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Susan S. Taylor - One of the best experts on this subject based on the ideXlab platform.
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nmr structural functional characterization of an oncogenic mutant of camp dependent Protein Kinase a prkaca dnajb1
Biophysical Journal, 2016Co-Authors: Adak Karamafrooz, Susan S. Taylor, Geoffrey Li, Sanford M Simon, Gianluigi VegliaAbstract:Cyclic AMP (cAMP)-dependent Protein Kinase A (cAMP-PKA) is involved in regulating a multitude of biological processes including cell growth and division, cell differentiation, as well as metabolism and immune responsiveness, as such misregulation of PKA has been implicated in tumorigenesis. Recent genomic studies have identified that the single driver of the progression of fibrolamellar hepatocellular carcinoma (FL-HCC) is a mutant of the catalytic subunit of cAMP-dependant Protein Kinase A (PKA) fused with the DNAJB1 chaperons on the N-terminus (PKA-DNAJB1). This result in upregulation of Kinase activity in vivo; however, the underlying molecular mechanism for the progression of the FL-HCC cancer by PKA-DNAJB1 is unknown. Our activity assays demonstrate that there is no significant difference in activity between the wild type enzyme and PKA-DNAJB1, however there is a substantial difference in affinity by the heat stable Protein Kinase A inhibitor (PKI). To investigate this difference we study the structure and dynamics of the wild type and PKA-DNAJB1 using NMR spectroscopy. We find that the Kinase core is largely intact, but there are allosteric changes propagated to the DNAJB1 fusion upon ligand binding. Nuclear spin relaxation measurements show that for PKA-DNAJB1 the two constructs move independently in the ps-ns timescale. Our future work will focus on understanding how differential conformational dynamics in the μs-ms timescale leads to differential specificity of binding of PKI, providing potential explanation for the constitutive activity of PKA in FL-HCC.
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role of n terminal myristylation in the structure and regulation of camp dependent Protein Kinase
Journal of Molecular Biology, 2012Co-Authors: Adam Christopher Bastidas, Michael S Deal, Jon M Steichen, Malik M Keshwani, Susan S. TaylorAbstract:The catalytic (C) subunit of cAMP-dependent Protein Kinase [Protein Kinase A (PKA)] is a major target of cAMP signaling, and its regulation is of fundamental importance to biological processes. One mode of regulation is N-myristylation, which has eluded structural and functional characterization so far because most crystal structures are of the non-myristylated enzyme, are phosphorylated on Ser10, and generally lack electron density for the first 13 residues. We crystallized myristylated wild-type (WT) PKA and a K7C mutant as binary (bound to a substrate peptide) and ternary [bound to a substrate peptide and adenosine-5′-(β,γ-imido)triphosphate] complexes. There was clear electron density for the entire N-terminus in the binary complexes, both refined to 2.0 A, and K7C ternary complex, refined to 1.35 A. The N-termini in these three structures display a novel conformation with a previously unseen helix from residues 1 to 7. The K7C mutant appears to have a more stable N-terminus, and this correlated with a significant decrease in the B-factors for the N-terminus in the myr-K7C complexes compared to the WT binary complex. The N-terminus of the myristylated WT ternary complex, refined to 2.0 A, was disordered as in previous structures. In addition to a more ordered N-terminus, the myristylated K7C mutant exhibited a 53% increase in kcat. The effect of nucleotide binding on the structure of the N-terminus in the WT Protein and the kinetic changes in the K7C Protein suggest that myristylation or occupancy of the myristyl binding pocket may serve as a site for allosteric regulation in the C-subunit.
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signaling through camp and camp dependent Protein Kinase diverse strategies for drug design
Biochimica et Biophysica Acta, 2008Co-Authors: Susan S. Taylor, Choel Kim, Cecilia Y Cheng, Simon H J Brown, Natarajan KannanAbstract:The catalytic subunit of cAMP-dependent Protein Kinase has served as a prototype for the Protein Kinase superfamily for many years while structures of the cAMP-bound regulatory subunits have defined the conserved cyclic nucleotide binding (CNB) motif. It is only structures of the holoenzymes, however, that enable us to appreciate the molecular features of inhibition by the regulatory subunits as well as activation by cAMP. These structures reveal for the first time the remarkable malleability of the regulatory subunits and the CNB domains. At the same time, they allow us to appreciate that the catalytic subunit is not only a catalyst but also a scaffold that mediates a wide variety of Protein:Protein interactions. The holoenzyme structures also provide a new paradigm for designing isoform-specific activators and inhibitors of PKA. In addition to binding to the catalytic subunits, the regulatory subunits also use their N-terminal dimerization/docking domain to bind with high affinity to A Kinase Anchoring Proteins using an amphipathic helical motif. This targeting mechanism, which localizes PKA near to its Protein substrates, is also a target for therapeutic intervention of PKA signaling.
