The Experts below are selected from a list of 13023 Experts worldwide ranked by ideXlab platform
Richard A Young - One of the best experts on this subject based on the ideXlab platform.
-
ca150 a nuclear protein associated with the rna polymerase ii Holoenzyme is involved in tat activated human immunodeficiency virus type 1 transcription
Molecular and Cellular Biology, 1997Co-Authors: Carlos Sune, Richard A Young, Takuma Hayashi, Yi Liu, William S Lane, Mariano A GarciablancoAbstract:Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II Holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II Holoenzyme. Furthermore, we found that functional Tat associates with the Holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II Holoenzyme that contains CA150.
-
brca1 is a component of the rna polymerase ii Holoenzyme
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Ralph Scully, Richard A Young, David M. Chao, Stephen Anderson, Wanjiang Wei, David M Livingston, Jeffrey D. ParvinAbstract:The familial breast–ovarian tumor suppressor gene product BRCA1 was found to be a component of the RNA polymerase II Holoenzyme by several criteria. BRCA1 was found to copurify with the Holoenzyme over multiple chromatographic steps. Other tested transcription activators that could potentially contact the Holoenzyme were not stably associated with the Holoenzyme as determined by copurification. Antibody specific for the Holoenzyme component hSRB7 specifically purifies BRCA1. Immunopurification of BRCA1 complexes also specifically purifies transcriptionally active RNA polymerase II and transcription factors TFIIF, TFIIE, and TFIIH. Moreover, a BRCA1 domain, which is deleted in about 90% of clinically relevant mutations, participates in binding to the Holoenzyme complex in cells. These data are consistent with recent data identifying transcription activation domains in the BRCA1 protein and link the BRCA1 tumor suppressor protein with the transcription process as a Holoenzyme-bound protein.
-
A mammalian SRB protein associated with an RNA polymerase II Holoenzyme
Nature, 1996Co-Authors: David M. Chao, Ellen L. Gadbois, Peter J. Murray, Stephen Anderson, Michelle S. Sonu, Jeffrey D. Parvin, Richard A YoungAbstract:A LARGE multisubunit complex containing RNA polymerase II, general transcription factors and SRB regulatory proteins initiates transcription of class II genes in yeast cells1–4. The SRB proteins are a hallmark of this RNA polymerase II Holoenzyme as they are found only in this complex, where they contribute to the response to regulators4–8. We have now isolated a human honiologue of the yeast SRB7 gene and used antibodies against human SRB7 protein to purify and characterize a mammalian RNA polymerase II Holoenzyme containing the general transcription factors TFIIE and TFIIH. This Holoenzyme is more responsive to transcriptional activators than core RNA polymerase II when assayed in the presence of coactivators.
-
rna polymerase ii Holoenzyme contains swi snf regulators involved in chromatin remodeling
Cell, 1996Co-Authors: C D Wilson, David M. Chao, Anthony N Imbalzano, Gavin R Schnitzler, Robert E Kingston, Richard A YoungAbstract:The RNA polymerase II Holoenzyme contains RNA polymerase II, a subset of general transcription factors and SRB regulatory proteins. We report here that SWI and SNF gene products, previously identified as global gene regulators whose functions include remodeling chromatin, are also integral components of the yeast RNA polymerase II Holoenzyme. The SWI/SNF proteins are components of the SRB complex, also known as the mediator, which is tightly associated with the RNA polymerase II C-terminal repeat domain. The SWI/SNF components provide the Holoenzyme with the capacity to disrupt nucleosomal DNA and thus facilitate stable binding of various components of the transcription initiation complex at promoters.
-
Association of an activator with an RNA polymerase II Holoenzyme.
Genes & development, 1995Co-Authors: Christoph J. Hengartner, Anthony J. Koleske, Craig.m Thompson, David M. Chao, Sha-mei Liao, Jianhua Zhang, Sara Okamura, Richard A YoungAbstract:RNA polymerase II Holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and four SRB proteins. The SRB proteins, which were identified through a selection for genes involved in transcription initiation by RNA polymerase II in vivo, are a hallmark of the Holoenzyme. We report here the isolation and characterization of additional SRB genes. We show that the products of all nine SRB genes identified thus far are components of the RNA polymerase II Holoenzyme and are associated with a Holoenzyme subcomplex termed the mediator of activation. The Holoenzyme is capable of responding to a transcriptional activator, suggesting a model in which activators function, in part, through direct interactions with the Holoenzyme. Immunoprecipitation experiments with anti-SRB5 antibodies demonstrate that the acidic activating domain of VP16 specifically binds to the Holoenzyme. Furthermore, the Holoenzyme and the mediator subcomplex bind to a VP16 affinity column. These results provide a more complete description of the RNA polymerase II Holoenzyme and suggest that this form of the transcription apparatus can be recruited to promoters via direct interactions with activators.
