The Mechanism of Dephosphorylation of Extracellular Signal-regulated Kinase 2 by Mitogen-activated Protein Kinase Phosphatase 3*

The mitogen-activated protein (MAP) kinase phospha-tase-3 (MKP3) is a dual specificity phosphatase that spe-cifically inactivates one subfamily of MAP kinases, the extracellular signal-regulated kinases (ERKs). Inactivation of MAP kinases occurs by dephosphorylation of Thr(P) and Tyr(P) in the T X Y kinase activation motif. To gain insight into the mechanism of ERK2 inactivation by MKP3, we have carried out an analysis of the MKP3-catalyzed dephosphorylation of the phosphorylated ERK2. We find that ERK2/pTpY dephosphorylation by MKP3 involves an ordered, distributive mechanism in which MKP3 binds the bisphosphorylated ERK2/pTpY, dephosphorylates Tyr(P) first, dissociates and releases the monophosphorylated ERK2/pT, which is then subjected to dephosphorylation by a second MKP3, yielding the fully dephosphorylated ERK2. The bisphosphorylated ERK2 is a highly specific substrate for MKP3 with a k cat / K m of 3.8 (cid:1) 10 6 M (cid:2) 1 s (cid:2) 1 , which is more than 6 orders of magnitude higher than that for small molecule aryl phosphates and an ERK2-derived phosphopeptide en-compassing the pTEpY motif. This strikingly high substrate specificity displayed

responds vigorously to growth factors and certain hormones, JNK and p38 react primarily to cytokines and stress stimuli. MAP kinases are activated by MAP kinase kinases (MEKs), which are dual specificity kinases that phosphorylate the conserved Tyr and Thr located within the TXY motif in the activation loop of the MAP kinase (2)(3)(4). Although the mechanism for MAP kinase activation has been well characterized, much less is known about its subsequent inactivation. Increasing evidence suggests that multiple phosphatases are involved in MAP kinase inactivation (5).
The MAP kinase phosphatases (MKPs) are dual specificity phosphatases capable of dephosphorylating both Tyr(P) and Thr(P) in the activation loop of MAP kinases (6,7). MKPs belong to the protein-tyrosine phosphatase (PTPase) superfamily, which is defined by the PTPase signature motif (H/ V)C(X) 5 R(S/T). Mechanistic studies on PTPases have mostly been conducted with small molecule aryl phosphates, such as p-nitrophenyl phosphate (pNPP), and phosphopeptides. These studies have shown that the tyrosine-specific PTPases and the dual specificity phosphatases share a common catalytic mechanism to effect catalysis (8). The PTPases utilize the Cys in the signature motif as a nucleophile in the formation of a thiophosphoryl covalent enzyme intermediate (9,10). The invariant Arg residue in the PTPase signature motif functions in substrate binding and in transition state stabilization (11)(12)(13). The initial phosphoryl transfer from the substrate to the enzyme is assisted by a conserved Asp on a surface loop, which protonates the leaving group (14,15), thereby acting as a general acid catalyst. The phosphoenzyme intermediate undergoes hydrolysis in a second step, which is catalyzed by the same Asp residue acting as a general base (16).
Although studies with small molecule substrates have yielded useful information about PTPase catalysis, further understanding of the specific functional role of PTPases in cellular signaling requires detailed investigation of PTPases with physiological substrates. Activated MAP kinases are physiological substrates for MKPs. In mammalian cells, at least nine MKPs have been identified. Previous studies suggest that these MKPs display distinct in vivo substrate preferences for the various MAP kinases (6,7). For example, MKP3 is predominantly localized in the cytoplasm and is highly specific in deactivating ERK1/2 (17)(18)(19)(20). Although it has been shown that the N-terminal domain of MKP3 can physically associate with ERK1/2 (21) and purified recombinant ERK2 stimulates the phosphatase activity of MKP3 against artificial substrate pNPP (22), little is known about how MKP3 catalyzes the dephosphorylation of the activated ERKs. In addition, MKP3 is a dual specificity phosphatase that can hydrolyze both Thr(P) 183 and Tyr(P) 185 in ERK2 (19,23). Because activated ERK2 contains two phosphorylated residues in close proximity, it is unclear whether the MKP3-catalyzed inactivation of ERK2 involves a processive or a distributive dephosphorylation mechanism (Scheme 1).

EXPERIMENTAL PROCEDURES
Recombinant Proteins-The cDNA for the constitutively active MAP kinase kinase MEK1 mutant G7B (⌬N4/S218D/M219D/N221D/S221D) in pRSETa was kindly provided by Dr. Natalie Ahn (University of Colorado, Boulder, CO). Recombinant human MEK1/G7B was expressed as a N-terminal His 6 -tagged protein in Escherichia coli BL21(DE3)pLysS and purified as described (24). The pET15b plasmid containing rat ERK2 was a generous gift from Dr. Chao-Feng Zheng. The mutants ERK2/K52R and ERK2/D319N were generated by polymerase chain reactions according to the standard procedure of the Quick-Change site-directed mutagenesis kit (Stratagene) using the pET15b-His 6 -ERK2 plasmid as a template. The N-terminal His 6tagged wild-type ERK2, ERK2/K52R, and ERK2/D319N mutant proteins were expressed in E. coli and purified to near homogeneity by nickel chelate chromatography. GST-MKP3 was expressed and purified as described (21). The C-terminal His 6 -tagged MKP3 was derived by subcloning the coding sequence of MKP3 into pET21a vector (Novagen) and purified as described (25).
Preparation of 32 P-Labeled Bisphosphorylated ERK2-The in vitro phosphorylation of the His 6 -tagged ERK2/K52R was carried out in a 500-l total volume by incubation of 1 mg/ml purified ERK2/K52R with 0.1 mg/ml His 6 -tagged MEK1/G7B in the presence of 10 mM HEPES, pH 7.4, 20 mM Mg(OAc) 2 , 100 mM NaCl, 2 mM dithiothreitol, 0.5 mM [␥-32 P]ATP (5000 cpm/pmol, PerkinElmer Life Sciences). The reaction mixture was incubated at 30°C for 90 min (26) with occasional stirring. The phosphorylated ERK2 was separated from free ATP by first passing through a high capacity (3 ml) Sephadex G-50 spin column (Roche). Then an equal volume of Ni 2ϩ -nitrilotriacetic acid-agarose suspension (Qiagen) (100 l of bed volume/0.5 mg of ERK2) was added to the reaction mixture and shaken gently at 4°C for at least 40 min. The beads were collected by centrifugation at 500 ϫ g and washed three times with 1 ml of wash buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% Triton X-100). The 32 P-labeled ERK2/K52R was then eluted by addition of 200 l of 300 mM imidazole in wash buffer. ERK2 elution was repeated one more time, and the 32 P-labeled ERK2/K52R from both elution steps was combined and passed through a high capacity Sephedex G-50 spin column again and stored on ice. The phosphorylated ERK2 was quantitated by SDS-PAGE using purified unphosphorylated ERK2 as a standard. Typical yields for ERK2/pTpY preparation were better than 80%. The stoichiometry of ERK2 phosphorylation was determined by tryptic peptide mapping. Because the bis-and monophosphorylated ERK2 peptides migrate at different positions in thin layer chromatography (Fig. 5A) and the unphosphorylated ERK2 was undetectable after the phosphorylation reaction, the relative amounts of the different species of the phosphorylated ERK2s can be quantified by PhosphorImager analysis. The stoichiometry of ERK2 phosphorylation was then calculated by dividing the relative concentration of the phosphate by that of ERK2 and found to be greater than 1.8 mol of phosphate/mol of ERK2. Furthermore, using the same procedure and with cold ATP, the stoichiometry of ERK2 phosphorylation was determined to be close to 2 mol of phosphate/mol of ERK2 by electrospray mass spectrometry. ERK2/D319N was phosphorylated in the same manner; however, the stoichiometry of phosphorylation for ERK2/D319N mutant was 0.8 -0.9 mol of phosphate/mol of protein.
