were prepared 20-24 h following transfection, and luciferase activity was determined as described previously(36) .

Western Blotting of PC12 Proteins

PC12 cells were lysed in 200 µl of a 1% Nonidet P-40 lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl, 10% glycerol, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 2 mM vanadate). Protein concentrations were determined by the method of Bradford(37) . One hundred µg of total protein was resolved on a 12% SDS-polyacrylamide gel and transferred onto Immobilon P membrane. The membranes were probed with an MKP-1 antibody (diluted at 1:2000) (Santa Cruz Biotech., Inc.). A horseradish peroxidase-conjugated secondary antibody was used to allow detection of the appropriate bands using enhanced chemiluminescence (Amersham Corp.).

Bacterial Expression of MKP-2

The catalytic domain of MKP-2 encoded within a carboxyl-terminal 690-bp fragment (C-MKP-2), extending from amino acids 163-393, was subcloned into the PET 23b vector (Novagen) using specific PCR oligonucleotides. This vector provides an amino-terminal epitope tag derived from the T7 capsid protein (T7 tag) that can be detected using specific antibodies (T7 antibody, Novagen). The frame of the resultant cDNA was confirmed by sequencing. This plasmid and the vector alone were used to transform BL21 bacteria (Novagen). A protein of the expected size (28 kDa) was induced upon incubation with 0.4 mM isopropyl-1-thio--D-galactopyranoside and was detected using anti-T7 capsid antibodies. Prior to phosphatase assays, bacterial extracts were prepared in lysis buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA) and sonicated. Insoluble debris was pelleted and the supernatant assayed directly.

ERK2 Dephosphorylation Assay

Activated ERK2 was prepared by incubating 10 ng of recombinant ERK2 (kindly provided by Dr. Edwin Krebs, University of Washington, Seattle, Washington) with 0.1 µg of active MAP kinase kinase (MEK) (Santa Cruz Biotechnology, Inc.) in 1 MEK buffer (25 mM Hepes, pH 7.5, 10 mM MgCl, 1 mM dithiothreitol, and 50 µM [-P]ATP) at 30 °C for 30 min. The activation of ERK2 was confirmed by Western blot analysis using an antibody directed against phosphotyrosine(30) . Ten ng of activated ERK2 was incubated with 10 µg of bacterial lystates from MKP-2 expressing and nonexpressing cells in 1 phosphatase buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10 mM dithiothreitol) for 15 min at 30 °C. The reaction was stopped by the addition of an equal volume of 2 Laemmli sample buffer, and the samples were separated by 13% SDS-polyacrylamide gel electrophoresis. The dephosphorylation of ERK2 was confirmed by Western blotting with the phosphotyrosine antibody using enhanced chemiluminescence for detection of the signal.

Phosphotyrosine Phosphatase Assay

The synthetic peptide Raytide (Oncogene Sciences) was phosphorylated on a unique tyrosine using Src tyrosine kinase activity immunoprecipitated from C3H10T1/2 cells (kindly provided by Sally Parsons, University of Virginia) as described(38) . Bacterial extracts containing 10-60 µg of bacterial proteins were incubated at 30 °C for 30 min with labeled peptide (10 cpm) in 100 µl of 1 phosphatase buffer (described above). Additional reagents (10 µM vanadate, 20 nM microcystin-leucine-arginine, and 1 µM okadaic acid) were added without prior incubation. The reaction was terminated by the addition of 0.75 ml of stop solution (2 mM NaHP0, 90 mM sodium pyrophosphate, 0.9 M HCl, 4% (v/v) Norit A). Following brief centrifugation, 400 µl of the supernatant was added to 2.5 ml of scintillant, and the released counts from phosphatase activity were measured on a scintillation counter. All phosphatase assays were performed in duplicate.