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identification of chchd3 as a novel substrate of the camp dependent Protein Kinase pka using an analog sensitive catalytic subunit
Journal of Biological Chemistry, 2007Co-Authors: Sharmin Schauble, Charles C King, Manjula Darshi, Antonius Koller, Kavita Shah, Susan S. TaylorAbstract:Due to the numerous Kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific Kinase. To identify novel substrates of cAMP-dependent Protein Kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N(6)-substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant Protein was active and fully phosphorylated on Thr(197) and Ser(338). Based on its kinetic properties, the engineered C-subunit preferred N(6)(benzyl)-ATP and N(6)(phenethyl)-ATP over other ATP analogs, but still retained a 30 microm K(m) for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One Protein, a novel mitochondrial ChChd Protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria Proteins.
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enhanced dephosphorylation of camp dependent Protein Kinase by oxidation and thiol modification
Journal of Biological Chemistry, 2005Co-Authors: Kenneth M Humphries, Michael S Deal, Susan S. TaylorAbstract:The catalytic subunit of cAMP-dependent Protein Kinase (PKA) is phosphorylated at threonine 197 and serine 338. Phosphorylation of threonine 197, located in the activation loop, is required for coordinating the active site conformation and optimal enzymatic activity. However, this phosphorylation has not been widely appreciated as a regulatory site because of the apparent constitutive nature of the phosphorylation and the general resistance of the Kinase to phosphatase treatment. We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197. Experiments performed in vitro demonstrated that mutation (cysteine 199 to alanine), oxidation, such as by glutathionylation or internal disulfide bond formation, or alkylation of the C-subunit enhanced its ability to be dephosphorylated. Furthermore, rephosphorylation of reduced C-subunit by PDK1 created a cycle whereby the inactive Kinase could be reactivated. To demonstrate that thiol modification of PKA can lead to enhanced dephosphorylation in vivo, PC12 cells were treated with N-ethylmaleimide (NEM). Such treatment resulted in complete PKA inactivation and dephosphorylation of threonine 197. This effect of NEM was contingent upon prior treatment of the cells with PKA activators, demonstrating the resistance of the holoenzyme to thiol alkylation-mediated dephosphorylation. Our results also demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the Protein Kinase Calpha activation loop, suggesting a common mechanism of regulation among members of the AGC family of Kinases.
Nguyenhuu Xuong - One of the best experts on this subject based on the ideXlab platform.
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dynamic features of camp dependent Protein Kinase revealed by apoenzyme crystal structure
Journal of Molecular Biology, 2003Co-Authors: Pearl Akamine, Susan S. Taylor, Nguyenhuu Xuong, Lynn Ten F EyckAbstract:Abstract To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3′,5′-cyclic monophosphate (cAMP)-dependent Protein Kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This “greasy” patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and β strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other Protein Kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.
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dynamic features of camp dependent Protein Kinase revealed by apoenzyme crystal structure
Journal of Molecular Biology, 2003Co-Authors: Pearl Akamine, Susan S. Taylor, Nguyenhuu Xuong, Lynn Ten F EyckAbstract:To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent Protein Kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other Protein Kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.
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crystal structure of a transition state mimic of the catalytic subunit of camp dependent Protein Kinase
Nature Structural & Molecular Biology, 2002Co-Authors: Pearl Akamine, Nguyenhuu Xuong, Susan S. TaylorAbstract:To understand the molecular mechanism underlying phosphoryl transfer of cAMP-dependent Protein Kinase, the structure of the catalytic subunit in complex with ADP, aluminum fluoride, Mg2+ ions and a substrate peptide was determined at 2.0 A resolution. Aluminum fluoride was modeled as AlF3 in a planar geometry; it is positioned 2.3 A from both the donor oxygen of ADP and the hydroxyl group of the recipient Ser residue. In this configuration, the aluminum atom forms a trigonal bipyramidal coordination with the oxygen atoms of the donor and recipient groups at the apical positions. This arrangement suggests that aluminum fluoride mimics the transition state and provides the first direct structural evidence for the in-line mechanism of phosphoryl transfer in a Protein Kinase.
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crystal structure of a polyhistidine tagged recombinant catalytic subunit of camp dependent Protein Kinase complexed with the peptide inhibitor pki 5 24 and adenosine
Biochemistry, 1997Co-Authors: Narendra Narayana, Susan S. Taylor, Sarah J Cox, Shmuel Shaltiel, Nguyenhuu XuongAbstract:The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent Protein Kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable Protein Kinase inhibitor PKI(5−24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The “active” enzyme adopts a “closed” conformation as found in rC:PKI(5−24) [Knighton et al. (1991a,b) Science 253, 407−414, 414−420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the Protein Kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415)...