Phosphatase Assay-The MKP3 phosphatase activity measurement was performed as described (27) with modifications. Briefly, the reactions (up to 100-l volume) containing various concentrations of 32 Plabeled ERK2 were set up in 1.5-ml tubes in phosphatase assay buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA), started by addition of MKP3, incubated at 30°C for appropriate amount of time, and stopped by adding charcoal termination mix (0.5 ml). The reaction mixture was vortexed thoroughly, let sit for at least 5 min at room temperature, and then centrifuged at 12, 000 ϫ g for 5 min. The released inorganic phosphate remained in the supernatant. A portion of the supernatant (0.3 ml) was removed carefully and added to a scintillation vial containing 3 ml of scintillation fluid, and the radioactivity was measured. Assays were performed in duplicate or triplicate. Reactions containing ERK2 substrate and buffer without MKP3 served as blank controls. The concentration of the released phosphate was determined from the ratio of radioactivity associated with phosphate over the specific activity of ATP used in ERK2 labeling reaction times an appropriate dilution factor. Steady state kinetic parameters were determined by non-linear least square analysis of initial velocity data obtained from six to eight different concentrations of phosphorylated ERK2. The data were fitted to the Michaelis-Menten equation using Origin (Microcal).
Phosphoamino Acid Analysis-Proteins blotted onto Immobilon-P membranes were subjected to hydrolysis in 5.7 M HCl (Pierce) at 110°C for 80 min. The hydrolysates were dried using a Speed-Vac (repeated three times) and analyzed by two-dimensional thin layer electrophoresis as described (28) with slight modifications (1.5 kV, 10 min at pH 1.9; 1.6 kV, 17 min at pH 3.5).
Tryptic Peptide Mapping-Blotted 32 P-labeled ERK2 was subjected to digestion with tosylphenylalanine chloromethyl ketone (TPCK)treated trypsin (Worthington) as described (29,30) with modifications. The 32 P-labeled ERK2 was fractionated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was rinsed with water after transfer and wrapped in Saran Wrap to keep the membrane moist. The band containing ERK2 was detected by autoradiography and excised. The membrane slices containing ERK2 were soaked immediately in 0.5% polyvinylpyrrolidone, M r 360,000 (PVP-360, Sigma) in 100 mM acetic acid for at least 30 min at 37°C. The liquid was then aspirated, and the membrane slices were washed extensively with H 2 O (5 times, 1 ml each) and then with freshly made 0.05 M NH 4 HCO 3 twice. The SCHEME 1. Potential mechanisms for the MKP3-catalyzed ERK2/pTpY dephosphorylation. In the processive mechanism, MKP3 binds the bisphosphorylated ERK2, dephosphorylates it once, slides to the second phosphorylation site in ERK2 and dephosphorylates it a second time, and then dissociates from the completely dephosphorylated ERK2. In the distributive mechanism, MKP3 binds the bisphosphorylated ERK2, dephosphorylates it once, and releases the monophosphorylated ERK2. The monophosphorylated ERK2 subsequently rebinds MKP3 and becomes dephosphorylated a second time to yield the unphosphorylated ERK2. ERK2/pTpY, ERK2 phosphorylated on both Thr 183 and Tyr 185 ; ERK2/pT and ERK2/pY, ERK2 phosphorylated on Thr 183 or Tyr 183 , respectively; P i , inorganic phosphate. membrane slices were incubated in 100 -150 l of 0.05 M NH 4 HCO 3 with 10 g of TPCK-treated trypsin (Worthington) for 4 h at 37°C, and then overnight with an additional 10 g of fresh trypsin. At the end of the digestion, 300 l of H 2 O was added to the sample(s) and centrifuged in a microcentrifuge for 5 min. The liquid was transferred to a new 1.5-ml Eppendorf tube and dried on a Speed-Vac. This was repeated twice to remove any traces of ammonium bicarbonate. The tryptic digests were dissolved in 10 l of H 2 O and subjected to one-dimensional thin layer electrophoresis at pH 8.9 for separation (fresh 1% ammonium carbonate) for 35 min at 1 kV. The tryptic peptides containing 32 Plabeled Thr(P), Tyr(P), or both were detected by autoradiography and quantitated by PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA). Quantification of each labeled species was calculated as relative proportions of the total counts in a given lane.

RESULTS
The Bisphosphorylated ERK2 Is a Highly Specific Substrate for MKP3-MKP3 is a dual specificity phosphatase, and it can remove the phosphate from both Thr(P) 183 and Tyr(P) 185 in ERK2 (19,23). However, it is not known how efficiently MKP3 can dephosphorylate and inactivate ERK2. In order to fully characterize the kinetic properties of the MKP3-catalyzed dephosphorylation of the bisphosphorylated ERK2, we prepared milligram quantities of 32 P-labeled, ERK2/pTpY (ERK2 phosphorylated on both Thr 183 and Tyr 185 ) by in vitro phosphorylation of recombinant ERK2 with a constitutively active form of MEK1 (MEK1/G7B, MEK1/⌬44 -51/S218D/M219D/N221D/ S222D) (24) and [␥-32 P]ATP (for details, see "Experimental Procedures"). To avoid possible complications from phosphorylation of MEK1 by ERK2, we used the catalytically impaired K52R mutant ERK2 for the preparation of the bisphosphorylated ERK2. ERK2/K52R has undetectable autophosphorylation activity, is unable to phosphorylate MEK1, and displays low ATPase activity (31,32). For these reasons, ERK2/K52R is also commonly used as a substrate for MEK1 kinetic studies (24,29). In our experience, we found that it was easier to achieve higher phosphorylation stoichiometry with ERK2/ K52R than with the wild-type ERK2, possibly because of the lower ATPase activity of the mutant. Analysis of the MKP3catalyzed dephosphorylation of the bisphosphorylated wildtype ERK2 and the K52R mutant yielded similar kinetic constants. Furthermore, using a quantitative procedure (25), we determined that the affinity of ERK2/K52R for MKP3 and its ability to activate the MKP3 phosphatase activity against pNPP were indistinguishable from those of the wild-type ERK2 (data not shown). Thus, most results described in this report were obtained using the kinase-impaired ERK2/K52R mutant as a MKP3 substrate.