RESULTS

PC12 Cells Express Multiple MKPs: Identification and Cloning of a Novel MKP

To identify potential MKPs that are expressed in PC12 cells, a screening strategy involving reverse transcriptase PCR amplification was employed. Alignment of the sequences of the known members of the MKP family (human PAC-1, mouse PAC-1, mouse 3CH134, and VH1) showed areas of high sequence homology particularly surrounding the catalytic core consensus site (HCXAGXXR, where X is any amino acid) (Ref. 23 and Fig. 2). Degenerate primers were designed to the conserved amino acid sequence WFNEAI (5` primer) and the conserved amino acid sequence NFSFMG (3` primer) surrounding the catalytic core site (Fig. 2). Reverse transcriptase PCR on total RNA from PC12 cells with these two degenerate primers revealed the expected 204-bp product that contained a representative population of MKPs expressed in PC12 cells (data not shown). This PCR product was gel-purified, digested with restriction enzymes engineered at the primer ends, and subcloned into the appropriate sites in Bluescript. Five positive clones were obtained that were analyzed by sequencing. One clone was identified as the rat homolog of MKP-1 or 3CH134(19) . The remaining four clones were identical and showed some unique features in comparison with the other known MKPs. To analyze this clone further, two additional primers were designed. The 3` primer was directed to a unique stretch in the cloned novel sequence (MKKRVR) (Fig. 2). The 5` degenerate primer was designed to another stretch of conserved sequence between the MKP family members (YDQGGP), 5` to the sequence already obtained (Fig. 2). Subsequent amplification using these two primers resulted in a 336-bp amplicon (data not shown). Sequencing this fragment confirmed the novelty of this cDNA sequence (Fig. 1B and 2). These results demonstrate that PC12 cells express at least two potential MKPs, MKP-1 and the novel cDNA that we have called MKP-2.

These reverse transcriptase PCR partial fragments of the novel MKP-2 cDNA were labeled and used as probes to screen an oligo(dT)-primed PC12 cDNA library in gt10. This library contained 5 10 individual recombinants that had been size-selected prior to ligation for clones greater than 2 kb. The three positive clones obtained with this screen were plaque-purified, and the phage DNA obtained was digested with EcoRI to release the insert. Upon digestion, two of the three positive clones each revealed two insert fragments of approximately 4 kb and 800 bp, while the other clone contained a single 3-kb insert fragment. The 3-kb insert was never successfully subcloned, and therefore was not analyzed further. The two contiguous insert fragments (4 kb and 800 bp) were cloned separately into Bluescript and characterized by restriction analysis (Fig. 1A) and sequencing (Fig. 1B). The 5`-untranslated region and the first 135 amino acids of the novel MKP were contained within the 800-bp fragment, while the remaining 3` end of the clone was contained in the 4-kb fragment. The cDNA encoding MKP-2 contained at least 377 bp of 5`-untranslated region and a translation start site with a 11/13 match with the Kozak consensus sequence (Fig. 1B). The open reading frame extends 1182 bp and encodes a protein product of 393 amino acids with a predicted molecular mass of 42.6 kDa and an isoelectric point of 7.86. The open reading frame was followed by an unusually large 3`-untranslated region of greater than 3.0 kb. In comparison, the size of full-length mouse MKP-1 (3CH134, Erp) and its human homolog (CL100) is only 2.4 kb(19, 22, 26) , similar to the recently cloned member of this family, B23, which is 2.5 kb(24) . The large 3`-untranslated region of MKP-2 contains several AU sequence motifs (Fig. 1B) that are thought to regulate mRNA stability and are also present in MKP-1(26) .

The putative 393-amino acid MKP-2 protein shares 58.8% identity to the 367-amino acid MKP-1 protein, 62.3% identity to the 314 amino acid mPAC protein, and 33% identity to the 397-amino acid human B23 protein (Fig. 2). The similarities are greatest at the 3` end near the catalytic domain. In contrast, however, the 5` end has significant sequence differences compared with the other members of this family. The N-terminal half of MKP-2 (amino acids 1-187) shares only 33% identity to MKP-1 while the C-terminal half(188-393) shows much greater homology (76%) with nearly 100% identity around the catalytic core (Fig. 2). The complete coding region of MKP-2 was subcloned into Bluescript to generate a full-length cDNA (MKP2FL-6) to allow transcription from the T7 promoter. In vitro transcription and translation of MKP-1 and MKP-2 cDNA revealed the expected 39.4- and 42.6-kDa proteins, respectively (Fig. 3A). Antisera directed to an MKP-1 C-terminal peptide sequence (amino acids 348-366) detected proteins of sizes similar to MKP-1 and -2 from PC12 cell lysates (Fig. 3B). This cross-reactivity is likely the result of the significant homology between MKP-1 and -2 in this region (Fig. 2).


