The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Susan S Taylor - One of the best experts on this subject based on the ideXlab platform.
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an isoform specific Myristylation switch targets type ii pka holoenzymes to membranes
Structure, 2015Co-Authors: Ping Zhang, Feng Ye, Adam C Bastidas, Alexandr P Kornev, Jian Wu, Mark H Ginsberg, Susan S TaylorAbstract:Summary Cyclic AMP-dependent protein kinase (PKA) is regulated in part by N-terminal Myristylation of its catalytic (C) subunit. Structural information about the role of Myristylation in membrane targeting of PKA has been limited. In mammalian cells there are four functionally non-redundant PKA regulatory subunits (RIα, RIβ, RIIα, and RIIβ). PKA is assembled as an inactive R 2 C 2 holoenzyme in cells. To explore the role of N-Myristylation in membrane targeting of PKA holoenzymes, we solved crystal structures of RIα:myrC and RIIβ 2 :myrC 2 , and showed that the N-terminal Myristylation site in the myrC serves as a flexible "switch" that can potentially be mobilized for membrane anchoring of RII, but not RI, holoenzymes. Furthermore, we synthesized nanodiscs and showed by electron microscopy that membrane targeting through the myristic acid is specific for the RII holoenzyme. This membrane-anchoring Myristylation switch is independent of A Kinase Anchoring Proteins (AKAPs) that target PKA to membranes by other mechanisms.
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influence of n Myristylation and ligand binding on the flexibility of the catalytic subunit of protein kinase a
Biochemistry, 2013Co-Authors: Adam C Bastidas, Levi C Pierce, Ross C Walker, David A Johnson, Susan S TaylorAbstract:The catalytic (C) subunit of protein kinase A is regulated in part by cotranslational N-Myristylation and ligand binding. Using a combination of time-resolved fluorescence anisotropy and molecular dynamics (MD) simulations, we characterized the effect of N-Myristylation and ligand binding on C-subunit dynamics. Five single-site cysteine-substitution mutants of the C-subunit were engineered with and without N-terminal Myristylation and labeled with fluorescein maleimide, and time-resolved fluorescence anisotropy decays were measured to assess the flexibility of the labeled regions in the presence and absence of ligands. A parallel set of in silico experiments were performed to complement the experimental findings. These experiments showed that Myristylation produces both local and global effects on C-subunit dynamics. The local effects include stabilization of the N-terminus and myristate pocket, and the global effects include small increases in mobility along the C-tail at residue C343. Additionally, liga...
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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 C 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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Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Milind Gangal, Susan S Taylor, Teresa Clifford, Jason Deich, Xiaodong Cheng, David A JohnsonAbstract:Although the catalytic (C) subunit of cAMP-dependent protein kinase is N-myristylated, it is a soluble protein, and no physiological role has been identified for its myristyl moiety. To determine whether the interaction of the two regulatory (R) subunit isoforms (RI and RII) with the N-myristylated C subunit affects its ability to target membranes, the effect of N-Myristylation and the RI and RII subunit isoforms on C subunit binding to phosphatidylcholine/phosphatidylserine liposomes was examined. Only the combination of N-Myristylation and RII subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the RII subunit also increased the conformational flexibility of the C subunit N terminus, the effect of N-Myristylation and the RI and RII subunits on the rotational freedom of the C subunit N terminus was measured. Specifically, fluorescein maleimide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit, and the time-resolved emission anisotropy was determined. The interaction of the RII subunit, but not the RI subunit, significantly increased the backbone flexibility around the site of mutation and labeling, strongly suggesting that RII subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the N-myristate. RII subunit thus appears to serve as an intermolecular switch that disrupts of the link between the N-terminal and core catalytic domains of the C subunit to expose the N-myristate and poise the holoenzyme for interaction with membranes.
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Crystal structures of the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed conformations.
Protein Science, 1993Co-Authors: Jianhua Zheng, Susan S Taylor, D. R. Knighton, Janusz M. Sowadski, Nguyen-huu Xuong, L. F. Ten EyckAbstract:Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of cAMP-dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32, 2154-2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 A resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C-terminal 30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surrounding the glycine-rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the Myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N-terminal 40 residues that lie outside the conserved catalytic core are anchored by the N-terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and Myristylation, contribute directly to the stable structure of this enzyme.
