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1.
Proc Natl Acad Sci U S A ; 116(33): 16347-16356, 2019 08 13.
Article in English | MEDLINE | ID: mdl-31363049

ABSTRACT

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, RIß, RIIα, RIIß), 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 RIIß, 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 RIIß holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.


Subject(s)
Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/chemistry , Cyclic AMP-Dependent Protein Kinases/chemistry , Protein Structure, Quaternary , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/genetics , Allosteric Regulation/genetics , Amino Acid Sequence/genetics , Crystallography, X-Ray , Cyclic AMP/chemistry , Cyclic AMP/genetics , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/genetics , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/genetics , Cyclic AMP-Dependent Protein Kinases/genetics , Gene Expression Regulation, Enzymologic/genetics , Holoenzymes/chemistry , Holoenzymes/genetics , Humans , Protein Binding/genetics , Protein Subunits/chemistry , Protein Subunits/genetics , Signal Transduction/genetics
2.
Proc Natl Acad Sci U S A ; 114(39): 10414-10419, 2017 09 26.
Article in English | MEDLINE | ID: mdl-28893983

ABSTRACT

Protein phosphorylation by cyclic AMP-dependent protein kinase (PKA) underlies key cellular processes, including sympathetic stimulation of heart cells, and potentiation of synaptic strength in neurons. Unrestrained PKA activity is pathological, and an enduring challenge is to understand how the activity of PKA catalytic subunits is directed in cells. We developed a light-activated cross-linking approach to monitor PKA subunit interactions with temporal precision in living cells. This enabled us to refute the recently proposed theory that PKA catalytic subunits remain tethered to regulatory subunits during cAMP elevation. Instead, we have identified other features of PKA signaling for reducing catalytic subunit diffusion and increasing recapture rate. Comprehensive quantitative immunoblotting of protein extracts from human embryonic kidney cells and rat organs reveals that regulatory subunits are always in large molar excess of catalytic subunits (average ∼17-fold). In the majority of organs tested, type II regulatory (RII) subunits were found to be the predominant PKA subunit. We also examined the architecture of PKA complexes containing RII subunits using cross-linking coupled to mass spectrometry. Quantitative comparison of cross-linking within a complex of RIIß and Cß, with or without the prototypical anchoring protein AKAP18α, revealed that the dimerization and docking domain of RIIß is between its second cAMP binding domains. This architecture is compatible with anchored RII subunits directing the myristylated N terminus of catalytic subunits toward the membrane for release and recapture within the plane of the membrane.


Subject(s)
Catalytic Domain/physiology , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Myocytes, Cardiac/metabolism , A Kinase Anchor Proteins/metabolism , Animals , Cell Line , Cyclic AMP/chemistry , HEK293 Cells , Humans , Male , Mass Spectrometry , Membrane Proteins/metabolism , Myocardium/metabolism , Phosphorylation/physiology , Protein Binding , Protein Structure, Secondary , Rats , Rats, Sprague-Dawley
3.
Structure ; 23(9): 1563-1572, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-26278174

ABSTRACT

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, RIß, RIIα, and RIIß). PKA is assembled as an inactive R2C2 holoenzyme in cells. To explore the role of N-myristylation in membrane targeting of PKA holoenzymes, we solved crystal structures of RIα:myrC and RIIß2:myrC2, 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.


Subject(s)
Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/metabolism , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/metabolism , Myristic Acid/metabolism , Catalytic Domain , Crystallography, X-Ray , Holoenzymes/chemistry , Holoenzymes/metabolism , Humans , Models, Molecular , Molecular Docking Simulation , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Structure, Secondary
4.
Nat Commun ; 5: 5680, 2014 Dec 05.
Article in English | MEDLINE | ID: mdl-25477193

ABSTRACT

We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA.