Figure 3: In vitro translation of MKP-1 and MKP-2 cDNAs. A, autoradiogram of [S]cysteine-labeled products of a coupled in vitro transcription-translation reaction are shown using cDNAs encoding MKP-1 (lane1) and MKP-2 (lane2). B, Western blot analysis of PC12 cell lysates (100 µg of total protein) using antisera directed to a MKP-1 peptide with significant identity to the C-terminal residues of MKP-2 (Santa Cruz Biotech). A 46-kDa protein molecular mass marker is indicated.



The MKP-2 Protein Contains Phosphotyrosine Phosphatase Activity

A carboxyl-terminal fragment of MKP-2 (amino acids 163-393) comprising the entire catalytic domain was subcloned into the bacterial expression vector PET-23b and expressed in the Escherichia coli strain BL21 (LysS). Induction of bacterially expressed truncated MKP-2 by isopropyl-1-thio--D-galactopyranoside was confirmed by Western blotting using an antibody directed to the T7 capsid epitope (data not shown). Bacterial extracts expressing the truncated MKP-2 fusion protein displayed phosphotyrosine phosphatase activity that was inhibited by vanadate but not by okadaic acid nor microcystin-LR, inhibitors of serine/threonine phosphatases(39, 40) (Fig. 4A). These extracts, but not extracts expressing vector alone, could dephosphorylate activated ERK2 protein in vitro as monitored by phosphotyrosine Western blotting (Fig. 4B). This dephosphorylation was blocked by vanadate (Fig. 4B).


Figure 4: MKP-2 contains phosphotyrosine phosphatase activity. A, dephosphorylation of the synthetic peptide Raytide by bacterial lysates expressing a truncated MKP-2 protein (amino acids 163-393). U, untreated extracts from bacteria expressing MKP-2; V, vanadate-treated extracts; O, okadaic acid-treated extracts; M, microcystin-treated extracts; C, control extracts from bacteria expressing vector alone. The activity is represented as percent of maximal stimulation. B, dephosphorylation of ERK2 by MKP-2 is shown. Activated ERK2 (pp42) (see ``Experimental Procedures'') was incubated with phosphatase buffer alone (lane1), bacterial lysates expressing vector alone (lane2), bacterial lysates expressing truncated MKP-2 protein (lane3), bacterial lysates espressing MKP-2, assayed in the presence of vanadate (lane4). Phosphorylation was assayed by Western blotting with a phosphotyrosine antibody as described.



MKP-2 Blocked MAP Kinase-dependent Activation of the Elk-1 Transactivation Domain

To demonstrate biologically significant activity toward ERKs in vivo, the full-length cDNA encoding MKP-2 (MKP-2FL-6 cDNA) was subcloned into a CMV-driven expression vector (CMV-MKP-2) and expressed in PC12 cells with a Gal4-Elk-1 fusion protein used to direct expression of a Gal4-luciferase reporter. Transcriptional activation by the Elk-1 transactivation domain requires activated MAP kinase(41) . In these cells, a constitutively active Raf-1 mutant, Raf BxB, induced Elk-1-dependent transcription of the luciferase reporter by greater than 100-fold (Fig. 5). Co-transfection with a MKP-1 expression vector under the control of a CMV promoter (CMV-MKP-1) blocked the activation by greater than 60% of maximally stimulated levels. Co-transfection with similar amounts of CMV-MKP-2 reduced expression by greater than 90% of maximal levels (Fig. 5). These studies demonstrate that MKP-2 can potently inhibit transcriptional activation that is dependent on MAP kinase. Unstimulated activation of Elk-1 was assayed in the presence of serum. Both MKPs inhibited this ``basal'' expression of luciferase activity to undetectable levels, presumably by blocking serum-induced MAP kinase activity.


Figure 5: MKP-2 blocks MAP kinase-dependent gene transcription. PC12 cells were transfected with 6 µg of either Rous sarcoma virus-globin (control) (white bars) or Raf BxB (constitutively active Raf) (gray bars) in the presence of 30 µgs of vector alone (CMV), CMV-MKP-1 (MKP-1) or CMV-MKP-2 (MKP-2). In addition, all cells received 3 µg of the reporter 5xGal4-E1B luciferase and 3 µg of Gal4-Elk-1. Activity is shown as light units/100 µg of protein. Note the activities of cells transfected with Rous sarcoma virus-globin and either MKP are below the limits of detection.