To determine the kinetic parameters for the MKP3-catalyzed dephosphorylation of 32 P-labeled ERK2/pTpY, initial velocities were determined by following the production of radioactive inorganic phosphate. A typical set of initial velocities versus ERK2/pTpY concentration is shown in Fig. 1A. Direct curvefitting of the data to the Michaelis-Menten equation yielded k cat and K m , which were 0.084 Ϯ 0.009 s Ϫ1 and 22 Ϯ 5 nM, respectively, for the dephosphorylation of ERK2/pTpY by MKP3 at pH 7.4 and 30°C (Table I). The observed K m value for activated ERK2 is within the range of intracellular ERK2 concentration (33). The substrate specificity constant k cat /K m (3.8 ϫ 10 6 M Ϫ1 s Ϫ1 ) for the MKP3-catalyzed ERK2/pTpY dephosphorylation is more than 6 orders of magnitude higher than those of the MKP3-catalyzed pNPP hydrolysis (2.0 M Ϫ1 s Ϫ1 ) and the MKP3-catalyzed dephosphorylation of the ERK2derived phosphopeptide containing both Thr(P) 183 and Tyr(P) 185 (DHTGFLpTEpYVATR) (5 M Ϫ1 s Ϫ1 ) (34). These results indicate that ERK2/pTpY is a highly specific substrate of MKP3 and that substrate specificity determinants involve other regions of ERK2 in addition to the activation loop harboring the pTEpY motif.
ERK2 can form a tight physical complex with MKP3 (21,22). Because ERK2 is a product of the MKP3 reaction and has high affinity for MKP3, we evaluated the ability of the unphosphorylated ERK2 to inhibit the MKP3-catalyzed dephosphorylation of ERK2/pTpY. As shown in Fig. 1B, the effect of ERK2 on the MKP3-catalyzed ERK2/pTpY dephosphorylation displayed the characteristic intersecting line pattern indicative of competitive inhibition. Thus, ERK2 acts as a competitive inhibitor of the ERK2/pTpY dephosphorylation by MKP3 with a K i value of 0.08 M, which is comparable to its K d (0.17 M) for MKP3 The reaction was carried out at 30°C in duplicate, and the product generated (inorganic phosphate) was kept below 5% of the initial substrate concentrations.

ERK2 Dephosphorylation by MKP3
determined by an activation-based binding assay (25). The MKP3-catalyzed Dephosphorylation of ERK2 Involves a Cysteinyl Phosphoenzyme Intermediate-Biochemical studies with artificial substrates (pNPP or Tyr(P)-containing peptides) have shown that the PTPase-catalyzed reaction proceeds through a double displacement mechanism, in which the phosphoryl group from the substrate is first transferred to the active site Cys residue forming a thiophosphate enzyme intermediate, which is subsequently hydrolyzed by water in the second step (9, 10, 35). It is not known whether the PTPasecatalyzed hydrolysis of a physiological protein substrate also involves a thiophosphate intermediate.
To determine whether MKP3 employs the same thiophosphate intermediate for catalysis when dephosphorylating its physiological protein substrate ERK2, 32 P-labeled ERK2/pTpY was incubated with MKP3 GST fusion protein. At appropriate time points, an aliquot of the reaction mixture was withdrawn and quenched with an equal volume of 2ϫ Laemmli SDS sample buffer. The denatured reaction mixtures were then analyzed by SDS-PAGE, and the transfer of the 32 P label from ERK2/pTpY to GST-MKP3 was visualized by autoradiography. GST-MKP3 was used in this experiment instead of His 6 -tagged MKP3 because the latter co-migrates with ERK2/pTpY on SDS-PAGE (MKP3 and ERK2 have similar molecular weight), making it difficult to detect 32 P-labeled MKP3. Additionally, the GST tag does not affect the catalytic activity of MKP3 (36). Fig.  2 shows that MKP3 was able to form a phosphoenzyme intermediate during ERK2/pTpY dephosphorylation. When the catalytic Cys 293 was mutated to a Ser, the ability of MKP3 to dephosphorylate ERK2/pTpY was completely abrogated. Furthermore, no 32 P-labeled GST-MKP3 was generated when MKP3/C293S was mixed with 32 P-labeled ERK2/pTpY (Fig. 2, lane 9). These results indicate that Cys 293 is essential for the formation of the MKP3 phosphoenzyme intermediate and that the same active site in MKP3 is responsible for the hydrolysis of both Thr(P) and Tyr(P) in ERK2.
The Monophosphorylated ERK2s Are Also Highly Efficient Substrates for MKP3-In addition to the bisphosphorylated ERK2/pTpY, both the monophosphorylated ERK2/pT and ERK2/pY have been detected in living cells (37). Monophosphorylated ERK2s can be produced by the action of MEK1, which phosphorylates ERK2 by a distributive mechanism, where MEK1 dissociates from ERK2 between the first and second phosphorylation events (29,38,39). Monophosphorylated ERK2s can also be generated through the action of serine/ threonine protein phosphatase PP2A (40) and tyrosine-specific PTP-SL (41) and HePTP (42) on bisphosphorylated ERK2. Will the monophosphorylated ERK2s serve as substrates for MKP3? We prepared 32 P-labeled ERK2/pT and ERK2/pY by treating 32 P-labeled ERK2/pTpY with tyrosine-specific HePTP and ser-ine/threonine-specific PP2A, respectively (see "Experimental Procedures"). Table II shows that the kinetic constants for the MKP3-catalyzed dephosphorylation of ERK2/pT and ERK2/pY are comparable to those of the bisphosphorylated ERK2 (Table  I). Thus, both forms of the monophosphorylated ERK2 can serve as effective MKP3 substrates, indicating that dephosphorylation of one phosphoamino acid by MKP3 does not require the presence of the other in the ERK2 substrate.
The MKP3-catalyzed ERK2/pTpY Dephosphorylation Involves an Ordered, Distributive Mechanism-In order to determine whether dephosphorylation of the bisphosphorylated ERK2 by MKP3 is processive or distributive, we set out to measure the order of ERK2/pTpY dephosphorylation by phosphoamino acid analysis. We also determined the level of the bisphosphorylated and monophosphorylated species of ERK2 during the MKP3 reaction by tryptic peptide analysis. Our results suggest that dephosphorylation of ERK2/pTpY by MKP3 involves an ordered, distributive mechanism.