J T Holt - One of the best experts on this subject based on the ideXlab platform.
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Myristylation of fbr v fos dictates the differentiation pathways in malignant osteosarcoma
Journal of Cell Biology, 1996Co-Authors: R M Jotte, J T HoltAbstract:Myristylation of FBR v-fos, a c-fos retroviral homologue that causes osteosarcomas in mice, determines many of its transcriptional properties in vitro. To determine whether Myristylation of FBR v-fos contributes to in vivo tumorigenicity, we examined its transforming capability in comparison to a nonmyristylated FBR v-fos (G2A-R). Retroviral infections with FBR v-fos and G2A-R transform BALB/c-3T3 cells. The number, size, and cellular morphology of foci generated by both FBR and G2A-R are indistinguishable. However, marked biological differences were found in transgenic mice expressing either the myristylated FBR v-fos or the nonmyristylated G2A-R. 11 of 26 FBR v-fos transgenic mice died as a result of gross tumor burden. None of the 28 G2A-R transgenic mice died from tumor burden, and only two of the G2A-R mice developed bone tumors. Histologic examination of the tumors reveals that the FBR v-fos bone tumors contain malignant cells with features of four cell lineages (osteocytes, chondrocytes, myocytes, and adipocytes) in an environment rich in extracellular matrix (ECM). However, the G2A-R tumors exist in an environment devoid of ECM and display malignant cells with features of adipocytes. Masson staining reveals that the ECM of the FBR tumors stains strongly for collagen. Immunohistochemical staining with collagen III antibody demonstrates an abundance of collagen III expression in this ECM. While NH2-terminal Myristylation is not required for FBR immortalization and transformation, it is essential in determining the degree of differentiation and tumorigenicity of malignant cells.
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Myristylation dependent transactivation by fbr v fos is regulated by c ebp
Journal of Biological Chemistry, 1994Co-Authors: R M Jotte, N Kamata, J T HoltAbstract:Abstract Viral oncogenes are generally believed to cause transformation through disregulated mimicry of their cellular homologues. However, here we show that FBR v-fos, unlike c-fos, transcriptionally activates unique genes in retrovirally induced chondro-osseous sarcomas. We show that FBR v-fos transactivates the collagen III and stromelysin promoters and that the unique transcriptional properties of transforming FBR depend upon its N-terminal Myristylation and the differentiation state of the cell. Deletion or mutation of the Myristylation site results in a loss of FBR v-fos transactivation in HeLa and undifferentiated 3T3-L1 preadipocyte cell lines. FBR v-fos transactivation of collagen III maps to a negative regulatory site which binds a key regulator of adipocyte differentiation, C/EBP alpha. Cotransfection of C/EBP alpha abolishes FBR v-fos transactivation of the alpha 1(III) collagen promoter. Furthermore, FBR v-fos cannot transactivate collagen III subsequent to adipocyte differentiation. We also demonstrate that collagen III transcription is reduced during adipocyte differentiation as the transcriptional activity of C/EBP alpha is concomitantly induced. Our results indicate that FBR v-fos transactivation depends upon its cotranslational Myristylation and maps to a negative regulatory region which binds C/EBP alpha.
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inhibitory effect of Myristylation on transrepression by fbr gag fos protein
Molecular and Cellular Biology, 1992Co-Authors: N Kamata, J T HoltAbstract:Abstract The myristylated v-fos product, FBR murine sarcoma virus (Gag-Fos) protein, exhibits a lower level of transrepression of the serum response element (SRE) than does c-fos protein (Fos). Mutation of the N-terminal Myristylation site in FBR protein restored SRE transrepression. Replacement of N-terminal viral Gag sequences with the Fos N terminus also restored this activity, providing additional evidence that Myristylation inhibits transrepression by FBR protein. However, the myristylated Gag domain did not inhibit SRE transrepression when fused to Fos, indicating that Myristylation of a fos protein is not by itself sufficient to prevent SRE transrepression and that C-terminal mutation is necessary to inhibit transrepression by N Myristylation. Comparison of transfection results with Fos C-terminal deletion mutants and the Fos/FBR chimeric mutant revealed that the FBR C terminus retained the potential for transrepression despite deletion of the normal Fos C terminus, whereas similar Fos deletion mutants did not. These results indicate that both N- and C-terminal mutations are required to inhibit transrepression by FBR protein and that multiple structural mutations accompanied by posttranslational protein modification alter gene regulation by FBR protein.