MKP-2 Is Expressed in Most Tissues and Cell Lines Examined: Distinct Expression Compared with MKP-1

To determine the distribution of MKP-2 in various tissues, Northern blot analysis using a 336-bp MKP-2 specific riboprobe (described under ``Experimental Procedures'') revealed expression in most tissues obtained from 10-12-week-old rats (Fig. 6A). MKP-2 mRNA was detected in most tissues including brain, spleen, and testes with the highest expression in the heart and lung and lower expression in skeletal muscle and kidney. No MKP-2 expression was detected in the liver. The same blot was stripped and reprobed with a riboprobe made to the entire coding region of MKP-1 (Fig. 6B). This probe did not cross-react with the 6-kb MKP-2 transcript. A 2.4-kb mRNA corresponding to MKP-1 was detected in all tissues but testes. The highest expression of MKP-1 was seen in the lung, as previously reported(19, 26) , and in the heart (similar to MKP-2). In the testes, MKP-2 is abundantly expressed, but MKP-1 is not. In the liver, the inverse expression pattern is found. These opposite expression patterns in two different tissues suggests different physiological roles for the members of the MKP family. These results show a fairly abundant basal expression of MKP-1 and MKP-2 in most tissues with distinct tissue distribution patterns between MKP-1 and MKP-2. MKP-2, as expected from the 4.8-kb cDNA, which was missing a portion of the 5`-untranslated region, encodes an approximate 6-kb transcript distinct from the 2.4-kb MKP-1 mRNA detected in the same tissues.


Figure 6: Expression of MKPs in rat tissues. A filter containing 2 µg of Poly(A) mRNA isolated from the indicated tissues (Clonetech Lab, Inc., Palo Alto, CA) was probed with a MKP-2-specific riboprobe (panel A), stripped, and reprobed with a MKP-1-specific riboprobe (panel B). The molecular mass markers are indicated to the right.



RNA was also isolated from cell lines of different lineages, and 10 µg of total RNA from various cell lines was analyzed by Northern analysis using the 336-bp MKP-2 specific riboprobe (Fig. 7A). A single 6-kb MKP-2 transcript was detected in all rat- and mouse-derived cell lines of different lineages, while a single 4-kb MKP-2 transcript was detected in all cells of human origin. The same blot was stripped and reprobed with an MKP-1 riboprobe (Fig. 7B). No MKP-1 was detected in cells of human origin. This may reflect the inability of the mouse MKP-1 riboprobe to hybridize across species to cells of human origin under the stringent conditions used. A rat MKP-1 probe was also used with similar results (data not shown).


Figure 7: Expression of MKPs in cell lines. 10 µg of total RNA was isolated from each of the cell lines indicated and probed with an MKP-2 specific riboprobe (panelA), stripped, and reprobed with a MKP-1 specific riboprobe (panelB). The migration of the ribosomal bands is indicated to the right. hMKP-2 refers to an MKP-2 specific transcript detected only in cells of human origin. Abbreviations for the cell lines include: RIN, rat insulinoma; GHC1, rat pituitary tumor; AtT20, mouse pituitary tumor; W2, rat medullary thyroid carcinoma; LTK, mouse fibroblasts; PC12, rat adrenal medullary tumor; MiaPACA, human pancreatic carcinoma; MOLT48, human lymphoblasts; HepG2, human hepatoblastoma; SKNMC, human neuroblastoma.



Distribution of MKP-2 in the Central Nervous System

It has been shown that ERK1 mRNA is expressed in all areas of the brain with the strongest expression in the hippocampus and piriform cortex while there was no overlapping expression of CL100 (human MKP-1) in those same areas(42) . In order to determine the distribution of MKP-2 mRNA in the brain, we performed in situ hybridization analysis on rat brain sections with an S-labeled antisense and sense MKP-2 riboprobe. MKP-2 appeared to be expressed in many areas of the brain with very strong staining of the hippocampus, piriform cortex, and the suprachiasmatic nucleus (Fig. 8). The sense riboprobe did not show any specific staining (data not shown). These results suggest the co-localization of an ERK isoform, ERK1, and a MKP isoform, MKP-2, in specific areas of the brain where MKP-1 is not expressed.


