Transition state analysis and requirement of Asp-262 general acid/base catalyst for full activation of dual-specificity phosphatase MKP3 by extracellular regulated kinase

Dual-specificity phosphatase MKP3 down-regulates mitogenic signaling through dephosphorylation of extracellular regulated kinase (ERK). Unlike a simple substrate-enzyme interaction, the noncatalytic, amino-terminal domain of MKP3 can bind efficiently to ERK, leading to activation of the phosphatase catalytic domain by as much as 100-fold toward exogenous substrates. It has been suggested that ERK activates MKP3 through the stabilization of the active phosphatase conformation, enabling general acid catalysis. Here, we investigated whether Asp-262 of MKP3 is the bona fide general acid and evaluated its contribution to the catalytic steps activated by ERK. Using site-directed mutagenesis, pH rate and Br?nsted analyses, kinetic isotope effects, and steady-state and rapid reaction kinetics, Asp-262 was identified as the authentic general acid catalyst, donating a proton to the leaving group oxygen during P-O bond cleavage. Kinetic isotope effects [(18)(V/K)(bridge), (18)(V/K)(nonbridge), and (15)(V/K)] were evaluated for the effect of ERK and of the D262N mutation on the transition state of the phosphoryl transfer reaction. The patterns of the three isotope effects for the reaction with native MKP3 in the presence of ERK are indicative of a reaction where the leaving group is protonated in the transition state, whereas in the D262N mutant, the leaving group departs as the anion. Even without general acid catalysis, the D262N mutant reaction is activated by ERK through increased phosphate affinity ( approximately 8-fold) and the partial stabilization of the transition state for phospho-enzyme intermediate formation ( approximately 4-fold). Based on these analyses, we estimate that dephosphorylation of phosphorylated ERK by the D262N mutant is >1000-fold lower than by native, activated MKP3. Also, the kinetic results suggest that Asp-262 functions as a general base during thiol-phosphate intermediate hydrolysis.     

Mechanism of mitogen-activated protein kinase phosphatase-3 activation by ERK2

The mitogen-activated protein kinase phosphatase 3 (MKP3)-catalyzed hydrolysis of aryl phosphates in the absence and presence of extracellular signal-regulated kinase 2 (ERK2) was investigated in order to provide insights into the molecular basis of the ERK2-induced MKP3 activation. In the absence of ERK2, the MKP3-catalyzed hydrolysis of simple aryl phosphates does not display any dependence on pH, viscosity, and the nature of the leaving group. Increased catalytic activity and enhanced affinity for oxyanions are observed for MKP3 in the presence of ERK2. In addition, normal bell-shaped pH dependence on the reaction catalyzed by MKP3 is restored in the presence of ERK2. Collectively, these results suggest that the rate-limiting step in the absence of ERK2 for the MKP3 reaction corresponds to a substrate-induced conformational change in MKP3 involving active site rearrangement and general acid loop closure. The binding of ERK2 to the N-terminal domain of MKP3 facilitates the repositioning of active site residues and speeds up the loop closure in MKP3 such that chemistry becomes rate-limiting in the presence of ERK2. Remarkably, it is found that the extent of ERK2-induced MKP3 activation is substrate dependent, with smaller activation observed for bulkier substrates. Unlike simple aryl phosphates, the MKP3-catalyzed hydrolysis of bulky polycyclic substrates exhibits bell-shaped pH rate profiles in the absence of ERK2. Furthermore, it is found that glycerol can also activate the MKP3-catalyzed reaction, increase the affinity of MKP3 for oxyanion, and restore the bell-shaped pH rate profile for the MKP3-catalyzed reaction. Thus, the rate of repositioning of catalytic groups and the reorienting of the electrostatic environment in the MKP3 active site can be enhanced not only by ERK2 but also by high affinity substrates or by glycerol.     

MAP kinase phosphatase 3 (MKP3) interacts with and is phosphorylated by protein kinase CK2alpha

