Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate<sup>,</sup>

Robinson, Megan J.; Harkins, P.C.; Zhang, Jiandong; Baer, Richard; Haycock, John W.; Cobb, Melanie H.; Goldsmith, Elizabeth J.

doi:10.1021/bi952723e

Cited by 134 publications

(134 citation statements)

References 40 publications

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“…This lysine is conserved in most kinases where it interacts with the α and β phosphates of ATP. Mutants of this lysine in other kinases, including lck (42), erk2 (41) and PKA (54), are also capable of binding ATP but are catalytically impaired. In PKR, the K296R mutation decreases adenosine nucleotide K d values by 3-6 fold but, in erk2 the analogous lysine → arginine mutation slightly increases the K m for ATP, suggesting that the lysine -ATP interaction does not contribute strongly to the overall binding affinity.…”

Section: Discussionmentioning

confidence: 99%

Unactivated PKR Exists in an Open Conformation Capable of Binding Nucleotides

et al. 2006

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The dsRNA-activated protein kinase, PKR, plays a pivotal role in the cellular antiviral response. PKR contains an N-terminal dsRNA binding domain (dsRBD) and a C-terminal kinase domain. An autoinhibition model has been proposed in which latent PKR exists in a closed conformation where the substrate binding cleft of the kinase is blocked by the dsRBD. Binding to dsRNA activates the enzyme by inducing an open conformation and enhancing dimerization. We have tested this model by characterizing the affinity and kinetics of nucleotide substrate binding to PKR. The fluorescent nucleotide mant-AMPPNP binds to unactivated PKR with K d ~ 30 μM and the affinity is not strongly affected by autophosphorylation or binding to dsRNA. Biphasic binding kinetics are observed where the fast phase depends on nucleotide concentration but the slow phase is ligand-independent. The kinetic data fit to a two-step model of ligand binding followed by a slow conformation change. The kinetics are also not strongly affected by phosphorylation state or dsRNA binding. Thus, the equilibrium and kinetic data indicate that substrate accessibility of the kinase is not modulated by PKR activation state as predicted by the autoinhibition model. In atomic force microscopy images, monomers of the latent protein are resolved with three separate regions linked by flexible, bridge-like structures. Resolution of the individual domains in the images supports a model in which unactivated PKR exists in an open conformation where the kinase domain is accessible and capable of binding substrate.PKR is a soluble protein kinase which is activated by dsRNA (1). PKR is constitutively expressed in most mammalian cell types but it is induced by type 1 interferons and plays a central role in the innate immunity defense against viral infection (2). In addition, PKR has been implicated in a variety of cellular signal transduction pathways (3). PKR is a member of the family of protein kinases which phosphorylate eIF2α, resulting in a blockage of translational initiation (4). Production of dsRNA during viral infection leads to PKR activation and inhibition of protein synthesis and apoptosis (5). PKR contains an N-terminal double-stranded RNA binding domain (dsRBD) and a C-terminal kinase domain. The dsRBD consists of two tandem copies of a widely distributed double stranded RNA binding * To whom correspondence may be addressed: (860) 486-4333 (telephone), james.cole@uconn.edu. Table S1: Relative contribution of the fast phase for association of mant-AMPNP with PKR. This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Information NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 2. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript motif (6). In an NMR structure of a PKR dsRBD construct, each of the dsRNA binding motifs adopts the canonical αβββα fold with a ~20 amino acid intervening region that is unstructured in the absence of dsRNA (7). The PKR kinas...

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Section: Discussionmentioning

confidence: 99%

Unactivated PKR Exists in an Open Conformation Capable of Binding Nucleotides

et al. 2006

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“…We prepared bisphosphorylated ERK2 with both the wild-type and the catalytically impaired K52R mutant ERK2 by in vitro phosphorylation with a constitutively active form of MEK1 (MEK1/ G7B) (9,27). We found that it was easier to achieve full phosphorylation stoichiometry with ERK2/K52R than with the wild-type ERK2, possibly because of the lower ATPase activity of the mutant (39). The homogeneity and phosphorylation stoichiometry of the bisphosphorylated ERK2 preparations were verified by SDS-PAGE and liquid chromatography-mass spectrometry as described previously (4,7,9).…”