Figure 8: Localization of MKP-2 mRNA in the rat brain using in situ hybridization. Lightfield (A, C, and E are thionin counterstains) and darkfield (B, D, and F) photomicrographs showing representative distribution of MKP-2 mRNA (32). DG, dentate gyrus of the hippocampus; Pir, piriform cortex; 3V, third ventricle; Sch, suprachiasmatic nucleus of the hypothalamus. Sense MKP-2 riboprobe did not hybridize (data not shown).



Regulation of MKP-1 and MKP-2 in PC12 Cells

MKPs have been shown to be immediate early genes and are transcriptionally regulated by a variety of agents. For example, MKP-1 mRNA appears to be induced by bombesin, EGF, 12-O-tetradecanoylphorbol-13-acetate, cAMP, and fibroblast growth factor to different extents and with different kinetics, suggesting the involvement of this phosphatase in various signaling pathways(26) . A recent member of the MKP family, B23, has also been shown to be regulated by serum in human skin fibroblasts(24) . In order to determine the involvement of MKP-2 in different neuronal signal transduction cascades in PC12 cells, Northern blot analysis was performed to determine the levels of MKP-1 and -2 mRNA following growth factor stimulation.

To determine whether MKP-2 mRNA is serum-inducible, total RNA was isolated from PC12 cells that were serum-starved overnight and then stimulated with media containing 15% serum for various times. Ten µg of RNA from each treatment was analyzed by Northern analysis using an MKP-2-specific riboprobe as described above. Both MKP-1 and MKP-2 were expressed in unstimulated PC12 cells (Fig. 7, A and B, and Fig. 9). Serum stimulation caused a biphasic stimulation of MKP-2 with a gradual increase by 1 h followed by a transient decrease and then a subsequent increase in expression of MKP-2 by 4 h. The same blot was stripped and reprobed with MKP-1, and the MKP-1 transcript followed a similar but less robust stimulation (Fig. 9, A and B, left panel). All quantitations were normalized to the 18 S ribosomal RNA as an internal control.


Figure 9: Effect of serum and growth factors on MKP-1 and MKP-2 mRNA. A, PC12 cells were serum-starved for 24 h (U, untreated control) and were treated with medium containing 15% serum for the indicated times (left panel) or with NGF (100 ng/ml) or EGF (20 ng/ml) for the indicated times (right panel). 10 µg of total RNA from each treatment was analyzed by Northern blot analysis using an MKP-2-specific riboprobe, the filter was stripped and reprobed with MKP-1, stripped again, and reprobed with a 18 S ribosomal RNA probe. The results from each probe are shown. B, quantitative representation of MKP-1 and -2 mRNA levels normalized to the 18 S ribosomal RNA from an average of two independent experiments, one of which is shown in A. Presented values for all treatments represent -fold induction compared with untreated cells (laneU), which was normalized as 1. The columns are aligned so as to be directly underneath the treatments indicated in A. Black boxes represent MKP mRNA levels in PC12 cells serum stimulated for the indicated times; gray boxes represent MKP mRNA levels from serum-starved PC12 cells treated with NGF for the indicated times; and the white boxes represent MKP mRNA levels from serum-starved PC12 cells treated with EGF for the indicated times.



To address whether differences in the induction of distinct MKPs may account for the differences observed in the kinetics of MAP kinase activation seen following NGF and EGF treatment of PC12 cells(5) , PC12 cells were serum-starved and treated with NGF or EGF for various times. Ten µg of total RNA from each of these treatments was analyzed by Northern blot analysis for MKP-1 and -2 expression (Fig. 9A, right panel). All quantitations were normalized to 18 S ribosomal RNA, which was used as an internal control for the amount of RNA loaded (Fig. 9B, right panel). NGF induced expression of MKP-1 and MKP-2 by 1 h. Continued stimulation by NGF resulted in a maximum of 5-fold increase in MKP-2 expression by 2 h and a 3-fold increase in MKP-1 expression by 1 h. Both MKP-1 and -2 were maintained at slightly elevated levels upon NGF stimulation. 1 h of EGF also induced expression of MKP-1 by 2.7-fold and MKP-2 by 4-fold. In contrast to NGF, this induction however was inhibited after 2 h of EGF treatment in both cases. Additional incubation with EGF resulted in a second induction of both MKP-1 (2.6-fold) and MKP-2 (5.7-fold) by 4 h. This biphasic kinetic pattern was also seen following stimulation with serum, with a minimum RNA level seen at 2-2.5 h, as well (Fig. 9B). These results suggest that growth factors that activate members of the MAP kinase cascade also result in the transcriptional activation of MKPs. The significance of the different kinetic pattern of MKP induction by NGF and EGF has yet to be determined.

