AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA

Götz, Frank; Roske, Yvette; Schulz, Maike Svenja; Autenrieth, Karolin; Bertinetti, Daniela; Faelber, Katja; Zühlke, Kerstin; Kreuchwig, Annika; Kennedy, Eileen J.; Krause, Gerd; Daumke, Oliver; Heinemann, Udo; Klussmann, Enno

doi:10.1042/bcj20160242

Cited by 28 publications

(19 citation statements)

References 57 publications

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“…Many AKAPs also localize to the cell and organellar membranes (15). AKAPs present amphipathic helices that bind to the dimerization and docking (D/D) domain formed by the first 45 amino acids of RII subunits (16)(17)(18). However, the first ∼100 amino acids of RII are not visible in the electron density of the most complete crystal structures of RII-C (13,19), and therefore, it is not clear whether association with membrane-tethered AKAPs is compatible with inserting C subunits into the membrane.…”

mentioning

confidence: 99%

Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches

Walker-Gray

Stengel

Gold

2017

Proc. Natl. Acad. Sci. U.S.A.

103

110

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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 RIIβ and Cβ, with or without the prototypical anchoring protein AKAP18α, revealed that the dimerization and docking domain of RIIβ 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.

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confidence: 99%

Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches

Walker-Gray

Stengel

Gold

2017

Proc. Natl. Acad. Sci. U.S.A.

103

110

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“…This finding correlates with recent results that suggest an extended conserved hydrophobic core may be critical for the AKAP:RII interaction interface. 21 On the other hand, the hydrophilic face of the amphipathic helix which is expected to be solvent exposed, including positions K2, K5, F6, S9 and K13 results in minimal perturbation of K D values for both RII isoforms, thereby highlighting that these positions are not crucial for AKAP binding on the RII surface as expected.…”

Section: Resultsmentioning

confidence: 71%

“…Several AKAPs contain charged polar residues in the N-terminus such as AKAP18 which contains a glutamate at the first two N-terminal positions that correlate with STAD-2. 21 The crystal structure of AKAP18 and RIIα reveal that one of these glutamate residues (Glu 74) is engaged in salt bridges with Arg 22 of the D/D domain for RIIα. Indeed, it will be interesting to test future derivatives of STAD-2 such as those lacking amino acids in positions 1 and 17 or longer derivatives that contain additional or differential N- and C-terminal residues such as glutamate to determine whether isoform specificity can be further refined.…”

Section: Discussionmentioning

confidence: 99%

Investigating PKA-RII specificity using analogs of the PKA:AKAP peptide inhibitor STAD-2

Bendzunas

Dörfler

Autenrieth

et al. 2018

Bioorganic & Medicinal Chemistry

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Generation of the second messenger molecule cAMP mediates a variety of cellular responses which are essential for critical cellular processes. In response to elevated cAMP levels, cAMP dependent protein kinase (PKA) phosphorylates serine and threonine residues on a wide variety of target substrates. In order to enhance the precision and directionality of these signaling events, PKA is localized to discrete locations within the cell by A-kinase anchoring proteins (AKAPs). The interaction between PKA and AKAPs is mediated via an amphipathic α-helix derived from AKAPs which binds to a stable hydrophobic groove formed in the dimerization/docking (D/D) domain of PKA-R in an isoform-specific fashion. Although numerous AKAP disruptors have previously been identified that can inhibit either RI- or RII-selective AKAPs, no AKAP disruptors have been identified that have isoform specificity for RIα versus RIβ or RIIα versus RIIβ. As a strategy to identify isoform-specific AKAP inhibitors, a library of chemically stapled protein-protein interaction (PPI) disruptors was developed based on the RII-selective AKAP disruptor, STAD-2. An alanine was substituted at each position in the sequence, and from this library it was possible to delineate the importance of longer aliphatic residues in the formation of a region which complements the hydrophobic cleft formed by the D/D domain. Interestingly, lysine residues that were added to both terminal ends of the peptide sequence to facilitate water solubility appear to contribute to isoform specificity for RIIα over RIIβ while having only weak interaction with RI. This work supports current hypotheses on the mechanisms of AKAP binding and highlights the significance of particular residue positions that aid in distinguishing between the RII isoforms and may provide insight into future design of isoform-selective AKAP disruptors.

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“…2E). Conversely, side chains between positions 282-286 of AKAP18γ are located on the hydrophobic face of the helix (19,40) (Fig. 2E).…”

Section: Resultsmentioning

confidence: 99%

Single nucleotide polymorphisms alter kinase anchoring and the subcellular targeting of A-kinase anchoring proteins

Smith

Omar

Nygren

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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A-kinase anchoring proteins (AKAPs) shape second-messenger signaling responses by constraining protein kinase A (PKA) at precise intracellular locations. A defining feature of AKAPs is a helical region that binds to regulatory subunits (RII) of PKA. Mining patient-derived databases has identified 42 nonsynonymous SNPs in the PKA-anchoring helices of five AKAPs. Solid-phase RII binding assays confirmed that 21 of these amino acid substitutions disrupt PKA anchoring. The most deleterious side-chain modifications are situated toward C-termini of AKAP helices. More extensive analysis was conducted on a valine-to-methionine variant in the PKA-anchoring helix of AKAP18. Molecular modeling indicates that additional density provided by methionine at position 282 in the AKAP18γ isoform deflects the pitch of the helical anchoring surface outward by 6.6°. Fluorescence polarization measurements show that this subtle topological change reduces RII-binding affinity 8.8-fold and impairs cAMP responsive potentiation of L-type Ca2+ currents in situ. Live-cell imaging of AKAP18γ V282M-GFP adducts led to the unexpected discovery that loss of PKA anchoring promotes nuclear accumulation of this polymorphic variant. Targeting proceeds via a mechanism whereby association with the PKA holoenzyme masks a polybasic nuclear localization signal on the anchoring protein. This led to the discovery of AKAP18ε: an exclusively nuclear isoform that lacks a PKA-anchoring helix. Enzyme-mediated proximity-proteomics reveal that compartment-selective variants of AKAP18 associate with distinct binding partners. Thus, naturally occurring PKA-anchoring-defective AKAP variants not only perturb dissemination of local second-messenger responses, but also may influence the intracellular distribution of certain AKAP18 isoforms.

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AKAP18:PKA-RIIα structure reveals crucial anchor points for recognition of regulatory subunits of PKA

Cited by 28 publications

References 57 publications

Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches

Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches

Investigating PKA-RII specificity using analogs of the PKA:AKAP peptide inhibitor STAD-2

Single nucleotide polymorphisms alter kinase anchoring and the subcellular targeting of A-kinase anchoring proteins

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