Section: Effect Of Mutations In Heptp On Erk2 Dephosphorylation-to Gamentioning

confidence: 99%

Molecular Determinants of Substrate Recognition in Hematopoietic Protein-tyrosine Phosphatase

Huang

Zhou

Zhang

2004

Journal of Biological Chemistry

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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 Thr 183 and Tyr 185 in the activation loop. Tyr 185 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 k cat /K m of 2.6 ؋ 10 6 M ؊1 s ؊1 . In contrast, the k cat /K m 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 Thr 106 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 (ϳ20-fold increase in k cat /K m ) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (ϳ20-fold increase in k cat /K m ) by organizing the catalytic residues with respect to Tyr(P) 185 for optimal phosphoryl transfer.The mitogen-activated protein (MAP) 1 kinases are central components in cellular signaling (1-3). The extent and duration of MAP kinase activation in response to extracellular stimuli are tightly regulated in a cell type-and stimulus-dependent manner, because of the coordinated action of protein kinases and phosphatases. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the activation loop by specific MAP kinase kinases. Downregulation of MAP kinase activity is carried out by multiple protein phosphatases, including Ser/Thr-specific, Tyr-specific, and dual specificity phosphatases (4). This would inevitably lead to the formation of monophosphorylated MAP kinases. Indeed, recent evidence indicates that both forms of the monophosphorylated extracellular signal-regulated kinase 2 (ERK2), a founding member of the MAP kinase family, exist in living cells, in addition to the bisphosphorylated and unphosphorylated forms (5, 6). Interestingly, the activity of the monophosphorylated ERK2/pY and ERK2/pT is ϳ2 and 3 orders of magnitude higher than that of the unphosphorylated ERK2 and is only 1 and 2 orders of magnitude lower than that of the fully active bisphosphorylated ERK2/pTpY (7). ...

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“…O domínio C-terminal consiste principalmente de hélices-alfa, além de quatro fitas-beta curtas que contêm muitos dos resíduos envolvidos na catálise. 15,16 MAPK requerem fosforilação de resíduo de tirosina e treonina, ambas catalisadas por MEK (quinase ativadora da MAP quinase), para se tornarem ativas. 17 Como conseqüência, essas quinases são inativadas pelos três maiores grupos de fosfatases: todas as que removem fosfato de serina/treonina ou de tirosina e as fosfatases dualespecíficas, que removem fosfato de serina, treonina e tirosina.…”

Section: Mapk (Proteínas Quinases Ativadas Por Mitógenos)unclassified

Proteínas quinases: características estruturais e inibidores químicos

et al. 2009

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Recebido em 30/4/08; aceito em 29/8/08; publicado na web em 5/2/09 KINASE PROTEIN: STRUCTURAL FEATURES AND CHEMICAL INHIBITORS. Protein kinases are one of the largest protein families and they are responsible for regulation of a great number of signal transduction pathways in cells, through the phosphorylation of serine, threonine, or tyrosine residues. Deregulation of these enzymes is associated with several diseases including cancer, diabetes and inflammation. For this reason, specific inhibition of tyrosine or serine/threonine kinases may represent an interesting therapeutic approach. The most important types of protein kinases, their structural features and chemical inhibitors are discussed in this paper. Emphasis is given to the small-molecule drugs that target the ATP-binding sites of these enzymes.

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Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate^,

Cited by 134 publications

References 40 publications

Unactivated PKR Exists in an Open Conformation Capable of Binding Nucleotides

Unactivated PKR Exists in an Open Conformation Capable of Binding Nucleotides

Molecular Determinants of Substrate Recognition in Hematopoietic Protein-tyrosine Phosphatase

Proteínas quinases: características estruturais e inibidores químicos

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Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate,

Cited by 134 publications

References 40 publications

Unactivated PKR Exists in an Open Conformation Capable of Binding Nucleotides

Unactivated PKR Exists in an Open Conformation Capable of Binding Nucleotides

Molecular Determinants of Substrate Recognition in Hematopoietic Protein-tyrosine Phosphatase

Proteínas quinases: características estruturais e inibidores químicos

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Mutation of Position 52 in ERK2 Creates a Nonproductive Binding Mode for Adenosine 5‘-Triphosphate^,