Regulation of Hippocampal Expression

Due to the high expression of MKP-2 mRNA in the hippocampus (Fig. 8), we analyzed MKP-2 expression after adult rats (250-300 g) had been subjected to the global seizure inducing drug, kainic acid (8 mg/kg). RNA from the hippocampus of rats subjected to kainic acid was extracted 0.5 and 1 h after drug treatment and analyzed by Northern blot analysis (Fig. 10A). Quantitations were normalized to the amount of cyclophilin in each case. MKP-2 mRNA was induced 5.1-fold by 1 h of kainic acid treatment, while MKP-1 mRNA was induced 3.2-fold (Fig. 10B). This kainic acid induced transcriptional stimulation of MKPs suggests the involvement of these genes in the stress-induced pathways in the brain.


Figure 10: Induction of MKPs in the hippocampus by Kainic acid. 10 µg of total hippocamal RNA was isolated from control rats (C) or rats treated with 8 mg/kg kainic acid for the indicated times (kindly provided by Drs. James Douglass and Pastor Couceyro). A, northern blot analysis was performed with a MKP-2-specific riboprobe, the filter was stripped and probed with MKP-1, stripped again, and reprobed with cyclophilin as an internal control. B, the blot was quantitated, and the results were normalized to cyclophilin and are shown graphically. The black box is the amount of MKP-1 mRNA, and the gray box represents the levels of MKP-2 mRNA.




DISCUSSION

We have identified a second widely-expressed MAP kinase phosphatase we term MKP-2. It is co-expressed with MKP-1 in a large number of tissues but also shows distinct differences. In contrast, the MKP PAC-1, is expressed only in lymphoid cells(23) . Like MKP-1 and PAC-1, MKP-2 shows homology to other tyrosine phosphatases. Its sequence is most conserved with MKP-1 and PAC-1 within the C terminus and less so within the N terminus (Fig. 2). All three share identity within the catalytic core (VHCQAGISR) and display phosphotyrosine phosphatase activity against synthetic peptides and purified MAP kinase. The dual specificity of MKP-2 toward a phosphothreonine has yet to be proven. However, we have shown that both MKP-2 and MKP-1 block MAP kinase-dependent gene transcription in vivo, as been shown for PAC-1(15) . All four MKPs contain the CH2 domains (cdc25 homology 2), which are regions present in members of the cdc25 family that flank the catalytic domain(42) . In MKPs, the catalytic domain is situated at the C terminus and not at the N terminus where the CH2 domains are found (Fig. 2). Whether these CH2 domains are functional has yet to be proven, but they have been speculated to either increase substrate selectivity or to be involved in localizing proteins to nuclear or cytoskeletal locations(42) .

The large (>3 kb) 3`-untranslated region in MKP-2 differs from the shorter (697 bp) 3`-untranslated region present in MKP-1 and may play a regulatory role in post-transcriptional events such as transcript stability. Several AUUUA motifs are found in the 3`-untranslated region of both MKP-1 (19, 26) and MKP-2 (Fig. 1B). MKP-2 also has a 25-nucleotide stretch of AU sequences, which might also contribute to posttranscriptional control. These AU sequence motifs have been implicated in the short half-life (10 min) of MKP-1 mRNA(19) . Such runs of AU sequences occur in the 3`-untranslated regions of lymphokines, cytokines, and proto-oncogenes and are thought to be recognition signals for selective mRNA degradation(43) . The role of these motifs in MKP-2 regulation has yet to be determined.