To determine whether there is any preference for Tyr(P) or Thr(P) hydrolysis in the MKP3-catalyzed ERK2 dephosphorylation, we first performed a single-turnover experiment, which allows the direct observation of the conversion of substrates to intermediates and products in a single pass of the reactants through the enzyme active site. This is a type of experiment in which MKP3 is used in excess over 32 P-labeled ERK2/pTpY such that substrate binding and product release cannot be rate-limiting. We performed a single turnover experiment at pH 7.4 and 0°C (on ice), with 8.5 M MKP3 and 1.8 M ERK2/ pTpY. The reaction was manually quenched in 2ϫ SDS sample buffer, and the disappearance of Thr(P) and Tyr(P) was examined by phosphoamino acid analysis (Fig. 3). At 20 s, over 95% of Tyr(P) in ERK2/pTpY were hydrolyzed, whereas less than 30% of Thr(P) were hydrolyzed. Thus, it appears that MKP3 has a preference for Tyr(P) over Thr(P) under single-turnover conditions.
We also followed the MKP3-catalyzed dephosphorylation of ERK2/pTpY under steady state conditions at pH 7.4 and 30°C (2 M ERK2/pTpY and 50 nM MKP3) by two-dimensional phosphoamino acid analysis. The time course of ERK2/pTpY dephosphorylation showed that, under steady state condition, the initial rate for Tyr(P) hydrolysis is ϳ2-fold faster than that of Thr(P) hydrolysis (Fig. 4). 2 This is in accord with the 2-fold higher k cat value observed for ERK2/pY than ERK2/pT with MKP3 (Table II; note that the initial rate measured at high substrate concentration, i.e. Ͼ ϾK m , is proportional to k cat ). If MKP3 hydrolyzes the bisphosphorylated ERK2 via a single collision, processive mechanism, the rate of dephosphorylation of Tyr(P) 185 and Thr(P) 183 would be expected to be the same under steady state condition. Thus, our results are consistent with an ordered, distributive mechanism for ERK2/pTpY de-

ERK2 Dephosphorylation by MKP3
phosphorylation, in which MKP3 hydrolyzes Tyr(P) first to generate ERK2/pT, dissociates from ERK2/pT, and then a second MKP3 binds ERK2/pT and hydrolyzes the Thr(P) to produce unphosphorylated ERK2 (Scheme 2). To further substantiate that the MKP3-catalyzed ERK2/ pTpY dephosphorylation is ordered and distributive, we set out to determine quantitatively the amount of ERK2/pTpY, ERK2/ pT, ERK2/pY, and ERK2 as a function of time during MKP3 turnover by tryptic peptide analysis of the phospho-ERK2s. This technique has been used successfully to quantify the amounts of ERK2 in different phosphorylation states in the constitutively activated MEK1-catalyzed ERK2 phosphorylation reaction (29). We reasoned that, if MKP3 dephosphorylates the bisphosphorylated ERK2 via a distributive mechanism, monophosphorylated ERK2 should be formed in excess of the MKP3 concentration in the reaction mixture. Otherwise, in a processive mechanism, the level of monophosphorylated ERK2 species should not exceed the amount of MKP3 present in the reaction.
We quantitated the amount of bis-and monophosphorylated ERK2s during the MKP3 reaction by exhaustive tryptic peptide mapping (29). At each time point, the reaction was stopped by 2ϫ SDS sample buffer. The 32 P-labeled ERK2 was resolved by SDS-PAGE, transferred to a nitrocellulose membrane, the band containing phospho-ERK2 excised, and subjected to exhaustive trypsin digestion. The resulting tryptic peptides were separated by one-dimensional thin layer electrophoresis at pH 8.9, detected by autoradiography, and quantitated by Phosphor-Imager. As shown in Fig. 5A, the bisphosphorylated ERK2 yielded a major radiolabeled spot and two minor spots, designated as spot 1, spot 2, and spot 3, respectively. Exhaustive dephosphorylation of ERK2/pTpY by PP2A or HePTP produced monophosphorylated ERK2/pY or ERK2/pT, respectively, which were confirmed by phosphoamino acid analysis. The spot generated by PP2A-treated ERK2/pTpY had the same electrophoretic mobility as spot 2 in Fig. 5A, whereas the spot generated by HePTP-treated ERK2/pTpY co-migrated with spot 3. Therefore, spots 1, 2, and 3 correspond to bis-, mono-Tyr(P), and mono-Thr(P) phosphorylated peptides, respectively. These assignments are similar to those reported previously (29).
The amount of bis-, mono-and unphosphorylated ERK2 as a function of time during the MKP3-catalyzed dephosphorylation reaction is displayed in Fig. 5. As shown in Fig. 5B, the starting material contained minor amounts of monophosphorylated species. However, the amount of ERK2/pY did not accumulate with time, whereas the amount of ERK2/pT in the reaction mixture accumulated and quickly exceeded the amount of MKP3 present in the reaction (20 nM) (Fig. 5, B and C). The level of ERK2/pT reached a maximum at 5 min and then decreased as the double phosphorylated ERK2 began to disappear. The fact that ERK2/pY does not accumulate suggests that the MKP3catalyzed dephosphorylation of ERK2/pTpY is ordered, in which Tyr(P) 185 is preferentially dephosphorylated by MKP3. The fact that ERK2/pT accumulates and exceeds the amount of MKP3 present during the reaction suggests that MKP3 dissociates from ERK2/pT after dephosphorylating Tyr(P) 185 , consistent with a distributive mechanism. Because both ERK2/pY and ERK2/pT are efficient substrates for MKP3 (Table II), the monophosphorylated ERK2/pT can be further processed efficiently by MKP3 to produce the unphosphorylated ERK2. Collectively, these results support the conclusion that the MKP3catalyzed dephosphorylation of ERK2/pTpY involves an ordered, two-collision, distributive mechanism (Scheme 2). In this mechanism, Tyr(P) 185 in ERK2/pTpY is first hydrolyzed, followed by the release of ERK2/pT from MKP3. The preference of MKP3 for Tyr(P) hydrolysis in the context of ERK2/pTpY leads to the accumulation of ERK2/pT, which subsequently re-associates with MKP3 to undergo the second dephosphorylation event, yielding the fully dephosphorylated ERK2.
The N-terminal Domain of MKP3 Is Important for Efficient ERK2 Dephosphorylation-All MKPs share two common structural features: a conserved catalytic domain that contains the PTPase active site signature motif (H/V)CX 5 R(S/T), and an N-terminal noncatalytic domain. The N-terminal domain of MKP3 can physically associate with ERK2, and it has been proposed that the N-terminal domain may play a role in controlling MKP3 substrate specificity (21). Interestingly, the Nterminal domain is not required for the MKP3-catalyzed pNPP hydrolysis (25,34). Using a quantitative binding assay that we developed, we were able to show that the binding affinity of MKP3/⌬N151 (an N-terminally truncated MKP3, lacking the first 1-151 amino acids) for ERK2 was 138-fold lower than that of the wild-type MKP3 (25). To quantitatively assess the contribution of the N-terminal domain to the MKP3-catalyzed ERK2 dephosphorylation, we measured the kinetic parameters of MKP3/⌬N151 toward ERK2/pTpY (Table I). The k cat of MKP3/⌬N151-catalyzed ERK2/pTpY dephosphorylation was 0.023 s Ϫ1 , which is comparable to that of the wild-type MKP3. However, its k cat /K m value was more than 2 orders of magnitude (135-fold) lower than that of the wild-type MKP3, which is due largely to a dramatic increase in K m . Thus, the ability of MKP3 to bind ERK2 through its N-terminal domain contributes directly to efficient ERK2 dephosphorylation by MKP3.