Vladimir N Podust - One of the best experts on this subject based on the ideXlab platform.
-
replication factor c disengages from proliferating cell nuclear antigen pcna upon sliding clamp formation and pcna itself tethers dna polymerase δ to dna
Journal of Biological Chemistry, 1998Co-Authors: Vladimir N Podust, Nikhil Tiwari, Scott Stephan, Ellen FanningAbstract:Abstract Replication factor C (RF-C) and proliferating cell nuclear antigen (PCNA) assemble a complex, called sliding clamp, onto DNA. The clamp in turn loads DNA polymerases (pol) δ and e to form the corresponding Holoenzymes, which play an essential role in replication of eukaryotic chromosomal DNA and in several DNA repair pathways. To determine the fate of RF-C after loading of PCNA onto DNA, we tagged the RF-C subunit p37 with a protein kinase A recognition motif, so that the recombinant five-subunit RF-C complex could be32P-labeled and quantitatively detected in femtomolar amounts. Nonspecific binding of RF-C to DNA was minimized by replacing the p140 subunit with an N-terminally truncated p140 subunit lacking the previously identified nonspecific DNA binding domain. Neither of these modifications impaired the clamp loading activity of the recombinant RF-C. Using gel filtration techniques, we demonstrated that RF-C dissociated from the DNA after clamp loading or pol δ Holoenzyme assembly, while PCNA or PCNA·pol δ complex remained bound to DNA. PCNA catalytically loaded onto the template-primer was sufficient by itself to tether pol δ and stimulate DNA replication. The readdition of RF-C to the isolated PCNA·DNA complex did not further stimulate pol δ DNA synthesis. We conclude that pol δ Holoenzyme consists of PCNA and pol δ core and that RF-C serves only to load PCNA clamp.
-
dna polymerase delta Holoenzyme action on single stranded dna and on double stranded dna in the presence of replicative dna helicases
Biochemistry, 1995Co-Authors: Vladimir N Podust, Friedemann Mueller, Larissa M Podust, Ulrich HuebscherAbstract:: DNA polymerase delta requires proliferating cell nuclear antigen and replication factor C to form a Holoenzyme efficient in DNA synthesis. We have analyzed three different aspects of calf thymus DNA polymerase delta Holoenzyme: (i) analysis of pausing during DNA synthesis, (ii) replication of double-stranded DNA in the absence of additional factors, and (iii) replication of double-stranded DNA in the presence of the two known replicative DNA helicases from simian virus 40 and bovine papilloma virus. DNA polymerase delta Holoenzyme replicated primed single-stranded DNA at a rate of 100-300 nucleotides/min, partially overcoming multiple pause sites on DNA. While Escherichia coli single-strand DNA binding protein helped DNA polymerase delta pass through pause sites, the DNA polymerase delta itself appeared to dissociate from the template in the absence of synthesis or when encountering pause sites. Proliferating cell nuclear antigen likely remained on the template. DNA polymerase delta Holoenzyme could perform limited strand displacement synthesis on double-stranded gapped circular DNA, and this reaction was not stimulated either by replication protein A or by E. coli single-strand DNA binding protein. DNA polymerase delta Holoenzyme could efficiently cooperate with replicative DNA helicases from simian virus 40 (large T antigen) and bovine papilloma virus 1 (protein E1) in replication through double-stranded DNA in a reaction that required replication protein A or E. coli single-strand DNA binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)
-
assembly of dna polymerase delta and epsilon Holoenzymes depends on the geometry of the dna template
Nucleic Acids Research, 1994Co-Authors: Larissa M Podust, Vladimir N Podust, Christian Floth, Ulrich HubscherAbstract:Abstract To study in details the assembly of DNA polymerases delta and epsilon Holoenzymes a circular double-stranded DNA template containing a gap of 45 nucleotides was constructed. Both replication factor C and proliferating cell nuclear antigen were absolutely required and sufficient for assembly of DNA polymerase delta Holoenzyme complex on DNA. On such a circular DNA substrate replication protein A (or E. coli single-strand DNA binding protein) was neither required for assembly of DNA polymerase delta Holoenzyme complex nor for the gap-filling reaction. A circular structure of the DNA substrate was found to be absolutely critical for the ability of auxiliary proteins to interact with DNA polymerases. The linearization of the circular DNA template resulted in three dramatic effects: (i) DNA synthesis by DNA polymerase delta Holoenzyme was abolished, (ii) the inhibition effect of replication factor C and proliferating cell nuclear antigen on DNA polymerase alpha was relieved and (iii) DNA polymerase epsilon could not form any longer a Holoenzyme with replication factor C and proliferating cell nuclear antigen. The comparison of the effect of replication factor C and proliferating cell nuclear antigen on DNA polymerases alpha, delta and epsilon indicated that the auxiliary proteins appear to form a mobile clamp, which can easily slide along double-stranded DNA.