In contrast to the expression of PAC-1, which is limited to lymphoid cells(23) , MKP-1 and MKP-2 show expression in a broad range of tissues with distinct differences. These distinct tissue distribution profiles may dictate unique roles of the members of this family in the regulation of MAP kinases and might reflect their co-expression with certain MAP kinase isoforms. MKP-1, ERK-1(42) , and MKP-2 mRNA are expressed in discrete areas of the brain. ERK2 expression is also prominent in neuronal cell bodies and dendrites particularly within the superficial layer of the neocortex, the hippocampal CA3 region, dendate gyrus, and cerebellar Purkinje cells(44) . The co-localization of MKP-2 and the ERK isoforms in certain discrete areas of the brain suggests that ERK1 and ERK2 may be physiological substrates for MKP-2. However, MKP-1 and MKP-2 also show overlapping expression, which suggests that co-expression of MKPs and ERKs is not the only criterion for substrate specificity.

The function of MAP kinase in postmitotic neuronal cells is unclear. MAP kinases within the developing and adult central nervous system (45) are activated by both neurotrophic growth factors and neurotransmitters. For example, activation of the N-methyl-D-aspartate receptor leads to increased tyrsoine phosphorylation of an ERK isozyme in hippocampal cultures(46) . Kainate is an N-methyl-D-aspartate receptor antagonist that induces seizures and immediate-early genes within the hippoocampus(47, 48) . The induction of CL100 (human MKP-1) and B23 mRNA by oxidative stress and heat shock has been reported(22, 24) , and the induction of MKP-1 and -2 observed with kainic acid treatment may represent a regulatory role that these genes might play in response to cellular stress. It is not known whether the stress-activated kinases are substrates for the MKPs(49) .

Basal expression of MKPs may be important for the resting cell. Vanadate stimulates MAP kinase activity in resting PC12 cells (data not shown). This suggests that a constitutive tyrosine phosphatase activity may inhibit basal MAP kinase activity. In addition, the introduction of dominant negative mutants of MKP-1 into unstimulated COS cells activates MAP kinases in the absence of serum(13) . Therefore MKPs may be active in resting cells and might function to minimize the level of basal MAP kinase activity in the resting cell.

In PC12 cells, both NGF and EGF stimulate a receptor-tyrosine kinase to phosphorylate and activate similar intracellular substrates including MAP kinase, whose action is required for both proliferative and differentiating responses(3, 4, 50) . It has been suggested that the duration of MAP kinase activation determines the biological response to growth factor stimulation(5, 51) . Proliferation is associated with a transient MAP kinase activation(5, 51, 52) , while agents that induce a differentiating response produce a sustained activation. It is not known whether regulation of MKPs establishes the time courses of MAP kinase inactivation. We demonstrate that both NGF and EGF induced a rapid increase in MKP-1 and MKP-2 mRNA levels with a more substantial increase in MKP-2 compared with MKP-1. Whether this induction of MKPs is responsible for the rapid inactivation of MAP kinase by EGF is not known. A recent report demonstrates that inactivation of MAP kinase by EGF is independent of MKP-1 induction(53) . Our results would agree with their findings as NGF, which sustains the level of active MAP kinase for a longer duration than EGF, also resulted in elevated levels of MKP-1 and -2 for at least 4 h of drug exposure. Therefore, it is possible that the MKPs are regulated post-transcriptionally by these agents. The differences in transcriptional regulation of MKP-1 and MKP-2 by NGF and EGF suggest that they have different mechanisms of regulating MKP activity.

In conclusion, PC12 cells express at least two related MKPs, MKP-1 and MKP-2. The identification of a family of MKPs that are expressed within the same cell suggests distinct roles for each member of this expanding family. Discrimination of the actions of these MKPs may occur through the divergent amino termini of these proteins. Further studies are required to identify the physiological roles of each member of this unique family of phosphatases in order to gain a better understanding of the mechanisms involved in cellular proliferation, differentiation, and stress.


FOOTNOTES

*
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: The Vollum Inst., L474, Oregon Health Sciences University, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-5494; Fax: 503-494-4976.

The abbreviations used are: MAP, mitogen-activated protein; EGF, epidermal growth factor; NGF, nerve growth factor; ERK, extracellular signal-regulated kinase; MKP, MAP kinase phosphatase; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pairs; CMV, cytomegalovirus.

Roberson, M. S., Misra-Press, A., Laurance, M. E., Stork, P. J. S., and Maurer, R. A. (1995) Mol. Cell. Biol., in press.


ACKNOWLEDGEMENTS

We thank Marty Mortrud and Drs. James Douglass, Pastor Couceyro, Malcolm Low, and Richard A. Maurer for providing scientific support and Sheri Medford for excellent secretarial assistance.


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