Subject(s)
Adrenal Cortex Neoplasms/enzymology , Adrenocortical Adenoma/enzymology , Cushing Syndrome/enzymology , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/genetics , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/metabolism , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/metabolism , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/metabolism , Adrenal Cortex Neoplasms/genetics , Adrenal Cortex Neoplasms/pathology , Adrenocortical Adenoma/genetics , Adrenocortical Adenoma/pathology , Catalytic Domain , Cell Line, Tumor , Cushing Syndrome/genetics , Cushing Syndrome/pathology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/chemistry , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/genetics , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/genetics , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Humans , Mutation , Protein Binding , Protein Stability
5.
J Biol Chem ; 289(41): 28505-12, 2014 Oct 10.
Article in English | MEDLINE | ID: mdl-25112875

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. 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 RIIß is structurally unique in that the type IIß holoenzyme is much more compact than the free RIIß 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 RIIß C-terminal deletion mutant (RIIß(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIß holoenzyme and the RIIß domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIß 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 RIIß 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 IIß isoform of PKA.


Subject(s)
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/chemistry , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Cyclic AMP/chemistry , Holoenzymes/chemistry , Animals , Catalytic Domain , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/genetics , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/metabolism , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/genetics , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Holoenzymes/genetics , Holoenzymes/metabolism , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Models, Molecular , Mutation , Neutron Diffraction , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Scattering, Small Angle , X-Ray Diffraction
6.
J Biol Chem ; 288(12): 8737-8749, 2013 Mar 22.
Article in English | MEDLINE | ID: mdl-23362281

ABSTRACT

Transforming growth factor ß (TGFß) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFß signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFß activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFß-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFß-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.


Subject(s)
Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit/metabolism , Smad4 Protein/metabolism , Transforming Growth Factor beta/metabolism , Animals , Binding Sites , Cell Line , Cell Movement , Cyclic AMP/chemistry , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit/physiology , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/physiology , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Enzyme Activation , Epithelial-Mesenchymal Transition , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Mink , Neoplasm Transplantation , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Sequence Deletion , Signal Transduction , Smad4 Protein/genetics , Transforming Growth Factor beta/physiology
7.
Science ; 335(6069): 712-6, 2012 Feb 10.
Article in English | MEDLINE | ID: mdl-22323819

ABSTRACT

In its physiological state, cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is a tetramer that contains a regulatory (R) subunit dimer and two catalytic (C) subunits. We describe here the 2.3 angstrom structure of full-length tetrameric RIIß(2):C(2) holoenzyme. This structure showing a dimer of dimers provides a mechanistic understanding of allosteric activation by cAMP. The heterodimers are anchored together by an interface created by the ß4-ß5 loop in the RIIß subunit, which docks onto the carboxyl-terminal tail of the adjacent C subunit, thereby forcing the C subunit into a fully closed conformation in the absence of nucleotide. Diffusion of magnesium adenosine triphosphate (ATP) into these crystals trapped not ATP, but the reaction products, adenosine diphosphate and the phosphorylated RIIß subunit. This complex has implications for the dissociation-reassociation cycling of PKA. The quaternary structure of the RIIß tetramer differs appreciably from our model of the RIα tetramer, confirming the small-angle x-ray scattering prediction that the structures of each PKA tetramer are different.


Subject(s)
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/chemistry , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/metabolism , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation , Allosteric Site , Amino Acid Sequence , Animals , Binding Sites , Crystallization , Crystallography, X-Ray , Cyclic AMP/metabolism , Holoenzymes/chemistry , Holoenzymes/metabolism , Hydrophobic and Hydrophilic Interactions , Mice , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Binding , Protein Folding , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , Rats
8.
J Biol Chem ; 285(46): 35910-8, 2010 Nov 12.
Article in English | MEDLINE | ID: mdl-20819953

ABSTRACT

cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human protein kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e. RIα or RIß but not by substrate inhibitors RIIα or RIIß. Detailed examination of the capacity of five PrKX-like kinases ranging from human to protozoa (Trypanosoma brucei) to form holoenzymes with human R subunits in living cells shows that this preference for pseudosubstrate inhibitors is evolutionarily conserved. To elucidate the molecular basis of this inhibitory pattern, we applied bioluminescence resonance energy transfer and surface plasmon resonance in combination with site-directed mutagenesis. We observed that the conserved αH-αI loop residue Arg-283 in PrKX is crucial for its RI over RII preference, as a R283L mutant was able to form a holoenzyme complex with wild type RII subunits. Changing the corresponding αH-αI loop residue in PKA Cα (L277R), significantly destabilized holoenzyme complexes in vitro, as cAMP-mediated holoenzyme activation was facilitated by a factor of 2-4, and lead to a decreased affinity of the mutant C subunit for R subunits, significantly affecting RII containing holoenzymes.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Mutation , Amino Acid Sequence , Animals , COS Cells , Catalytic Domain/genetics , Chlorocebus aethiops , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/genetics , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/metabolism , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/genetics , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/metabolism , Cyclic AMP-Dependent Protein Kinases/chemistry , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Humans , Kinetics , Mice , Models, Molecular , Molecular Sequence Data , Protein Binding , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Sequence Homology, Amino Acid , Surface Plasmon Resonance , Trypanosoma brucei brucei/enzymology
9.
J Mol Biol ; 393(5): 1070-82, 2009 Nov 13.
Article in English | MEDLINE | ID: mdl-19748511