Mitogen-activated protein (MAP) kinases play a central role in controlling a wide range of cellular functions following their activation by a variety of extracellular stimuli. MAP kinase phosphatases (MKPs) represent a subfamily of dual specificity phosphatases, which negatively regulate MAP kinases. Although ERK2 activity is regulated by its phosphorylation state, MKP3 is regulated by physical interaction with ERK2, independent of its enzymatic activity (Camps, M., Nichols, A., Gillieron, C., Antonsson, B., Muda, M., Chabert, C., Boschert, U., and Arkinstall, S., (1998) Science 280, 1262-1265; Farooq, A., Chaturvedi, G., Mujtaba, S., Plotnikova, O., Zeng, L., Dhalluin, C., Ashton, R., and Zhou, M. M. (2001), Mol. Cell 7, 387-399; Zhou, B., and Zhang, Z. Y. (1999) J. Biol. Chem. 274, 35526-35534). The interaction of ERK2 and MKP3 allows the reciprocal cross-regulation of their catalytic activity. Indeed, MKP3 acts as a negative regulator on ERK2-MAP kinase signal transduction activity, representing thus a negative feedback for this MAPK pathway. To identify novel proteins able to complex MKP3, we used the yeast two-hybrid system. Here we report that MKP3 and protein kinase CK2 form a protein complex, which can include ERK2. The phosphatase activity of MKP3 is then slightly increased in vitro, whereas in transfected cells, ERK2 dephosphorylation is reduced. In addition, we demonstrated that CK2 selectively phosphorylates MKP3, suggesting cross-regulation between CK2alpha and MKP3, as well as a modulation of ERK2-MAPK signaling by CK2alpha via MKP3.     

Mitogen-activated protein kinase (MAPK) phosphatase 3-mediated cross-talk between MAPKs ERK2 and p38alpha

MAPK phosphatase 3 (MKP3) is highly specific for ERK1/2 inactivation via dephosphorylation of both phosphotyrosine and phosphothreonine critical for enzymatic activation. Here, we show that MKP3 is able to effectively dephosphorylate the phosphotyrosine, but not phosphothreonine, in the activation loop of p38α in vitro and in intact cells. The catalytic constant of the MKP3 reaction for p38α is comparable with that for ERK2. Remarkably, MKP3, ERK2, and phosphorylated p38α can form a stable ternary complex in solution, and the phosphatase activity of MKP3 toward p38α substrate is allosterically regulated by ERK2-MKP3 interaction. This suggests that MKP3 not only controls the activities of ERK2 and p38α but also mediates cross-talk between these two MAPK pathways. The crystal structure of bisphosphorylated p38α has been determined at 2.1 Å resolution. Comparisons between the phosphorylated MAPK structures reveal the molecular basis of MKP3 substrate specificity.     

Multiple regions of MAP kinase phosphatase 3 are involved in its recognition and activation by ERK2

Mitogen-activated protein kinase phosphatase 3 (MKP3) is a specific regulator of extracellular signal-regulated protein kinase 2 (ERK2). Association of ERK2 with MKP3 results in a powerful increase in MKP3 phosphatase activity. To determine the molecular basis of the specific ERK2 recognition by MKP3 and the ERK2-induced MKP3 activation, we have carried out a systematic mutational and deletion analysis of MKP3. Using activation-based and competition-based assays, we are able to quantitatively evaluate the contributions that residues/regions within MKP3 make to ERK2 binding and ERK2-induced MKP3 activation. Our results show that recognition and activation of MKP3 by ERK2 involves multiple regions of MKP3. Thus, the kinase interaction motif (KIM; residues 61--75) in MKP3 plays a major role (135-fold) for high affinity ERK2 binding. The most important residue in the KIM sequence of MKP3 is Arg(65), which probably interacts with Asp(319) in ERK2. In addition to KIM, a unique sequence conserved in cytosolic MKPs (residues 161--177 in MKP3) also contributes to ERK2 binding (15-fold). However, these two regions are not essential for ERK2-induced MKP3 activation. A third ERK2 binding site is localized in the C terminus of MKP3 (residues 348--381). Although deletion of this region or mutation of the putative ERK specific docking sequence (364)FTAP(367) in this region reduces MKP3's affinity for ERK2 by less than 10-fold, this region is absolutely required for ERK2-induced MKP3 activation.     

Inhibition of mitogen-activated protein kinase phosphatase 3 activity by interdomain binding

Mitogen-activated protein (MAP) kinase phosphatase 3 (MKP3) is a cytoplasmic dual specificity phosphatase that functions to attenuate signaling via dephosphorylation and subsequent deactivation of its substrate and allosteric regulator, extracellular signal-regulated protein kinase 2 (ERK2). Expression of MKP3 has been shown to be under the control of ERK2, thus providing an elegant feedback mechanism for regulating the rate and duration of proliferative signals. Previously published studies suggest that MKP3 might serve as a tumor suppressor; however, significantly elevated, rather than reduced, levels of this protein have been reported in early lesions. Because overexpression of this phosphatase is counterintuitive to a proposed tumor suppressor function, the observed cellular tolerance suggested a self-inactivation mechanism. Using surface plasmon resonance, we have provided direct evidence of physical interaction between the N- and C-terminal domains. Kinetic analysis using dimethyl sulfoxide to activate the C-terminal fragment in the absence of ERK2 showed that the isolated C-terminal domain had higher catalytic efficiency than the similarly activated full-length protein. Furthermore, when the isolated N-terminal domain was added to the activated C-terminal domain, a dose-dependant inhibition of catalytic activity was observed. The similarity between the K(I) and K(D) values obtained indicate that interdomain binding stabilizes the inactive conformation of the catalytic site and implies that the N-terminal domain functions as an allosteric inhibitor of phosphatase activity. Finally, we have provided evidence for oligomerization of MKP3 in pancreatic cancer cells expressing elevated levels of this phosphatase.     