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Myristylation alters dna binding activity and transactivation of fbr gag fos protein
Molecular and Cellular Biology, 1991Co-Authors: N Kamata, R M Jotte, J T HoltAbstract:Abstract FBR murine sarcoma virus (gag-fos) protein, a virally transduced Fos protein, exhibits decreased gene transactivation in comparison with the cellular Fos protein. Biochemical analysis suggests that Myristylation of the virally encoded N-terminal gag region results in decreased DNA binding and transcriptional activation without affecting heterodimerization with Jun protein. These findings demonstrate that protein Myristylation can modulate gene regulation by a DNA-binding protein.
Keith A Wharton - One of the best experts on this subject based on the ideXlab platform.
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cell autonomous myristyl independent activity of the drosophila wnt wingless antagonist naked cuticle nkd
Developmental Biology, 2007Co-Authors: Chihchiang Chan, Tolga Cagatay, Shu Zhang, Keith A WhartonAbstract:Robust animal development, tissue homeostasis, and stem cell renewal requires precise control of the Wnt/β-catenin signaling axis. In the embryo of the fruit fly Drosophila melanogaster, the naked cuticle (nkd) gene attenuates signaling by the Wnt ligand Wingless (Wg) during segmentation. nkd mutants have been reported to exhibit abnormalities in wg transcription, Wg protein distribution and/or transport, and the intracellular response to Wg, but the relationship between each alteration and the molecular mechanism of Nkd action remains unclear. In addition, whether Nkd acts in a cell-autonomous or nonautonomous fashion in the embryo is not known. Mammalian Nkd homologs have N-terminal consensus sequences that direct the post-translational addition of a lipophilic myristoyl moiety, but fly and mosquito Nkd, while sharing N-terminal sequence homology, lack a myristoylation consensus sequence. Here we provide evidence that fly Nkd acts cell-autonomously in the embryo, with its N-terminus able to confer unique functional properties and membrane association that cannot be mimicked in vivo by heterologous myristoylation consensus sequences. In conjunction with our recent observation that Nkd requires nuclear localization for function, our data suggest that Nkd acts at more than one subcellular location within signal-receiving cells to attenuate Wg signaling.
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Cell-autonomous, myristyl-independent activity of the Drosophila Wnt/Wingless antagonist Naked cuticle (Nkd).
Developmental Biology, 2007Co-Authors: Chihchiang Chan, Tolga Cagatay, Shu Zhang, Keith A WhartonAbstract:Robust animal development, tissue homeostasis, and stem cell renewal requires precise control of the Wnt/β-catenin signaling axis. In the embryo of the fruit fly Drosophila melanogaster, the naked cuticle (nkd) gene attenuates signaling by the Wnt ligand Wingless (Wg) during segmentation. nkd mutants have been reported to exhibit abnormalities in wg transcription, Wg protein distribution and/or transport, and the intracellular response to Wg, but the relationship between each alteration and the molecular mechanism of Nkd action remains unclear. In addition, whether Nkd acts in a cell-autonomous or nonautonomous fashion in the embryo is not known. Mammalian Nkd homologs have N-terminal consensus sequences that direct the post-translational addition of a lipophilic myristoyl moiety, but fly and mosquito Nkd, while sharing N-terminal sequence homology, lack a myristoylation consensus sequence. Here we provide evidence that fly Nkd acts cell-autonomously in the embryo, with its N-terminus able to confer unique functional properties and membrane association that cannot be mimicked in vivo by heterologous myristoylation consensus sequences. In conjunction with our recent observation that Nkd requires nuclear localization for function, our data suggest that Nkd acts at more than one subcellular location within signal-receiving cells to attenuate Wg signaling.
Chihchiang Chan - One of the best experts on this subject based on the ideXlab platform.