-
DNA polymerase delta and epsilon Holoenzymes from calf thymus.
Chromosoma, 1992Co-Authors: Vladimir N Podust, Anthi Georgaki, Victor Mikhailov, Ulrich HubscherAbstract:Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) delta completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (or E. coli single-strand DNA binding protein, SSB). Pol epsilon core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol epsilon was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol epsilon by PCNA/RF-C/SSB appears to be the superposition of two events: pol epsilon Holoenzyme (pol epsilon, PCNA, RF-C) synthesized longer products than its pol epsilon core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol alpha/primase with pol delta or pol epsilon Holoenzymes on unprimed M13 DNA. While pol delta displayed higher dNMP incorporation than pol epsilon, when a single primer was preannealed to DNA, pol epsilon was more efficient in the utilization of the primers synthesized by pol alpha/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol epsilon Holoenzyme, than for pol delta. Our data support the hypothesis of pol delta as the leading and pol epsilon as the second lagging strand replication enzyme.
-
dna polymerase delta and epsilon Holoenzymes from calf thymus
Chromosoma, 1992Co-Authors: Vladimir N Podust, Anthi Georgaki, Victor Mikhailov, Ulrich HubscherAbstract:Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) δ completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (orE.coli singlestrand DNA binding protein, SSB). Pol ɛ core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol ɛ was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol ɛ by PCNA/RF-C/SSB appears to be the superposition of two events: pol, ɛ Holoenzyme (pol ɛ, PCNA, RF-C) synthesized longer products than its pol ɛ core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol α/primase with pol δ or pol ɛ Holoenzymes on unprimed M13 DNA. While pol δ displayed higher dNMP incorporation than pol ɛ, when a single primer was preannealed to DNA, pol ɛ was more efficient in the utilization of the primers synthesized by pol α/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol ɛ Holoenzyme, than for pol δ. Our data support the hypothesis of pol δ as the leading and pol ɛ as the second lagging strand replication enzyme.
Ulrich Hubscher - One of the best experts on this subject based on the ideXlab platform.
-
assembly of dna polymerase delta and epsilon Holoenzymes depends on the geometry of the dna template
Nucleic Acids Research, 1994Co-Authors: Larissa M Podust, Vladimir N Podust, Christian Floth, Ulrich HubscherAbstract:Abstract To study in details the assembly of DNA polymerases delta and epsilon Holoenzymes a circular double-stranded DNA template containing a gap of 45 nucleotides was constructed. Both replication factor C and proliferating cell nuclear antigen were absolutely required and sufficient for assembly of DNA polymerase delta Holoenzyme complex on DNA. On such a circular DNA substrate replication protein A (or E. coli single-strand DNA binding protein) was neither required for assembly of DNA polymerase delta Holoenzyme complex nor for the gap-filling reaction. A circular structure of the DNA substrate was found to be absolutely critical for the ability of auxiliary proteins to interact with DNA polymerases. The linearization of the circular DNA template resulted in three dramatic effects: (i) DNA synthesis by DNA polymerase delta Holoenzyme was abolished, (ii) the inhibition effect of replication factor C and proliferating cell nuclear antigen on DNA polymerase alpha was relieved and (iii) DNA polymerase epsilon could not form any longer a Holoenzyme with replication factor C and proliferating cell nuclear antigen. The comparison of the effect of replication factor C and proliferating cell nuclear antigen on DNA polymerases alpha, delta and epsilon indicated that the auxiliary proteins appear to form a mobile clamp, which can easily slide along double-stranded DNA.
-
DNA polymerase delta and epsilon Holoenzymes from calf thymus.
