DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

Abstract

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of themore » RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.« less

Authors:
 [1];  [2];  [2];  [2];  [3];  [2]
  1. Univ. of Utah, Salt Lake City, UT (United States)
  2. Univ. of California, San Diego, CA (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). High Flux Isotope Reactor (HFIR); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Spallation Neutron Source (SNS); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Structural Molecular Biology (CSMB)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1163593
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 289; Journal Issue: 41; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; cyclic AMP (cAMP); intrinsically disordered protein; protein domain; protein dynamic; protein kinase A (PKA); protein structure; small angle x-ray scattering (SAXS); small angle neutron scattering (SANS)

Citation Formats

Blumenthal, Donald K., Copps, Jeffrey, Smith-Nguyen, Eric V., Zhang, Ping, Heller, William T., and Taylor, Susan S. The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study. United States: N. p., 2014. Web. doi:10.1074/jbc.M114.584177.
Blumenthal, Donald K., Copps, Jeffrey, Smith-Nguyen, Eric V., Zhang, Ping, Heller, William T., & Taylor, Susan S. The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study. United States. https://doi.org/10.1074/jbc.M114.584177
Blumenthal, Donald K., Copps, Jeffrey, Smith-Nguyen, Eric V., Zhang, Ping, Heller, William T., and Taylor, Susan S. Mon . "The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study". United States. https://doi.org/10.1074/jbc.M114.584177. https://www.osti.gov/servlets/purl/1163593.
@article{osti_1163593,
title = {The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study},
author = {Blumenthal, Donald K. and Copps, Jeffrey and Smith-Nguyen, Eric V. and Zhang, Ping and Heller, William T. and Taylor, Susan S.},
abstractNote = {Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.},
doi = {10.1074/jbc.M114.584177},
journal = {Journal of Biological Chemistry},
number = 41,
volume = 289,
place = {United States},
year = {2014},
month = {8}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 1 work
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Recurrent somatic mutations underlie corticotropin-independent Cushing's syndrome
journal, May 2014


Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli : multiple isozymes reflect different phosphorylation states
journal, January 1993

  • Herberg, Friedrich W.; Bell, Sean M.; Taylor, Susan S.
  • "Protein Engineering, Design and Selection", Vol. 6, Issue 7
  • DOI: 10.1093/protein/6.7.771

Signaling through dynamic linkers as revealed by PKA
journal, August 2013

  • Akimoto, M.; Selvaratnam, R.; McNicholl, E. T.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 35
  • DOI: 10.1073/pnas.1312644110

Differential Effects of Substrate on Type I and Type II PKA Holoenzyme Dissociation
journal, May 2004

  • Vigil, Dominico; Blumenthal, Donald K.; Brown, Simon
  • Biochemistry, Vol. 43, Issue 19
  • DOI: 10.1021/bi0499157

Comparison of neutron and X-ray scattering of dilute myoglobin solutions
journal, April 1975


Structure and Allostery of the PKA RII  Tetrameric Holoenzyme
journal, February 2012


C Subunits Binding to the Protein Kinase A RIα Dimer Induce a Large Conformational Change
journal, April 2004

  • Heller, William T.; Vigil, Dominico; Brown, Simon
  • Journal of Biological Chemistry, Vol. 279, Issue 18
  • DOI: 10.1074/jbc.M313405200

Determination of the regularization parameter in indirect-transform methods using perceptual criteria
journal, August 1992


Activating hotspot L205R mutation in PRKACA and adrenal Cushing's syndrome
journal, April 2014


Localization and quaternary structure of the PKA RI  holoenzyme
journal, July 2012

  • Ilouz, R.; Bubis, J.; Wu, J.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 31
  • DOI: 10.1073/pnas.1209538109

Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors
journal, April 2014

  • Goh, Gerald; Scholl, Ute I.; Healy, James M.
  • Nature Genetics, Vol. 46, Issue 6
  • DOI: 10.1038/ng.2956

PRIMUS: a Windows PC-based system for small-angle scattering data analysis
journal, September 2003

  • Konarev, Petr V.; Volkov, Vladimir V.; Sokolova, Anna V.
  • Journal of Applied Crystallography, Vol. 36, Issue 5, p. 1277-1282
  • DOI: 10.1107/S0021889803012779

New developments in the ATSAS program package for small-angle scattering data analysis
journal, March 2012

  • Petoukhov, Maxim V.; Franke, Daniel; Shkumatov, Alexander V.
  • Journal of Applied Crystallography, Vol. 45, Issue 2
  • DOI: 10.1107/S0021889812007662

cAMP-dependent Protein Kinase Regulatory Subunit Type IIβ
journal, February 2004

  • Zawadzki, Kerri M.; Taylor, Susan S.
  • Journal of Biological Chemistry, Vol. 279, Issue 8
  • DOI: 10.1074/jbc.M310804200

Reduction and analysis of SANS and USANS data using IGOR Pro
journal, November 2006


MULCh : modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies
journal, January 2008

  • Whitten, Andrew E.; Cai, Shuzhi; Trewhella, Jill
  • Journal of Applied Crystallography, Vol. 41, Issue 1
  • DOI: 10.1107/S0021889807055136

Mutations of the gene encoding the protein kinase A type I-α regulatory subunit in patients with the Carney complex
journal, September 2000

  • Kirschner, Lawrence S.; Carney, J. Aidan; Pack, Svetlana D.
  • Nature Genetics, Vol. 26, Issue 1
  • DOI: 10.1038/79238

Conformational Differences Among Solution Structures of the Type Iα, IIα and IIβ Protein Kinase A Regulatory Subunit Homodimers: Role of the Linker Regions
journal, April 2004

  • Vigil, Dominico; Blumenthal, Donald K.; Heller, William T.
  • Journal of Molecular Biology, Vol. 337, Issue 5
  • DOI: 10.1016/j.jmb.2004.02.028

Constitutive Activation of PKA Catalytic Subunit in Adrenal Cushing's Syndrome
journal, March 2014

  • Beuschlein, Felix; Fassnacht, Martin; Assié, Guillaume
  • New England Journal of Medicine, Vol. 370, Issue 11
  • DOI: 10.1056/NEJMoa1310359

Bio-SANS—A dedicated facility for neutron structural biology at Oak Ridge National Laboratory
journal, November 2006


Solution Scattering Reveals Large Differences in the Global Structures of Type II Protein Kinase A Isoforms
journal, March 2006

  • Vigil, Dominico; Blumenthal, Donald K.; Taylor, Susan S.
  • Journal of Molecular Biology, Vol. 357, Issue 3
  • DOI: 10.1016/j.jmb.2006.01.006

ORNL_SAS : software for calculation of small-angle scattering intensities of proteins and protein complexes
journal, July 2007


UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

  • Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
  • Journal of Computational Chemistry, Vol. 25, Issue 13
  • DOI: 10.1002/jcc.20084

Quaternary Structures of a Catalytic Subunit-Regulatory Subunit Dimeric Complex and the Holoenzyme of the cAMP-dependent Protein Kinase by Neutron Contrast Variation
journal, November 1998

  • Zhao, Jinkui; Hoye, Elaine; Boylan, Sharon
  • Journal of Biological Chemistry, Vol. 273, Issue 46
  • DOI: 10.1074/jbc.273.46.30448

Assembly of allosteric macromolecular switches: lessons from PKA
journal, September 2012

  • Taylor, Susan S.; Ilouz, Ronit; Zhang, Ping
  • Nature Reviews Molecular Cell Biology, Vol. 13, Issue 10
  • DOI: 10.1038/nrm3432