The importance of binding affinity to catalytic efficiency is further corroborated by comparing the kinetic parameters of MKP3 toward ERK2/D319N and wild-type ERK2 (Table I). ERK2/D319N, a Sevenmaker type mutation in ERK2 (44), is much less sensitive to inactivation by MKPs than the wild-type ERK2 (45,46). Previous studies indicated that ERK2/D319N displays a reduced affinity for MKP3 (22,47), and the binding affinity of ERK2/D319N for MKP3 was determined to be 87-fold lower than that of the wild-type ERK2 (25). As expected, the k cat /K m for the MKP3-catalyzed dephosphorylation of ERK2/ D319N/pTpY was 50-fold lower than that with ERK2/pTpY as a substrate (Table I). In addition, MKP3 contains a "kinase interaction motif" (KIM, residues 61-75) that likely interacts directly with Asp 319 in ERK2 through Arg 65 in the KIM sequence of MKP3 (25,47). Deletion of the KIM sequence from MKP3 results in a 135-fold reduction in ERK2 binding affinity (25). Again as expected, MKP3/⌬KIM exhibited a k cat /K m similar to that of MKP3/⌬N151 with ERK2/pTpY as a substrate (data not shown), which was more than 2 orders of magnitude lower than that for the wild-type MKP3 (Table I). Interestingly, when structural elements responsible for binding to the Asp 319 region in ERK2 were removed from MKP3, the resulting MKP3/⌬N151 displayed identical k cat /K m values toward ERK2/ pTpY and ERK2/D319N/pTpY (Table I). Taken together, the results show that tight binding between ERK2 and the Nterminal domain of MKP3 dramatically decrease the apparent K m without any significant change on k cat for the MKP3-catalyzed ERK2/pTpY dephosphorylation. The results further support that this effect is largely mediated through the interaction of the KIM in MKP3 with Asp 319 in ERK2.
The N-terminal Domain Also Contributes to the Dual Specificity of MKP3-We next investigated whether the specific interaction between the N-terminal domain of MKP3 and ERK2 contributes to the dual specificity of MKP3. The amino acids Tyr and Thr differ drastically in both structure and intrinsic chemical reactivity. Furthermore, the pK a of the phenol group in Tyr (9.8) is estimated to be at least 5 orders of magnitude lower than that of the aliphatic hydroxyl group in Thr (15.0) in the context of a polypeptide (48). Therefore, Tyr(P) is intrinsically about 5 orders of magnitude more reactive than Thr(P). Thus, it is remarkable that MKP3 is capable of dephosphorylating both Tyr(P) and Thr(P) in ERK2 with almost equal efficiency (Table II).
Using monophosphorylated ERK2s as substrates, we determined the steady state kinetic parameters for the MKP3/ ⌬N151 reaction (Table II). We found that the k cat for the MKP3/ ⌬N151-catalyzed dephosphorylation of ERK2/pY is similar to that of ERK2/pT (Table II). However, the k cat /K m for ERK2/pY is 10-fold higher than that for ERK2/pT (Table II). The results suggest that MKP3/⌬N151 has a stronger preference for Tyr(P) over Thr(P) than does the wild-type MKP3 in ERK2 dephosphorylation. Indeed, it is clear that the decrease in affinity of ERK2 for MKP3/⌬N151 had a more deleterious effect on the ability of MKP3/⌬N151 to dephosphorylate Thr(P) (Fig. 6). When [ERK2/pTpY] ϭ 2 M and [MKP3/⌬N151] ϭ 0.05 M, the initial rate for Tyr(P) hydrolysis was estimated to be 5.5-fold faster than that of Thr(P) hydrolysis for the MKP3/⌬N151- FIG. 5. Time course of MKP3-catalyzed dephosphorylation of ERK2/pTpY followed by tryptic peptide mapping. A, autoradiogram of tryptic peptides derived from ERK2/pTpY and separated by thin layer electrophoresis. Lanes 1 and 2, ERK2/pTpY was treated with PP2A (0.4 unit) and HePTP (3.6 M), respectively, before trypsin digestion. Lane 3, ERK2/pTpY. The phosphoproteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The 32 Plabeled ERK2 band was excised, digested with TPCK-treated trypsin, and analyzed by thin layer electrophoresis at pH 8.9. B, thin layer electrophoresis of a tryptic digest of a time-course dephosphorylation of 32 P-labeled ERK2 by MKP3, allowing the relative proportions of ERK2/ pTpY, ERK2/pT, ERK2/pY, and ERK2 to be quantified. C, quantitative data derived from PhosphorImager analysis (average of two experiments). The concentration of MKP3 added to the reaction was 20 nM, and the total concentration of ERK2 was 2.1 M. catalyzed dephosphorylation of ERK2/pTpY (Fig. 6).
Time-course analysis of the MKP3/⌬N151-catalyzed ERK2/ pTpY reaction by tryptic peptide mapping (Fig. 7) showed that the amount of ERK2/pY did not accumulate with time, whereas the amount of ERK2/pT accumulated in large excess of the amount of MKP3/⌬151 present (100 nM). Unlike the wild-type MKP3 reaction (Fig. 5), the disappearance of ERK2/pT in the MKP3/⌬N151 reaction lagged significantly behind that for the bisphosphorylated ERK2/pTpY (Figs. 5 and 7). Thus the dephosphorylation of ERK2/pTpY by MKP3/⌬N151 is also ordered and distributive, and the rate of ERK2/pT hydrolysis by MKP3/⌬N151 is further decreased from that of ERK2/pY hydrolysis. Collectively, our results suggest that loss of the specific binding between the N-terminal domain of MKP3 and ERK2 has a 2-fold effect; it decreases the catalytic efficiency of the MKP3-catalyzed ERK2 dephosphorylation by over 2 orders of magnitude, and it compromises especially the ability of MKP3 to dephosphorylate Thr(P), leading to a more obviously distributive mechanism for the MKP3/⌬N152 reaction. Thus, specific binding of the MKP3 N-terminal domain to ERK2-P2 contributes to the dual specificity of MKP3 toward ERK2/pTpY.