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crystal structure of the catalytic subunit of camp dependent Protein Kinase complexed with magnesium atp and peptide inhibitor
Biochemistry, 1993Co-Authors: Jianhua Zheng, Susan S. Taylor, Nguyenhuu Xuong, D R Knighton, Lynn Ten F Eyck, Rolf Karlsson, Janusz M SowadskiAbstract:The structure of a ternary complex of the catalytic subunit of cAMP-dependent Protein Kinase, MgATP, and a 20-residue inhibitor peptide was determined at a resolution of 2.7 A using the difference Fourier technique starting from the model of the binary complex (Knighton et al., 1991a). The model of the ternary complex was refined using both X-PLOR and TNT to an R factor of 0.212 and 0.224, respectively. The orientation of the nucleotide and the interactions of MgATP with numerous conserved residues at the active site of the enzyme are clearly defined. The unique Protein Kinase nucleotide binding site consists of a five-stranded antiparallel beta-sheet with the base buried in a hydrophobic site along beta-strands 1 and 2 and fixed by hydrogen bonds to the N6 amino and N7 nitrogens. The small lobe secures the nucleotide via a glycine-rich loop and by ion pairing with Lys72 and Glu91. While the small lobe fixes the nontransferable alpha- and beta-phosphates in this inhibitor complex, the gamma-phosphate is secured by two Mg2+ ions and interacts both directly and indirectly with several residues in the large lobe--Asp184, Asn171, Lys168. Asp166 is positioned to serve as a catalytic base. The structure is correlated with previous chemical evidence, and the features that distinguish this nucleotide binding motif from other nucleotide binding Proteins are delineated.
Istvan Mody - One of the best experts on this subject based on the ideXlab platform.
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γ hydroxybutyrate induces cyclic amp responsive element binding Protein phosphorylation in mouse hippocampus an involvement of gabab receptors and camp dependent Protein Kinase activation
Neuroscience, 2006Co-Authors: X Ren, Istvan ModyAbstract:Abstract γ-Hydroxybutyrate is a widely used recreational drug. Its abuse has been associated with cognitive impairments and development of tolerance and dependence. However, the neural mechanisms underlying these effects remain unclear. In the present study we investigated the possible cellular signaling mechanisms that might mediate γ-hydroxybutyrate’s action. Acute administration of γ-hydroxybutyrate (500 mg/kg, i.p.) was found to cause a rapid and long-lasting increase in the phosphorylation level of the cAMP-responsive element-binding Protein in mouse (C57/BL6) hippocampus. Pretreatment with the specific GABA B receptor antagonist [3-[1-(R)-[(3-cyclohexylmethyl)hydroxyphosphinyl]-2-(S)-hydroxy-propyl]amino]ethyl]-benzoic acid (20 mg/kg, i.p.) prevented the action of γ-hydroxybutyrate, confirming a GABA B receptor-mediated mechanism. In addition, acute γ-hydroxybutyrate administration induced a significant increase in cytosolic cAMP-dependent Protein Kinase activity in the hippocampus, and pretreatment with the cAMP-dependent Protein Kinase inhibitor H-89 could prevent the effect of γ-hydroxybutyrate on cAMP-responsive element-binding Protein phosphorylation, indicating a direct involvement of cAMP-dependent Protein Kinase in γ-hydroxybutyrate-induced cAMP-responsive element-binding Protein phosphorylation. On the other hand, the increased expression of phosphorylated cAMP-responsive element-binding Protein was not observed in the hippocampus of mice subjected to repeated γ-hydroxybutyrate exposure, suggesting the development of a γ-hydroxybutyrate-induced desensitization of the signaling pathway leading to cAMP-responsive element-binding Protein activation. Since cAMP-responsive element-binding Protein activation has been implicated in a variety of neural plasticities, our findings may have revealed a new mechanism underlying γ-hydroxybutyrate-induced neuroadaptations.
Pearl Akamine - One of the best experts on this subject based on the ideXlab platform.
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dynamic features of camp dependent Protein Kinase revealed by apoenzyme crystal structure
Journal of Molecular Biology, 2003Co-Authors: Pearl Akamine, Susan S. Taylor, Nguyenhuu Xuong, Lynn Ten F EyckAbstract:To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3',5'-cyclic monophosphate (cAMP)-dependent Protein Kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This "greasy" patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and beta strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other Protein Kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.
