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 (RIanchoring 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. 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 (RIanchoring 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 (1Carnegie G.K. Smith F.D. McConnachie G. Langeberg L.K. Scott J.D. Mol. Cell. 2004; 15: 889-899Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 2Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (480) Google Scholar, 3Tasken K. Aandahl E.M. Physiol. Rev. 2004; 84: 137-167Crossref PubMed Scopus (620) Google Scholar). Many of these responses require activation of the cAMP-dependent protein kinase (PKA). 3The abbreviations used are: PKA, protein kinase A; RIAD, RI anchoring disruptor; scRIAD, scrambled RIAD; AKAP, A-kinase anchoring protein; cAMP, cyclic AMP; PDSM, position-dependent scoring matrix; PKI, protein kinase inhibitor; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ACTH, adrenocorticotropic hormone; StAR, steroid acute response protein, LAT, linker for activation of T cells; Csk, C-terminal Src kinase; Lck, lymphocyte-specific protein-tyrosine kinase; GFP, green fluorescent protein; HEK, human embryonic kidney. 3The abbreviations used are: PKA, protein kinase A; RIAD, RI anchoring disruptor; scRIAD, scrambled RIAD; AKAP, A-kinase anchoring protein; cAMP, cyclic AMP; PDSM, position-dependent scoring matrix; PKI, protein kinase inhibitor; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ACTH, adrenocorticotropic hormone; StAR, steroid acute response protein, LAT, linker for activation of T cells; Csk, C-terminal Src kinase; Lck, lymphocyte-specific protein-tyrosine kinase; GFP, green fluorescent protein; HEK, human embryonic kidney.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