The General Acid Asp 262 Is Essential for MKP3 Dual Specificity and Catalytic Efficiency-Elimination of the N-terminal domain of MKP3 increases its preference for Tyr(P) severalfold, yet MKP3/⌬N151 is still a reasonably good dual specific phosphatase. To identify additional structural elements responsible for the dual specificity, we then examined the role of the general acid in MKP3 reaction. Structural comparison between the catalytic domain of MKP3 and the dual specificity phosphatase, VHR (23), suggests that Asp 262 may serve as a general acid in MKP3, protonating the Tyr phenolate or the Thr alkoxide leaving group. To abolish the general acid functionality, we replaced Asp 262 with Asn. Using 32 P-labeled ERK2/ pTpY as a substrate, MKP3/D262N displayed a k cat value that was nearly 2 orders of magnitude slower than that of the wild-type MKP3 (0.001 s Ϫ1 versus 0.084 s Ϫ1 ). Phosphoamino acid analysis showed that MKP3/D262N was unable to dephosphorylate Thr(P) in ERK2 (Fig. 8). These results indicate that Asp 262 is essential for the dual specificity and catalytic efficiency of MKP3. DISCUSSION It is well known that both the magnitude and duration of ERK1/2 activation are important in determining cell fate (1,5). For example, in PC12 cells, transient induction of ERK1/2 activity promotes cell proliferation, whereas sustained activation of ERK1/2 drives cells into neuronal differentiation (49). The only known mechanism for ERK1/2 activation is through dual phosphorylation by MEK1/2 of the Thr and Tyr residues in the activation loop motif TEY (2)(3)(4). Because activated ERK1/2 contain both Thr(P) and Tyr(P), they could serve as substrates for all classes of protein phosphatases. Thus, ERK1/2 deactivation could occur through the action of serine/threonine protein phosphatase PP2A (40), tyrosine-specific PTPases PTP-SL and HePTP (41,42), or dual specificity MKPs (46,50,51). The fact that multiple phosphatases are involved in the inactivation of ERK1/2 suggests that phosphatases may play a crucial role in determining cellular responses to external stimuli and that the duration and extent of ERK activation may be controlled by the balanced activities of MEKs and various ERK-specific phosphatases.
At least nine different MKPs have been described (6,7). This implies a considerable complexity in the regulation of MAP kinase signaling by these phosphatases. However, despite the remarkable progress in the identification and characterization of new MKPs, the molecular basis by which MKPs distinguish and recognize various MAP kinases that they encounter in the cell remains elusive. MKP3 prefers ERK1/2 to JNK and p38 when overexpressed in mammalian cell culture (17,19). To begin to understand the molecular basis of specific ERK2 inactivation by MKP3, we have carried out a detailed mechanistic study of the MKP3-catalyzed ERK2 dephosphorylation. Our results shed light on several aspects of MKP3 catalysis, including the mechanism of ERK2 dephosphorylation and the molecular basis of substrate specificity and dual specificity. ERK2/pTpY Dephosphorylation by MKP3 Occurs by an Ordered, Distributive Mechanism-Four lines of evidence suggest that ERK2/pTpY dephosphorylation by MKP3 proceeds by an ordered, distributive mechanism. First, MKP3 displays a preference for Tyr(P) hydrolysis in bisphosphorylated ERK2 under both single-turnover and steady state conditions. Thus, dephosphorylation of Thr(P) always lags behind that of Tyr(P). If the mechanism were processive, one would expect an identical rate of hydrolysis for both Tyr(P) and Thr(P) under steady state conditions. Second, there is no accumulation of the monophosphorylated ERK2/pY during the MKP3-catalyzed ERK2/pTpY dephosphorylation (Fig. 5). This result indicates that Tyr(P) hydrolysis in ERK2/pTpY occurs first and that Thr(P) hydrolysis in ERK2/pTpY cannot happen unless Tyr(P) is already dephosphorylated. Third, monophosphorylated ERK2/pT accumulates and exceeds the amount of MKP3 in the MKP3-cata- lyzed dephosphorylation of ERK2/pTpY. This is consistent with the observation that, after the initial Tyr(P) hydrolysis, ERK2/pT falls off MKP3. In contrast, if the mechanism were processive, one would not expect to see accumulation of monophosphorylated species in excess of the catalytic amount of MKP3 present in the reaction. Finally, the monophosphorylated species generated from the first dephosphorylation event can be efficiently processed to yield the final product, the unphosphorylated ERK2, because the monophosphorylated species are equally good as substrates as the bisphosphorylated ERK2. Collectively, our results are consistent with an ordered, distributive mechanism in which MKP3 can bind ERK2/pTpY, dephosphorylate Tyr(P) first, dissociate, and release the monophosphorylated ERK2/pT. ERK2/pT can then be dephosphorylated by a second molecule of MKP3, yielding the fully dephosphorylated ERK2 (Scheme 2).
However, it is important to point out that, although our data do not support a complete processive model, they cannot exclude the possibility of a partially processive mechanism, in which some MKP3 molecules hydrolyze both Tyr(P) and Thr(P) before dissociating from ERK2. Because processivity is controlled by the relative rates at which the MKP3⅐ERK2/pT and MKP3⅐ERK2/pY complexes dissociate compared with the rates at which the MKP3⅐ERK2/pT and MKP3⅐ERK2/pY give rise to ERK2 (Scheme 1), it is conceivable that in vivo the dephosphorylation of ERK2/pTpY could become more or completely processive in the presence of appropriate scaffolding proteins or cofactors that enhance the affinity of MKP3 for ERK2. In contrast, mutations that reduce the affinity of MKP3 for ERK2 or slow down the chemistry would render the reaction more distributive, as observed in this study for MKP3/⌬N151 and MKP3/D262N. In any event, processive or distributive, our data show that MKP3 can carry out the dephosphorylation of both Thr(P) and Tyr(P) in ERK2 very efficiently. This may have significant biological implications.
It has been shown that constitutively active MEK1 carries out its dual phosphorylation of ERK2 by a distributive mechanism, and it has been argued that nonprocessive ERK2 phosphorylation is important for the fidelity and sensitivity of ERK2 signaling (29,39). The specificity of ERK2 activation should be greater under a distributive mechanism than under a processive mechanism, since activation depends upon two independent MEK1 binding events rather than one. Presumably, the action of PP2A and HePTP on ERK2/pTpY would generate monophosphorylated ERK2 species, which would allow MEK1 to activate ERK2 more rapidly under certain conditions. Efficient removal of the phosphate group from both Thr(P) and Tyr(P) of activated ERK2 by MKP3 may be important for prolonged inactivation and efficient repriming of the ERK signaling cycle.
Molecular Basis of the Dual Specificity of MKP3 toward ERK2-MKP3 is a dual specificity phosphatase and is capable of dephosphorylating efficiently both the Tyr(P) and the Thr(P) on ERK2. VHR is also a dual specificity phosphatase, and ERK2 has been suggested to be an authentic substrate for VHR (52). However, aside from the active site, the crystal structures of the catalytic domain of MKP3 (23) and VHR (53) do not provide any evidence for the existence of a second phosphoamino acid-binding site. It appears that the same active site is responsible for both Tyr(P) and Ser(P)/Thr(P) hydrolysis in dual specificity phosphatases (Ref. 35, and this work). The structural basis for the dual specificity has been attributed to the shallow active site cleft, with a depth of 5.5 Å in MKP3 (23) and 6 Å in VHR (53), which can accommodate both Tyr(P) and Thr(P). The much deeper active site pocket (9 Å) in PTPases selects exclusively Tyr(P)-containing substrates (54,55). How-ever, VHR was found to dephosphorylate only Tyr(P) in ERK2/ pTpY (52). We have confirmed that VHR can only hydrolyze Tyr(P) in ERK2/pTpY (data not shown). This suggests that the depth of the active site is not the sole determinant for dual specificity. In addition, under identical conditions used for MKP3, we determined the k cat and K m for the VHR-catalyzed dephosphorylation of 32 P-labeled ERK2/pTpY to be 0.004 Ϯ 0.001 s Ϫ1 and 3.8 Ϯ 0.9 M, respectively, by monitoring directly the release of 32 P-labeled phosphate. Thus, the k cat /K m for the VHR-catalyzed ERK2/pTpY dephosphorylation (1.1 ϫ 10 3 M Ϫ1 s Ϫ1 ) is Ͼ10 3 -fold lower than that for the MKP3 reaction (3.8 ϫ 10 6 M Ϫ1 s Ϫ1 ).