ABSTRACT

The cAMP-dependent protein kinase catalytic (C) subunit is inhibited by two classes of functionally nonredundant regulatory (R) subunits, RI and RII. Unlike RI subunits, RII subunits are both substrates and inhibitors. Because RIIbeta knockout mice have important disease phenotypes, the RIIbeta holoenzyme is a target for developing isoform-specific agonists and/or antagonists. We also know little about the linker region that connects the inhibitor site to the N-terminal dimerization domain, although this linker determines the unique globular architecture of the RIIbeta holoenzyme. To understand how RIIbeta functions as both an inhibitor and a substrate and to elucidate the structural role of the linker, we engineered different RIIbeta constructs. In the absence of nucleotide, RIIbeta(108-268), which contains a single cyclic nucleotide binding domain, bound C subunit poorly, whereas with AMP-PNP, a non-hydrolyzable ATP analog, the affinity was 11 nM. The RIIbeta(108-268) holoenzyme structure (1.62 A) with AMP-PNP/Mn(2+) showed that we trapped the RIIbeta subunit in an enzyme:substrate complex with the C subunit in a closed conformation. The enhanced affinity afforded by AMP-PNP/Mn(2+) may be a useful strategy for increasing affinity and trapping other protein substrates with their cognate protein kinase. Because mutagenesis predicted that the region N-terminal to the inhibitor site might dock differently to RI and RII, we also engineered RIIbeta(102-265), which contained six additional linker residues. The additional linker residues in RIIbeta(102-265) increased the affinity to 1.6 nM, suggesting that docking to this surface may also enhance catalytic efficiency. In the corresponding holoenzyme structure, this linker docks as an extended strand onto the surface of the large lobe. This hydrophobic pocket, formed by the alphaF-alphaG loop and conserved in many protein kinases, also provides a docking site for the amphipathic helix of PKI. This novel orientation of the linker peptide provides the first clues as to how this region contributes to the unique organization of the RIIbeta holoenzyme.


Subject(s)
Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Adenylyl Imidodiphosphate/chemistry , Amino Acid Sequence , Amino Acids , Animals , Crystallization , Crystallography, X-Ray , Enzyme Stability , Holoenzymes/chemistry , Isoenzymes/chemistry , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Protein Subunits/chemistry , Rats , Substrate Specificity
10.
J Mol Graph Model ; 27(4): 563-7, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18835798

ABSTRACT

Fluorescent imaging in vivo has became one of the most powerful tools to follow the temporal and spatial localization of a variety of intracellular molecular events. Genetically encoded fluorescent indicators using the FRET effect are routinely used although the molecular basis regulating their functioning is not completely known. Here, the structural and dynamics properties of a commonly used FRET sensor for the second messenger cAMP based on the cAMP-binding domains of the regulatory subunit of Protein Kinase A are presented. Molecular dynamics simulations allowed pinpointing the main features of cAMP driven conformational transition and dissecting the contributions of geometric factors governing the functioning of the biosensor. Simulations suggest that, although orientational factors are not fully isotropic, they are highly dynamic making the inter-chromophore distance the dominant feature, determining the functioning of the probes. It is expected that this computer-aided methodology may state general basis for rational design strategies of fluorescent markers for in vivo imaging.


Subject(s)
Fluorescent Dyes/chemistry , Computer Simulation , Cyclic AMP/chemistry , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit/metabolism , Isoenzymes/chemistry , Isoenzymes/metabolism , Models, Molecular , Molecular Conformation , Solvents
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