Intramolecular dephosphorylation of ERK by MKP3

The dual specificity mitogen-activated protein kinase phosphatase MKP3 downregulates mitogenic signaling through dephosphorylation of extracellular signal-regulated kinase (ERK). Like other MKPs, MKP3 consists of a noncatalytic N-terminal domain and a catalytic C-terminal domain. ERK binding to the N-terminal noncatalytic domain of MKP3 has been shown to increase (up to 100-fold) the catalytic activity of MKP3 toward small artificial substrates. Here, we address the function of the N-terminal domain of MKP3 in either inter- or intramolecular dephosphorylation of pERK (phosphorylated ERK) and the stoichiometry of the MKP3/pERK Michaelis complex. These are important mechanistic distinctions given the observation that ERK exists in a monomer/dimer equilibrium that is shifted toward the dimer when phosphorylated and given that MKP3 undergoes catalytic activation toward other substrates when bound to ERK. Wild-type and engineered mutants of ERK and MKP3, binding analyses, reaction kinetics, and chemical cross-linking studies were used to demonstrate that the monomer of MKP3 binds to the monomeric form of pERK and that MKP3 within the resulting heterodimer performs intramolecular dephosphorylation of pERK. This study provides the first direct evidence that MKP3 utilizes intramolecular dephosphorylation between a complex consisting of one molecule each of MKP3 and ERK. Catalytic activation and substrate tethering by MKP3 lead to a >or=4000-fold rate enhancement (k(cat)/K(m)) for dephosphorylation of pERK.     

Molecular determinants of substrate recognition in hematopoietic protein-tyrosine phosphatase

The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr183 and Tyr185 in the activation loop. Tyr185 dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with a kcat/Km of 2.6 x 10(6) m(-1) s(-1). In contrast, the kcat/Km values for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis of p-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr106 in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (approximately 20-fold increase in kcat/Km) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (approximately 20-fold increase in kcat/Km) by organizing the catalytic residues with respect to Tyr(P)185 for optimal phosphoryl transfer.     

pH Dependence of catalysis by Pseudomonas aeruginosa isochorismate-pyruvate lyase: implications for transition state stabilization and the role of lysine 42

An isochorismate-pyruvate lyase with adventitious chorismate mutase activity from Pseudomonas aerugionsa (PchB) achieves catalysis of both pericyclic reactions in part by the stabilization of reactive conformations and in part by electrostatic transition-state stabilization. When the active site loop Lys42 is mutated to histidine, the enzyme develops a pH dependence corresponding to a loss of catalytic power upon deprotonation of the histidine. Structural data indicate that the change is not due to changes in active site architecture, but due to the difference in charge at this key site. With loss of the positive charge on the K42H side chain at high pH, the enzyme retains lyase activity at ～100-fold lowered catalytic efficiency but loses detectable mutase activity. We propose that both substrate organization and electrostatic transition state stabilization contribute to catalysis. However, the dominant reaction path for catalysis is dependent on reaction conditions, which influence the electrostatic properties of the enzyme active site amino acid side chains.     

The mechanism of dephosphorylation of extracellular signal-regulated kinase 2 by mitogen-activated protein kinase phosphatase 3.

The mitogen-activated protein (MAP) kinase phosphatase-3 (MKP3) is a dual specificity phosphatase that specifically 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 TXY 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 x 10(6) m(-1) s(-1), which is more than 6 orders of magnitude higher than that for small molecule aryl phosphates and an ERK2-derived phosphopeptide encompassing the pTEpY motif. This strikingly high substrate specificity displayed by MKP3 may result from a combination of high affinity binding interactions between the N-terminal domain of MKP3 and ERK2 and specific ERK2-induced allosteric activation of the MKP3 C-terminal phosphatase domain.     