Chromosoma, 1992Co-Authors: Vladimir N Podust, Anthi Georgaki, Victor Mikhailov, Ulrich HubscherAbstract:Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) delta completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (or E. coli single-strand DNA binding protein, SSB). Pol epsilon core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol epsilon was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol epsilon by PCNA/RF-C/SSB appears to be the superposition of two events: pol epsilon Holoenzyme (pol epsilon, PCNA, RF-C) synthesized longer products than its pol epsilon core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol alpha/primase with pol delta or pol epsilon Holoenzymes on unprimed M13 DNA. While pol delta displayed higher dNMP incorporation than pol epsilon, when a single primer was preannealed to DNA, pol epsilon was more efficient in the utilization of the primers synthesized by pol alpha/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol epsilon Holoenzyme, than for pol delta. Our data support the hypothesis of pol delta as the leading and pol epsilon as the second lagging strand replication enzyme.
-
dna polymerase delta and epsilon Holoenzymes from calf thymus
Chromosoma, 1992Co-Authors: Vladimir N Podust, Anthi Georgaki, Victor Mikhailov, Ulrich HubscherAbstract:Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) δ completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (orE.coli singlestrand DNA binding protein, SSB). Pol ɛ core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol ɛ was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol ɛ by PCNA/RF-C/SSB appears to be the superposition of two events: pol, ɛ Holoenzyme (pol ɛ, PCNA, RF-C) synthesized longer products than its pol ɛ core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol α/primase with pol δ or pol ɛ Holoenzymes on unprimed M13 DNA. While pol δ displayed higher dNMP incorporation than pol ɛ, when a single primer was preannealed to DNA, pol ɛ was more efficient in the utilization of the primers synthesized by pol α/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol ɛ Holoenzyme, than for pol δ. Our data support the hypothesis of pol δ as the leading and pol ɛ as the second lagging strand replication enzyme.
Charles S Mchenry - One of the best experts on this subject based on the ideXlab platform.
-
the dnax binding subunits delta and psi are bound to gamma and not tau in the dna polymerase iii Holoenzyme
Journal of Biological Chemistry, 2000Co-Authors: Bradley P Glover, Charles S MchenryAbstract:Abstract The DnaX complex subassembly of the DNA polymerase III Holoenzyme is comprised of the DnaX proteins τ and γ and the auxiliary subunits δ, δ′, χ, and ψ, which together load the β processivity factor onto primed DNA in an ATP-dependent reaction. δ′ and ψ bind directly to DnaX whereas δ and χ bind to δ′ and ψ, respectively (Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13348–13357). Until now, it has been unclear which DnaX protein, τ or γ, in Holoenzyme binds the auxiliary subunits δ, δ′, χ,and ψ. Treatment of purified Holoenzyme with the homobifunctional cross-linker bis(sulfosuccinimidyl)suberate produces covalently cross-linked γ-δ′ and γ-ψ complexes identified by Western blot analysis. Immunodetection of cross-linked species with anti-δ′ and anti-ψ antibodies revealed that no τ-δ′ or τ-ψ cross-links had formed, suggesting that the δ′ and ψ subunits reside only on γ within Holoenzyme.
-
in vivo assembly of overproduced dna polymerase iii overproduction purification and characterization of the alpha alpha epsilon and alpha epsilon theta subunits
Journal of Biological Chemistry, 1996Co-Authors: Deok Ryong Kim, Charles S MchenryAbstract:The genes for the polymerase core (alphaepsilontheta) of the DNA polymerase III Holoenzyme map to widely separated loci on the Escherichia coli chromosome. To enable efficient overproduction and in vivo assembly of DNA polymerase III core, artificial operons containing the three structural genes, dnaE, dnaQ, and holE, were placed in an expression plasmid. The proteins alpha, alphaepsilon and alphaepsilontheta were overexpressed and assembled in E. coli and purified to homogeneity. The three purified polymerases had a similar specific activity of about 6.0 x 10(6) units/mg in a gap-filling assay. Kinetics studies showed that neither epsilon nor theta influenced the Km of alpha for deoxynucleotide triphosphate and only slightly decreased the Km of alpha for DNA, although epsilon was absolutely required for maximal DNA synthesis. The rate of DNA synthesis by alpha-reconstituted Holoenzyme using tau complex was about 5-fold less than that of alphaepsilon or alphaepsilontheta-reconstituted Holoenzyme as determined by a gel analysis. The processivity of alpha-reconstituted Holoenzyme was very similar to that of alphaepsilontheta-reconstituted Holoenzyme when tau complex was used as a clamp loader.
Ping Zhang - One of the best experts on this subject based on the ideXlab platform.
