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PKA RI? homodimer structure reveals an intermolecular interface with implications for cooperative cAMP binding and Carney complex disease.

ABSTRACT: The regulatory (R) subunit is the cAMP receptor of protein kinase A. Following cAMP binding, the inactive PKA holoenzyme complex separates into two active catalytic (C) subunits and a cAMP-bound R dimer. Thus far, only monomeric R structures have been solved, which fell short in explaining differences of cAMP binding for the full-length protein as compared to the truncated R subunits. Here we solved a full-length R-dimer structure that reflects the biologically relevant conformation, and this structure agrees well with small angle X-ray scattering. An isoform-specific interface is revealed between the protomers. This interface acts as an intermolecular sensor for cAMP and explains the cooperative character of cAMP binding to the RI? dimer. Mutagenesis of residues on this interface not only leads to structural and biochemical changes, but is also linked to Carney complex disease.

SUBMITTER: Bruystens JG

PROVIDER: S-EPMC3963464 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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PKA RIα homodimer structure reveals an intermolecular interface with implications for cooperative cAMP binding and Carney complex disease.

Bruystens Jessica G H JG Wu Jian J Fortezzo Audrey A Kornev Alexandr P AP Blumenthal Donald K DK Taylor Susan S SS

Structure (London, England : 1993) 20131205 1

The regulatory (R) subunit is the cAMP receptor of protein kinase A. Following cAMP binding, the inactive PKA holoenzyme complex separates into two active catalytic (C) subunits and a cAMP-bound R dimer. Thus far, only monomeric R structures have been solved, which fell short in explaining differences of cAMP binding for the full-length protein as compared to the truncated R subunits. Here we solved a full-length R-dimer structure that reflects the biologically relevant conformation, and this st ...[more]

PMID: 24316401

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Project description:BACKGROUND:Carney complex (CNC) is a multiple endocrine neoplasia syndrome due to inactivating mutations in the PRKAR1A gene that codes for type I? regulatory (RI?) subunit of protein kinase A. Most PRKAR1A mutations are subject to nonsense mRNA decay (NMD) and, thus, lead to haploinsufficiency. METHODS AND SETTING:Patient phenotyping for CNC features and DNA, RNA, protein, and transfection studies were carried out at a research center. RESULTS:We describe in unrelated kindreds with CNC four naturally occurring PRKAR1A mutations (1055del4, 1067del4ins5, 1076delTTins13, and 1142del4) that are predicted to escape NMD because they are located in the last coding exon of the gene. The phenotype of CNC was not different from that in other patients with the condition, although the number of patients was small. Each of the mutations caused a frameshift that led to a new stop codon into the 3' untranslated open reading frame, predicting an elongated protein that, however, was absent in patient-derived cells. After site-directed mutagenesis, in vitro transcription, and cell-free translation experiments, the expected size mutant proteins were present. However, when the mutant constructs were transfected in adrenal (NCI-295), testicular (N-TERA), and embryonic (HEK293) cells and despite the presence of the mutant mRNA, Western blot analysis indicated that there were no longer proteins. The subsequent application of proteasome inhibitors to cells transfected with the mutant constructs led to the detection of the aberrant proteins, although a compound that affects protein folding had no effect. The wild-type protein was also decreased in both patient-derived cells and/or tissues as well as in the in vitro systems used in this study. CONCLUSIONS:This was the first demonstration of proteasomal degradation of RI? protein variants leading to PRKAR1A haploinsufficiency and CNC, adding protein surveillance to NMD in the cellular mechanisms overseeing RI? synthesis. In agreement with the molecular data, CNC patients bearing PRKAR1A defects that extend the open reading frame did not have a different phenotype, although this has to be confirmed in a larger number of patients.

Dataset Information

PKA RI? homodimer structure reveals an intermolecular interface with implications for cooperative cAMP binding and Carney complex disease.

Publications

PKA RIα homodimer structure reveals an intermolecular interface with implications for cooperative cAMP binding and Carney complex disease.

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