Redox regulation of MAP kinase phosphatase 3

MAP kinase phosphatase 3 (MKP3) is a protein tyrosine phosphatase (PTP) for which in vivo evidence suggests that regulation can occur by oxidation and/or reduction of the active site cysteine. Using kinetics and mass spectrometry, we have probed the biochemical details of oxidation of the active site cysteine in MKP3, with particular focus on the mechanism of protection from irreversible inactivation to the sulfinic or sulfonic acid species. Like other PTPs, MKP3 was found to be rapidly and reversibly inactivated by mild treatment with hydrogen peroxide. We demonstrate that unlike the case for some PTPs, the sulfenic acid of the active site cysteine in MKP3 is not stabilized in the active site but instead is rapidly trapped in a re-reducible form. Unlike the case for other PTPs, the sulfenic acid in MKP3 does not form a sulfenyl-amide species with its neighboring residue or a disulfide with a single proximate cysteine. Instead, multiple cysteines distributed in both the N-terminal substrate-binding domain (Cys147 in particular) and the C-terminal catalytic domain (Cys218) are capable of rapidly and efficiently trapping the sulfenic acid as a disulfide. Our results extend the diversity of mechanisms utilized by PTPs to prevent irreversible oxidation of their active sites and expand the role of the N-terminal substrate recognition domain in MKP3 to include redox regulation.     

Crystal structure of the catalytic domain of human MAP kinase phosphatase 5: structural insight into constitutively active phosphatase

MAP kinase phosphatase 5 (MKP5) is a member of the mitogen-activated protein kinase phosphatase (MKP) family and selectively dephosphorylates JNK and p38. We have determined the crystal structure of the catalytic domain of human MKP5 (MKP5-C) to 1.6 A. In previously reported MKP-C structures, the residues that constitute the active site are seriously deviated from the active conformation of protein tyrosine phosphatases (PTPs), which are accompanied by low catalytic activity. High activities of MKPs are achieved by binding their cognate substrates, representing substrate-induced activation. However, the MKP5-C structure adopts an active conformation of PTP even in the absence of its substrate binding, which is consistent with the previous results that MKP5 solely possesses the intrinsic activity. Further, we identify a sequence motif common to the members of MKPs having low catalytic activity by comparing structures and sequences of other MKPs. Our structural information provides an explanation of constitutive activity of MKP5 as well as the structural insight into substrate-induced activation occurred in other MKPs.     

Over-expression and refolding of MAP kinase phosphatase 3

MAP kinase phosphatase 3 (MKP3, also known as DUSP6 and PYST1) is involved in extracellular signal receptor kinase (ERK) regulation and functions as a specific phosphatase to the activated (phosphorylated) forms of ERK1 and ERK2. MKP3 displays allosteric activation, which aids in tightly regulating its function to ERK substrates, but not other related MAPKs. Due to MKP3's specificity for the ERK signaling pathway, the development of specific activators or inhibitors to the enzyme have been suggested in order to expressly influence the ERK1 and ERK2 pathways. To produce the high yields of MKP3 protein necessary for physico-chemical characterization of MKP3 and for high throughput screening of its small-molecule activators and inhibitors, we have cloned, purified and, and identified refolding conditions suitable for producing full-length, human MKP3 from Escherichia coli inclusion bodies. Furthermore, we demonstrate the use of a 96-well plate format refolding assay in which the ERK-induced activity of MKP3 is simulated by 33% DMSO. The assay allowed for rapid detection of MKP3's function following a refolding screen in the absence of ERK and thus provides quick and inexpensive testing of MKP3 activity. Following screening, the refolded product was confirmed to be correctly folded by steady-state kinetic analysis and by the CD spectroscopy-determined secondary structure content. CD data were consistent with 36% helix and 14% sheet, which compared to an expected 32.9% helix and 12.4% sheet. These data indicated that MKP3 was properly folded, making it a suitable protein for use in functional studies.     

Mapping ERK2-MKP3 binding interfaces by hydrogen/deuterium exchange mass spectrometry

ERK2, a prototypic member of the MAPK family, plays a central role in regulating cell growth and differentiation. MKP3, an ERK2-specific phosphatase, terminates ERK2 signaling. To understand the molecular basis of ERK2 recognition by MKP3, we carried out hydrogen/deuterium exchange mass spectrometry experiments to map the interaction surfaces between the two proteins. The results show that the exquisite specificity of MKP3 for ERK2 is governed by two distinctive protein-protein interactions. To increase the "effective concentration" of the interacting molecules, the kinase interaction motif in MKP3 ((64)RRLQKGNLPVR(74)) and an MKP3-specific segment ((101)NSSDWNE(107)) bind the common docking site in ERK2 defined by residues in L(16), L(5), beta(7)-beta(8), and alpha(d)-L(8)-alpha(e), located opposite the kinase active site. In addition to this "tethering" effect, additional interactions between the (364)FTAP(367) sequence in MKP3 and the ERK2 substrate-binding site, formed by residues in the activation lip and the P+1 site (beta(9)-alpha(f) loop), L(13) (alpha(f)-alpha(g) loop), and the MAPK insert (L(14)-alpha(1L14)-alpha(2L14)), are essential for allosteric activation of MKP3 and formation of a productive complex whereby the MKP3 catalytic site is correctly juxtaposed to carry out the dephosphorylation of phospho-Thr(183)/phospho-Tyr(185) in ERK2. This bipartite protein-protein interaction model may be applicable to the recognition of other MAPKs by their cognate regulators and substrates.     