-
structural analyses of the pka riiβ Holoenzyme containing the oncogenic dnajb1 pkac fusion protein reveal protomer asymmetry and fusion induced allosteric perturbations in fibrolamellar hepatocellular carcinoma
PLOS Biology, 2020Co-Authors: Phillip C Aoto, Ping Zhang, Sanford M. Simon, Juihung Weng, Cole Nielsen, Jennifer N Cash, James A Hall, Michael A CianfroccoAbstract:When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA Holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA Holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ Holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ Holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.
-
two pka riα Holoenzyme states define atp as an isoform specific orthosteric inhibitor that competes with the allosteric activator camp
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Phillip C Aoto, Lalima G Ahuja, Juihung Weng, Nicholas Sun, Cecilia Y Cheng, Ping ZhangAbstract:Protein kinase A (PKA) Holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIβ, RIIα, RIIβ), RIα is most essential for regulating PKA activity in cells. Our 2 RIα2C2 Holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the Holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIβ, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-Holoenzyme dimer interface mediated by N3A–N3A′ motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIβ Holoenzyme demonstrate isoform-specific Holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.
-
RESEARCH ARTICLE Single Turnover Autophosphorylation Cycle
2016Co-Authors: Of The Pka Riiβ Holoenzyme, Ping Zhang, Matthias J Knape, Lalima G Ahuja, Malik M Keshwani, Mira Sastri, C. King, Friedrich W HerbergAbstract:To provide tight spatiotemporal signaling control, the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) Holoenzyme typically nucleates a macromolecular complex or a “PKA signalosome. ” Using the RIIβ Holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RIIβ subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RIIβ Holoenzyme could undergo a single turnover autophosphorylation with adenosine triphos-phate and magnesium (MgATP) and trap both products in the crystal lattice, we asked here whether calcium could trap an ATP:RIIβ Holoenzyme since the RIIβ Holoenzyme is located close to ion channels. The 2.8Å structure of an RIIβp2:C2:(Ca2ADP)2 Holoenzyme, sup-ported by biochemical and biophysical data, reveals a trapped single phosphorylation event similar to MgATP. Thus, calcium can mediate a single turnover event with either ATP or adenosine-5'-(β,γ-imido)triphosphate (AMP-PNP), even though it cannot support steady-state catalysis efficiently. The Holoenzyme serves as a “product trap ” because of the slow off-rate of the pRIIβ subunit, which is controlled by cAMP, not by phosphorylation of th
-
an isoform specific myristylation switch targets type ii pka Holoenzymes to membranes
Structure, 2015Co-Authors: Ping Zhang, Adam Christopher Bastidas, Alexandr P Kornev, Feng Ye, Jian WuAbstract: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.
-
Single Turnover Autophosphorylation Cycle of the PKA RIIβ Holoenzyme.
Public Library of Science (PLoS), 2015Co-Authors: Ping Zhang, Matthias J Knape, Lalima G Ahuja, Malik M Keshwani, Charles C King, Mira Sastri, Friedrich W HerbergAbstract:To provide tight spatiotemporal signaling control, the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) Holoenzyme typically nucleates a macromolecular complex or a "PKA signalosome." Using the RIIβ Holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RIIβ subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RIIβ Holoenzyme could undergo a single turnover autophosphorylation with adenosine triphosphate and magnesium (MgATP) and trap both products in the crystal lattice, we asked here whether calcium could trap an ATP:RIIβ Holoenzyme since the RIIβ Holoenzyme is located close to ion channels. The 2.8Å structure of an RIIβp2:C2:(Ca2ADP)2 Holoenzyme, supported by biochemical and biophysical data, reveals a trapped single phosphorylation event similar to MgATP. Thus, calcium can mediate a single turnover event with either ATP or adenosine-5'-(β,γ-imido)triphosphate (AMP-PNP), even though it cannot support steady-state catalysis efficiently. The Holoenzyme serves as a "product trap" because of the slow off-rate of the pRIIβ subunit, which is controlled by cAMP, not by phosphorylation of the inhibitor site. By quantitatively defining the RIIβ signaling cycle, we show that release of pRIIβ in the presence of cAMP is reduced by calcium, whereas autophosphorylation at the phosphorylation site (P-site) inhibits Holoenzyme reassociation with the catalytic subunit. Adding a single phosphoryl group to the preformed RIIβ Holoenzyme thus creates a signaling cycle in which phosphatases become an essential partner. This previously unappreciated molecular mechanism is an integral part of PKA signaling for type II Holoenzymes