Given the huge difference in intrinsic chemical reactivity (10 5 -fold) between Tyr(P) and Thr(P), it is remarkable that MKP3 can hydrolyze Thr(P) and Tyr(P) in ERK2 with similar efficiency. What is the molecular basis for the dual specificity of MKP3? VHR is a small protein (20 kDa) made of a single phosphatase domain that shares considerable sequence homology with the catalytic domain of MKP3 (37% identity and 49% similarity). MKP3 differs from VHR in that it also contains an N-terminal non-catalytic domain, which can interact with ERK2. We initially reasoned that the high affinity binding between the N-terminal domain of MKP3 and ERK2 might be responsible for the higher MKP3 activity and compensate for the difference of 5 orders of magnitude in the intrinsic reactivity between Tyr(P) and Thr(P), resulting in dual specificity of MKP3 toward ERK2. We found that disruption of the N-terminal domain binding with ERK2 either by deletion of residues 1-151 in MKP3 or introduction of a Sevenmaker type of mutation (D319N) in ERK2 resulted in a decrease by 2 orders of magnitude in k cat /K m compared with those of the wild-type MKP3 or ERK2. However, disruption of the N-terminal domain binding with ERK2 only had a modest effect (severalfold) on the ability of MKP3 to dephosphorylate Thr(P) relative to Tyr(P) in ERK2/pTpY. Thus, the N-terminal domain of MKP3 is not a major determinant for the dual specificity of MKP3 toward ERK2.
We next evaluated the role of the general acid Asp 262 in conferring dual specificity to MKP3. The function of the general acid in PTPase catalysis is to donate a proton to the phenolate/ alkoxide oxygen to facilitate the expulsion of the leaving group (8). Phosphate dianion monoester hydrolysis displays a strong dependence on the leaving group pK a with a ␤ lg of Ϫ1.27 in uncatalyzed solution reaction (56). In contrast, little leaving group dependence is observed for the PTPase reaction (8). Thus, in effect, the presence of general acid catalysis in PTPases "neutralizes" the intrinsic difference in chemical reactivity of the different phosphate esters. When the general acid functionality is impaired, large leaving group dependence is observed in PTPase reactions (16,57,58). Thus, one would expect that removal of the general acid functionality should lead to a more severe effect on Thr(P) hydrolysis than on Tyr(P). Indeed, mutation of Asp 262 to Asn lowered the k cat for the MKP3-catalyzed dephosphorylation of Tyr(P) in ERK2/ pTpY by 2 orders of magnitude while at the same time it rendered MKP3 incapable of dephosphorylating Thr(P) in ERK2/pTpY (Fig. 8). These results indicate that the general acid Asp 262 is essential for the dual specificity and catalytic efficiency of MKP3.
The corresponding general acid, Asp 92 , is intact in VHR, yet VHR is unable to dephosphorylate Thr(P) in ERK2. One likely explanation for VHR's lack of activity toward Thr(P) in ERK2 is that the action of the general acid is substrate-dependent. It is interesting to note that the k cat (0.004 s Ϫ1 ) for VHR-catalyzed dephosphorylation of Tyr(P) in ERK2/pTpY is similar to that of the general acid-deficient VHR/D92N-catalyzed hydrolysis of free Tyr(P) amino acid (57). Thus, Asp 92 may not be functional in the VHR⅐ERK2/pTpY complex, possibly due to incorrect positioning of the general acid with respect to the substrate. This leads to reduced turnover number for ERK2/pY and even more reduced still (in fact undetectable) for ERK2/pT hydrolysis. In fact, we are aware of several precedents in which perturbation of the exact positioning of the general acid in PTPases impairs its function (58,59). Along the same line, we also note that MKP3 can only dephosphorylate Tyr(P) but not Thr(P) in the context of the synthetic bisphosphorylated peptide (DHTG-FLpTEpYVATR) derived from the activation lip of ERK2 (34). Furthermore, MKP3 is ϳ100-fold less active toward activated p38 kinase than ERK2/pTpY and it can only dephosphorylate Tyr(P) in the activated p38 (19). These results are consistent with the notion that the general acid Asp 262 in MKP3 is not functional in the presence of nonphysiological substrates.
The superiority of ERK2/pTpY to the ERK2-derived phosphopeptide and the bisphosphorylated p38 as a MKP3 substrate most likely results from specific protein-protein interactions between ERK2 and MKP3 that are not available between the ERK2 phosphopeptide and MKP3 or between bisphosphorylated p38 and MKP3. Specific protein-protein interactions between ERK2 and MKP3 may be required to favorably orient the general acid and the leaving group for highly efficient ERK2 dephosphorylation. Binding of a small peptide substrate or a nonspecific substrate to MKP3 may not be sufficient to enable such a conformational change in MKP3 so that the general acid Asp 262 will not function in catalysis. We note that, even in the absence of the N-terminal domain, the catalytic domain of MKP3 is still able to dephosphorylate both Tyr(P) and Thr(P) in ERK2 (Ref. 23, and this work). Thus, it appears that binding of ERK2/pTpY to the MKP3 catalytic domain is sufficient to organize the structure within the MKP3 active site such that Asp 262 will be correctly positioned to participate in catalysis. In contrast, the interaction between VHR and ERK2/ pTpY may not allow the general acid to function properly. In the context of the above discussion and the fact that the value of k cat /K m for the VHR-catalyzed ERK/pTpY dephosphorylation is at least 1000-fold lower than that for the MKP3 reaction, it is debatable whether ERK2 is a physiological substrate for VHR.
When the data are taken together, the molecular basis for efficient dual dephosphorylation of ERK2/pTpY by MKP3 may lie in the specific interactions between ERK2 and MKP3 that are not possible between nonspecific substrates and MKP3. The specific interactions between ERK2 and MKP3 may induce a conformational change in MKP3 (see below) that is important for the precise orientation of the geometry of the general acid and other active site residues with respect to the phosphoamino acid for efficient dephosphorylation.