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dynamic features of camp dependent Protein Kinase revealed by apoenzyme crystal structure
Journal of Molecular Biology, 2003Co-Authors: Pearl Akamine, Susan S. Taylor, Nguyenhuu Xuong, Lynn Ten F EyckAbstract:Abstract To better understand the mechanism of ligand binding and ligand-induced conformational change, the crystal structure of apoenzyme catalytic (C) subunit of adenosine-3′,5′-cyclic monophosphate (cAMP)-dependent Protein Kinase (PKA) was solved. The apoenzyme structure (Apo) provides a snapshot of the enzyme in the first step of the catalytic cycle, and in this unliganded form the PKA C subunit adopts an open conformation. A hydrophobic junction is formed by residues from the small and large lobes that come into close contact. This “greasy” patch may lubricate the shearing motion associated with domain rotation, and the opening and closing of the active-site cleft. Although Apo appears to be quite dynamic, many important residues for MgATP binding and phosphoryl transfer in the active site are preformed. Residues around the adenine ring of ATP and residues involved in phosphoryl transfer from the large lobe are mostly preformed, whereas residues involved in ribose binding and in the Gly-rich loop are not. Prior to ligand binding, Lys72 and the C-terminal tail, two important ATP-binding elements are also disordered. The surface created in the active site is contoured to bind ATP, but not GTP, and appears to be held in place by a stable hydrophobic core, which includes helices C, E, and F, and β strand 6. This core seems to provide a network for communicating from the active site, where nucleotide binds, to the peripheral peptide-binding F-to-G helix loop, exemplified by Phe239. Two potential lines of communication are the D helix and the F helix. The conserved Trp222-Phe238 network, which lies adjacent to the F-to-G helix loop, suggests that this network would exist in other Protein Kinases and may be a conserved means of communicating ATP binding from the active site to the distal peptide-binding ledge.
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crystal structure of a transition state mimic of the catalytic subunit of camp dependent Protein Kinase
Nature Structural & Molecular Biology, 2002Co-Authors: Pearl Akamine, Nguyenhuu Xuong, Susan S. TaylorAbstract:To understand the molecular mechanism underlying phosphoryl transfer of cAMP-dependent Protein Kinase, the structure of the catalytic subunit in complex with ADP, aluminum fluoride, Mg2+ ions and a substrate peptide was determined at 2.0 A resolution. Aluminum fluoride was modeled as AlF3 in a planar geometry; it is positioned 2.3 A from both the donor oxygen of ADP and the hydroxyl group of the recipient Ser residue. In this configuration, the aluminum atom forms a trigonal bipyramidal coordination with the oxygen atoms of the donor and recipient groups at the apical positions. This arrangement suggests that aluminum fluoride mimics the transition state and provides the first direct structural evidence for the in-line mechanism of phosphoryl transfer in a Protein Kinase.
X Ren - One of the best experts on this subject based on the ideXlab platform.
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γ hydroxybutyrate induces cyclic amp responsive element binding Protein phosphorylation in mouse hippocampus an involvement of gabab receptors and camp dependent Protein Kinase activation
Neuroscience, 2006Co-Authors: X Ren, Istvan ModyAbstract:Abstract γ-Hydroxybutyrate is a widely used recreational drug. Its abuse has been associated with cognitive impairments and development of tolerance and dependence. However, the neural mechanisms underlying these effects remain unclear. In the present study we investigated the possible cellular signaling mechanisms that might mediate γ-hydroxybutyrate’s action. Acute administration of γ-hydroxybutyrate (500 mg/kg, i.p.) was found to cause a rapid and long-lasting increase in the phosphorylation level of the cAMP-responsive element-binding Protein in mouse (C57/BL6) hippocampus. Pretreatment with the specific GABA B receptor antagonist [3-[1-(R)-[(3-cyclohexylmethyl)hydroxyphosphinyl]-2-(S)-hydroxy-propyl]amino]ethyl]-benzoic acid (20 mg/kg, i.p.) prevented the action of γ-hydroxybutyrate, confirming a GABA B receptor-mediated mechanism. In addition, acute γ-hydroxybutyrate administration induced a significant increase in cytosolic cAMP-dependent Protein Kinase activity in the hippocampus, and pretreatment with the cAMP-dependent Protein Kinase inhibitor H-89 could prevent the effect of γ-hydroxybutyrate on cAMP-responsive element-binding Protein phosphorylation, indicating a direct involvement of cAMP-dependent Protein Kinase in γ-hydroxybutyrate-induced cAMP-responsive element-binding Protein phosphorylation. On the other hand, the increased expression of phosphorylated cAMP-responsive element-binding Protein was not observed in the hippocampus of mice subjected to repeated γ-hydroxybutyrate exposure, suggesting the development of a γ-hydroxybutyrate-induced desensitization of the signaling pathway leading to cAMP-responsive element-binding Protein activation. Since cAMP-responsive element-binding Protein activation has been implicated in a variety of neural plasticities, our findings may have revealed a new mechanism underlying γ-hydroxybutyrate-induced neuroadaptations.