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cell autonomous myristyl independent activity of the drosophila wnt wingless antagonist naked cuticle nkd
Developmental Biology, 2007Co-Authors: Chihchiang Chan, Tolga Cagatay, Shu Zhang, Keith A WhartonAbstract:Robust animal development, tissue homeostasis, and stem cell renewal requires precise control of the Wnt/β-catenin signaling axis. In the embryo of the fruit fly Drosophila melanogaster, the naked cuticle (nkd) gene attenuates signaling by the Wnt ligand Wingless (Wg) during segmentation. nkd mutants have been reported to exhibit abnormalities in wg transcription, Wg protein distribution and/or transport, and the intracellular response to Wg, but the relationship between each alteration and the molecular mechanism of Nkd action remains unclear. In addition, whether Nkd acts in a cell-autonomous or nonautonomous fashion in the embryo is not known. Mammalian Nkd homologs have N-terminal consensus sequences that direct the post-translational addition of a lipophilic myristoyl moiety, but fly and mosquito Nkd, while sharing N-terminal sequence homology, lack a myristoylation consensus sequence. Here we provide evidence that fly Nkd acts cell-autonomously in the embryo, with its N-terminus able to confer unique functional properties and membrane association that cannot be mimicked in vivo by heterologous myristoylation consensus sequences. In conjunction with our recent observation that Nkd requires nuclear localization for function, our data suggest that Nkd acts at more than one subcellular location within signal-receiving cells to attenuate Wg signaling.
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Cell-autonomous, myristyl-independent activity of the Drosophila Wnt/Wingless antagonist Naked cuticle (Nkd).
Developmental Biology, 2007Co-Authors: Chihchiang Chan, Tolga Cagatay, Shu Zhang, Keith A WhartonAbstract:Robust animal development, tissue homeostasis, and stem cell renewal requires precise control of the Wnt/β-catenin signaling axis. In the embryo of the fruit fly Drosophila melanogaster, the naked cuticle (nkd) gene attenuates signaling by the Wnt ligand Wingless (Wg) during segmentation. nkd mutants have been reported to exhibit abnormalities in wg transcription, Wg protein distribution and/or transport, and the intracellular response to Wg, but the relationship between each alteration and the molecular mechanism of Nkd action remains unclear. In addition, whether Nkd acts in a cell-autonomous or nonautonomous fashion in the embryo is not known. Mammalian Nkd homologs have N-terminal consensus sequences that direct the post-translational addition of a lipophilic myristoyl moiety, but fly and mosquito Nkd, while sharing N-terminal sequence homology, lack a myristoylation consensus sequence. Here we provide evidence that fly Nkd acts cell-autonomously in the embryo, with its N-terminus able to confer unique functional properties and membrane association that cannot be mimicked in vivo by heterologous myristoylation consensus sequences. In conjunction with our recent observation that Nkd requires nuclear localization for function, our data suggest that Nkd acts at more than one subcellular location within signal-receiving cells to attenuate Wg signaling.
Maxine L Linial - One of the best experts on this subject based on the ideXlab platform.
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efficient particle formation can occur if the matrix domain of human immunodeficiency virus type 1 gag is substituted by a Myristylation signal
Journal of Virology, 1994Co-Authors: Maxine L LinialAbstract:Lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), assemble at and bud through the cytoplasmic membrane. Both the matrix (MA) domain of Gag and its amino-terminal Myristylation have been implicated in these processes. We have created HIV-1 proviruses lacking the entire matrix domain of gag which either lack or contain an amino-terminal myristate addition sequence at the beginning of the capsid domain. Myristate- and matrix-deficient [myr(-)MA(-)] viruses produced after transient transfection are still able to assemble into particles, although the majority do not form at the plasma membrane or bud efficiently. Myristylation of the amino terminus of the truncated Gag precursor permits a much more efficient release of the mutant virions. While myr(-)MA(-) particles were inefficient in proteolytic processing of the Gag precursor, Myristylation enabled efficient proteolysis of the mutant Gag. All matrix-deficient viruses are noninfectious. Particles produced by matrix-deficient mutants contain low levels of glycoproteins, indicating the importance of matrix in either incorporation or stable retention of Env. Since matrix-deficient viruses contain a normal complement of viral genomic RNA, a role for MA in genomic incorporation can be excluded. Contrary to previous reports, the HIV-1 genome does not require sequences between the 5' splice donor site and the gag start codon for efficient packaging.