A Substrate-induced Fit Mechanism for MKP3 Activation and Specificity-MKP3 exhibits very low activity toward small aryl phosphates (e.g. pNPP and Tyr(P); Ref. 36) and the bisphosphorylated peptide derived from the activation lip of ERK2 (DHTGFLpTEpYVATR; Ref. 34). In fact, the k cat /K m for the MKP3-catalyzed hydrolysis of ERK2/pTpY (3.8 ϫ 10 6 M Ϫ1 s Ϫ1 ) is 10 6 -fold higher than those for the hydrolysis of pNPP or the ERK2-derived phosphopeptide. In addition, MKP3 is unable to dephosphorylate a range of phosphoproteins including myelin basic protein, casein, myosin light chain, and phosphorylase (19). This strikingly high substrate specificity displayed by MKP3 may be a consequence of both high affinity binding between ERK2 and MKP3 and specific ERK2-induced MKP3 activation.
Theoretically, high affinity interaction between MKP3 and ERK2 can increase the catalytic efficiency either by a recruit-ment mechanism, in which ERK2 is tethered so that the effective local concentration of the phosphorylated TEP motif is increased, or by an allosteric mechanism, in which binding interactions away from the active site causes conformational changes in the active site to enhance catalysis. In reality both mechanisms may be operative. Direct comparison between the mutants and the wild-type reactions provides a mechanistic basis for the understanding of how high affinity protein-protein interactions increase the catalytic efficiency in the MKP3-catalyzed ERK2/pTpY dephosphorylation.
The 10 6 -fold higher activity of MKP3 for ERK2/pTpY, as compared with that for the ERK2-derived phosphopeptide harboring the same pTEpY motif, indicates that there is no structural complementarity between the peptide substrate and MKP3 and that the peptide by itself is insufficient to induce full activation of MKP3. This also suggests that structural features outside the immediate vicinity of the pTEpY site of ERK2 play an important role for the high specificity of MKP3 for ERK2. Indeed, Asp 319 , which is located on the opposite side of ERK2 molecule where the pTEpY kinase activation loop resides (4), is important for high affinity ERK2/MKP3 interaction (22,25,47). In addition, we have shown that recognition and activation of MKP3 by ERK2 involves multiple regions of MKP3, some of which are important for high affinity binding while others are more important for the ERK2-induced MKP3 activation (25). The N-terminal domain of MKP3 contains a KIM motif (residues 61-75), which likely interacts with Asp 319 in ERK2. Disruption of this binding interaction (either by deletion of the KIM or the N-terminal domain in MKP3, or by mutation of Asp 319 to Asn in ERK2) reduces both the affinity of MKP3 for ERK2 (25) and the k cat /K m for ERK2 dephosphorylation by 2 orders of magnitude. However, disruption of the N-terminal domain binding with ERK2 had only a modest effect (severalfold) on the ability of MKP3/⌬N151 to dephosphorylate Thr(P) relative to Tyr(P) in ERK2/pTpY. Thus, the major function of the MKP3 N-terminal domain is to increase the "effective concentration" of the ERK2 pTEpY motif in the vicinity of the active site of MKP3 for dephosphorylation. This leads to a 100-fold increase in catalytic efficiency (k cat /K m ) for the MKP3catalyzed ERK2 dephosphorylation and only a modest effect on the ERK2-induced MKP3 activation, which is manifested in MKP3 dual specificity. This is consistent with the observation that, although the N-terminal domain of MKP3 is important (2 orders of magnitude) for high affinity ERK2 binding, MKP3/ ⌬151 can still be fully activated by ERK2 (25).
It is important to point out that, even in the absence of the N-terminal domain, the catalytic domain of MKP3 is still capable of dephosphorylating both Tyr(P) and Thr(P) in ERK2 (Ref. 23 and this work). Furthermore, the k cat /K m for the MKP3/⌬151-catalyzed ERK2/pTpY dephosphorylation is still 10 4 -fold higher than that for the hydrolysis of the ERK2-derived phosphopeptide. This indicates that binding of ERK2 to the MKP3 catalytic domain is sufficient to organize the structure within the MKP3 active site such that Asp 262 will be correctly positioned to participate in catalysis. Thus, in addition to the contribution from the "tethering" effect (100-fold) between MKP3 N-terminal domain and ERK2, the strikingly high substrate specificity displayed by MKP3 may also be a consequence of a substrate-induced activation mechanism (10 4fold) in which binding of the native substrate ERK2 to the C-terminal catalytic domain of MKP3 induces MKP3 to adopt a more reactive conformation, whereas binding of nonspecific substrates does not lead to MKP3 activation.
Structural and biochemical studies support the hypothesis that conformational reorganization in the active site is required for MKP3 to achieve full activity (23,36,60). The crystal structure of the catalytic domain of MKP3 in the absence of its substrate reveals a distorted active site structure that is incompetent for catalysis (23). Unlike other PTPase structures, the MKP3 active site residues (Cys 293 and Arg 299 ) are misaligned and the general acid (Asp 262 ) is positioned away from the active site. Interestingly, although phosphorylated ERK2 is a substrate for MKP3, binding of ERK2 to MKP3 triggers activation of MKP3 phosphatase activity toward pNPP (22). Kinetic evidence suggests that ERK2 binding to MKP3 elicit activation of MKP3 activity by facilitating the repositioning of active site residues and general acid loop closure in MKP3 (36).
Binding of a small peptide substrate or a nonspecific substrate to MKP3 may not be sufficient to enable such a conformational change in MKP3 so that the general acid Asp 262 will not function in catalysis.
Taken together, our results suggest that MKP3 substrate specificity result from a combination of tight binding of its physiological substrate and specific substrate-induced enzyme activation. Thus, MKP3 substrate specificity is linked to the ability of the substrate to induce productive orientation in the active site. This provides a powerful mechanism to ensure high fidelity in ERK2 inactivation by MKP3. In this mechanism, MKP3 exists in latent, inactive states and upon association with specific ERK2 substrate, MKP3 is activated leading to selective inactivation of ERK2.
In summary, we have shown that the MKP3-catalyzed ERK2 dephosphorylation goes through a cysteinyl phosphoenzyme intermediate. MKP3 dephosphorylates both Tyr(P) and Thr(P) in ERK2 efficiently albeit with preference for Tyr(P). MKP3 uses an ordered, distributive mechanism for ERK2 dephosphorylation in which MKP3 dissociates from ERK2 between each dephosphorylation event. Quantitative comparison of the kinetics of the wild-type MKP3 reaction with those of MKP3 mutants lacking N-terminal interaction with ERK2 suggest that this binding interaction may serve to increase the "effective concentration" of ERK2. Our date show that the bisphosphorylated ERK2 is a highly specific substrate for MKP3, with a k cat /K m of 3.8 ϫ 10 6 M Ϫ1 s Ϫ1 , which is more than 6 orders of magnitude higher than those of pNPP and the ERK2-derived phosphopeptide DHTGFLpTEpYVATR. The extremely high substrate specificity of MKP3 for ERK2 may result from both high affinity binding interactions between the N-terminal domain of MKP3 and ERK2 and an ERK2 induced allosteric activation in which specific interactions between MKP3 and ERK2 enable the attainment of optimal alignment of the catalytic general acid and other active site residues with respect to the substrate for efficient catalysis and specificity.