Probing the function of the conserved tryptophan in the flexible loop of the Yersinia protein-tyrosine phosphatase.

The involvement of the strictly conserved Trp354 residue in the catalysis of the Yersinia protein tyrosine phosphatase (PTPase) has been investigated by site-directed mutagenesis and kinetic studies. Crystallographic structural data have revealed that Trp354 interacts with the active site Arg409 and is located at one of the hinge positions of the flexible surface loop (WpD loop) which also harbors the general acid/base (Asp356) essential for catalysis [Schubert, H. L., Fauman, E. B., Stuckey, J. A., Dixon, J. E. & Saper, M. A. (1995) Protein Sci. 4, 1904-1913]. Two mutants were constructed and expressed that contained the Trp354-->Phe and Trp354-->Ala substitutions. The K(m) of the W354F and W354A mutants were not significantly different from that of the wild-type. However, a major decrease in the affinity for oxyanions was observed for the mutants, which is consistent with Trp354 playing a role in aligning Arg409 for oxyanion binding. In addition replacement of Trp354 with Phe or Ala caused a decrease in kcat of 200-fold and 480-fold, respectively, and impaired the ability of the mutant enzymes to stabilize the negative charge in the leaving group at the transition state. In fact, the W354F and W354A mutants exhibited catalytic efficiency and leaving group dependency similar to those observed for the general acid-deficient PTPase D356N. These results indicate that Trp354 is an important residue that keeps the WpD loop in a catalytically competent conformation and positions the general acid/base Asp356 in the correct orientation for proton transfer.     

Proton relay mechanism of general acid catalysis by DNA topoisomerase IB.

Type IB topoisomerases cleave and rejoin DNA through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes the attack of the tyrosine nucleophile (Tyr-274) at the scissile phosphodiester. Previous studies implicated Arg-223 and His-265 in transition state stabilization and Lys-167 in proton donation to the 5'-O of the leaving DNA strand. Here we find that Arg-130 also plays a major role in leaving group expulsion. The rate of DNA cleavage by vaccinia topoisomerase mutant R130K, which was slower than wild-type topoisomerase by a factor of 10(-4.3), was stimulated 2600-fold by a 5'-bridging phosphorothiolate at the cleavage site. The catalytic defect of the R130A mutant was also rescued by the 5'-S modification (190-fold stimulation), albeit to a lesser degree than R130K. We surmise that Arg-130 plays dual roles in transition state stabilization and general acid catalysis. Whereas the R130A mutation abolishes both functions, R130K permits the transition state stabilization function (via contact of lysine with the scissile phosphate) but not the proton transfer function. Our results show that the process of general acid catalysis is complex and suggest that Lys-167 and Arg-130 comprise a proton relay from the topoisomerase to the 5'-O of the leaving DNA strand.     

Crystal structure of the MAPK phosphatase Pyst1 catalytic domain and implications for regulated activation

The crystal structure of the catalytic domain from the MAPK phosphatase Pyst1 (Pyst1-CD) has been determined at 2.35 A. The structure adopts a protein tyrosine phosphatase (PTPase) fold with a shallow active site that displays a distorted geometry in the absence of its substrate with some similarity to the dual-specificity phosphatase cdc25. Functional characterization of Pyst1-CD indicates it is sufficient to dephosphorylate activated ERK2 in vitro. Kinetic analysis of Pyst1 and Pyst1-CD using the substrate p-nitrophenyl phosphate (pNPP) reveals that both molecules undergo catalytic activation in the presence of recombinant inactive ERK2, switching from a low- to high-activity form. Mutation of Asp 262, located 5.5 A distal to the active site, demonstrates it is essential for catalysis in the high-activity ERK2-dependent conformation of Pyst1 but not for the low-activity ERK2-independent form, suggesting that ERK2 induces closure of the Asp 262 loop over the active site, thereby enhancing Pyst1 catalytic efficiency.