Delineation of Type I Protein Kinase A-selective Signaling Events Using an RI Anchoring Disruptor

Abstract

Control of specificity in cAMP signaling is achieved by A-kinase anchoring proteins (AKAPs), which assemble cAMP effectors such as protein kinase A (PKA) into multiprotein signaling complexes in the cell. AKAPs tether the PKA holoenzymes at subcellular locations to favor the phosphorylation of selected substrates. PKA anchoring is mediated by an amphipathic helix of 14 -18 residues on each AKAP that binds to the R subunit dimer of the PKA holoenzymes. Using a combination of bioinformatics and peptide array screening, we have developed a high affinity-binding peptide called RIAD (RI anchoring disruptor) with >1000-fold selectivity for type I PKA over type II PKA. Cell-soluble RIAD selectively uncouples cAMP-mediated inhibition of T cell function and inhibits progesterone synthesis at the mitochondria in steroid-producing cells. This study suggests that these processes are controlled by the type I PKA holoenzyme and that RIAD can be used as a tool to define anchored type I PKA signaling events.The cAMP signaling pathway synchronizes a variety of physiological responses including cell proliferation and differentiation, microtubule dynamics, reproductive function, modulation of immune responses, and steroidogenesis (1-3). Many of these responses require activation of the cAMP-dependent protein kinase (PKA).3 The dormant PKA holoenzyme is a heterotetramer composed of two catalytic (C) subunits held in an inactive conformation by a regulatory (R) subunit dimer. Upon activation with cAMP, the C subunits are released from their interaction with the R subunit dimer and are free to phosphorylate a plethora of target substrates. Individual cells express a range of PKA isozymes differing in R (RI␣, RI␤, RII␣, RII␤) and C (C␣, C␤, C␥) subunit composition. The type I and type II PKA isozymes, which are classified on the basis of their R subunit dimer, possess slightly different physical and biological properties including a differential sensitivity to cAMP.Spatiotemporal regulation of PKA phosphorylation events is facilitated by A-kinase anchoring proteins (AKAPs). This family of structurally diverse but functionally related proteins act as molecular scaffolds to cluster PKA with specific substrates and signal termination enzymes such as phosphatases (4, 5) and cAMP-specific phosphodiesterases (6 -9). The number of AKAPs has been estimated to more than 75, of which ϳ50 have been identified to date (3, 10). Although the majority of AKAPs described to date bind type II PKA via the RII regulatory subunit, several dual function AKAPs are capable of interacting with both the type I and the type II PKA holoenzyme (11-15). There are also examples of anchoring proteins such as AKAPce (16), PAP7 (17), and merlin (18) that selectively interact with the RI subunit of PKA.The molecular basis for PKA anchoring is the interaction of an amphipathic helical motif of 14 -18 residues on the AKAP with a hydrophobic groove in the N-terminal dimerization domain of the R subunit. High affinity peptides mimicking the amphipathic helix bind to the R...

Inhibition of T Cell Activation by Cyclic Adenosine 5′-Monophosphate Requires Lipid Raft Targeting of Protein Kinase A Type I by the A-Kinase Anchoring Protein Ezrin

“…This apparent discrepancy might be explained by the Biacore analysis mainly measuring interaction of the R subunits with the amphipatic helix region of Ezrin. Additional experiments will be necessary to determine whether the region spanning residues 363-470 contains an independent RI␣ binding domain or provides additional determinants that augments RI binding to the amphipatic helix region within aa 404 -445, as suggested by our RI binding site analysis (43). Alternatively, the preference for RI in situ despite a higher affinity for RII could reflect the availability of PKA type I and II inside T cells.…”

Myosin Va cooperates with PKA RIα to mediate maintenance of the endplate in vivo

“…Their interaction is the result of a dimerization/docking (DD) domain and an amphipathic α-helix on PKA R and AKAP, respectively. PKA R isoformspecific AKAP disruptor peptides were developed, such as the RI anchoring disruptor (RIAD) peptide for PKA RI (33).…”

Pharmacological Interference With Protein-protein Interactions of Akinase Anchoring Proteins as a Strategy for the Treatment of Disease

“…RI-- and RII--preferring and RI--and RII--specific peptides are available now, e.g. the RI anchoring disruptor, RIAD [239] and SuperAKAP--IS for RII [214]; the dual--specific D--AKAP2 was used as template for the generation of RI--and RII--preferring peptides [240]. All peptides are available as membrane--permeant versions for cell--based studies.…”