Transition State Analysis and Requirement of Asp-262 General Acid/Base Catalyst for Full Activation of Dual-Specificity Phosphatase MKP3 by Extracellular Regulated Kinase

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Cdc25 phosphatases are dual specificity phosphatases that dephosphorylate and activate cyclin-dependent kinases (CDKs), thereby effecting the progression from one phase of the cell cycle to the next. Despite its central role in the cell cycle, relatively little is known about the catalytic mechanism of Cdc25. In order to provide insights into the catalytic mechanism of Cdc25, we have performed a detailed mechanistic analysis of the catalytic domain of human Cdc25A. Our kinetic isotope effect results, Bronsted analysis, and pH dependence studies employing a range of aryl phosphates clearly indicate a dissociative transition state for the Cdc25A reaction that does not involve a general acid for the hydrolysis of substrates with low leaving group pK a values (5.45-8.05). Interestingly, our Bronsted analysis and pH dependence studies reveal that Cdc25A employs a different mechanism for the hydrolysis of substrates with high leaving group pK a values (8.68 -9.99) that appears to require the protonation of glutamic acid 431. Mutation of glutamic acid 431 into glutamine leads to a dramatic drop in the hydrolysis rate for the high leaving group pK a substrates and the disappearance of the basic limb of the pH rate profile for the substrate with a leaving group pK a of 8.05, indicating that glutamic acid 431 is essential for the efficient hydrolysis of substrates with high leaving group pK a . We suggest that hydrolysis of the high leaving group pK a substrates proceeds through an unfavored but more catalytically active form of Cdc25A, and we propose several models illustrating this. Since the activity of Cdc25A toward small molecule substrates is several orders of magnitude lower than toward the physiological substrate, cyclin-CDK, we suggest that the cyclin-CDK is able to preferentially induce this more catalytically active form of Cdc25A for efficient phosphothreonine and phosphotyrosine dephosphorylation.

Section: Mutation Of Acidic and Hydrophobic Residues Within The Cdc25mentioning

confidence: 73%

Section: Mutation Of Acidic and Hydrophobic Residues Within The Cdc25mentioning

confidence: 99%

mentioning

confidence: 99%

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The Catalytic Mechanism of Cdc25A Phosphatase

McCain

Catrina

Hengge

et al. 2002

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“…In the absence of ERK, MKP3 has little activity, but in the presence of ERK, the ability of MKP3 to dephosphorylate pNPP increases by nearly 100-fold. This increase in catalytic activity upon ERK binding has been attributed to a structural reorganization that triggers the closure of a flexible loop region near the MKP3 active site (26,27,29,30). In this model, loop closure repositions an aspartic acid (Asp-262), placing it in proximity with the active site cysteine and arginine residues (Cys-293 and Arg-299), which enables the acid-catalyzed hydrolysis of phosphoamino acids.…”

mentioning

confidence: 99%

Inhibition of Mitogen-activated Protein Kinase Phosphatase 3 Activity by Interdomain Binding

Mark

Aubin

Smith

et al. 2008

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 signalregulated 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.

“…The ability of enzymes such as MKP-3, MKP-1, and MKP-2 to dephosphorylate different MAP kinase isoforms selectively is driven by protein-protein interactions that are mediated by MAP kinase interaction motifs (KIMs) within the amino-terminal domains of these enzymes (10,(15)(16)(17). Substrate binding is accompanied by catalytic activation of MKPs in vitro (12, 16 -18), and this is mediated by the movement of a conserved general acid residue into a more favorable conformation for catalysis (19,20). Based on the apparent universality of this phenomenon, catalytic activation is suggested to be a general mechanism regulating substrate selectivity and activity of the MKPs.…”

mentioning

confidence: 99%

Both Nuclear-Cytoplasmic Shuttling of the Dual Specificity Phosphatase MKP-3 and Its Ability to Anchor MAP Kinase in the Cytoplasm Are Mediated by a Conserved Nuclear Export Signal

Karlsson

Mathers

Dickinson

et al. 2004

119

MAP kinase phosphatase (MKP)-3 is a cytoplasmic dual specificity protein phosphatase that specifically binds to and inactivates the ERK1/2 MAP kinases in mammalian cells. However, the molecular basis of the cytoplasmic localization of MKP-3 or its physiological significance is unknown. We have used MKP-3-green fluorescent protein fusions in conjunction with leptomycin B to show that the cytoplasmic localization of MKP-3 is mediated by a chromosome region maintenance-1 (CRM1)-dependent nuclear export pathway. Furthermore, the nuclear translocation of MKP-3 seen in the presence of leptomycin B is mediated by an active process, indicating that MKP-3 shuttles between the nucleus and cytoplasm. The aminoterminal noncatalytic domain of MKP-3 is both necessary and sufficient for nuclear export of the phosphatase and contains a single functional leucine-rich nuclear export signal (NES). Even though this domain of the protein also mediates the binding of MKP-3 to MAP kinase, we show that mutations of the kinase interaction motif which abrogate ERK2 binding do not affect MKP-3 localization. Conversely, mutation of the NES does not affect either the binding or phosphatase activity of MKP-3 toward ERK2, indicating that the kinase interaction motif and NES function independently. Finally, we demonstrate that the ability of MKP-3 to cause the cytoplasmic retention of ERK2 requires both a functional kinase interaction motif and NES. We conclude that in addition to its established function in the regulated dephosphorylation and inactivation of MAP kinase, MKP-3 may also play a role in determining the subcellular localization of its substrate. Our results reinforce the idea that regulatory proteins such as MKP-3 may play a key role in the spatio-temporal regulation of MAP kinase activity.Dual specificity (Thr/Tyr) protein phosphatases play an important role in the regulation of signaling by mitogen-activated protein (MAP) 1 kinases in eukaryotic cells (1-3). These MAP kinase phosphatases (MKPs) act in direct opposition to the MAP kinase kinases (MKKs or MEKs) to dephosphorylate and inactivate the MAP kinase, thus regulating the duration and magnitude of activation and hence the biological outcome of signaling. Studies in Saccharomyces cerevisiae, Drosophila, and chicken embryos have also demonstrated that the expression of certain MKPs is induced in response to MAP kinase activation, indicating that they are components of negative feedback loops (4 -6). Furthermore, mathematical modeling of MAP kinase pathway dynamics emphasizes the role of MKPs in determining the timing and duration of MAP kinase activation in terms of bistable (switch-like) or monostable (proportional) response to agonists (7).There are 10 distinct MKPs in mammalian cells (3), and these enzymes feature a common structure comprising a carboxyl-terminal catalytic domain with sequence similarity to the prototypic VH1 dual specificity phosphatase of vaccinia virus (8) and an amino-terminal noncatalytic domain containing two short regions of homology with the cell ...