271 REVIEW Specificity and spatial dynamics of protein kinase A signaling organized by A-kinase-anchoring proteins Guillaume Pidoux and Kjetil Taske´n Biotechnology Centre of Oslo and Centre for Molecular Medicine, Nordic EMBL Partnership, University of Oslo, PO Box 1125, Blindern, N-0317 Oslo, Norway (Correspondence should be addressed to K Taske´n; Email: [email protected]) Abstract Protein phosphorylation is the most common post-translational modification observed in cell signaling and is controlled by the balance between protein kinase and phosphatase activities. The cAMP–protein kinase A (PKA) pathway is one of the most studied and well-known signal pathways. To maintain a high level of specificity, the cAMP–PKA pathway is tightly regulated in space and time. A-kinase-anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity in the mediation of biological effects controlled by the cAMP–PKA pathway. AKAPs also serve as scaffolding proteins that assemble PKA together with signal terminators such as phosphoprotein phosphatases and cAMP-specific phosphodiesterases as well as components of other signaling pathways into multiprotein-signaling complexes. Journal of Molecular Endocrinology (2010) 44, 271–284 Introduction spatiotemporal regulation of cellular signaling and constitutes a concept by which a common signaling In multicellular organisms, cell signaling constitutes pathway can perform many different functions. One of communication mechanisms between and inside cells the best-characterized signal transduction pathways is to ensure coordinated behavior of the organism and the cAMP-signaling pathway where ligand binding to G to regulate various biological processes such as cell protein-coupled receptors (GPCRs) via G proteins and proliferation, development, metabolism, gene adenylyl cyclase (AC) leads to an intracellular increase expression, and other more specialized functions of in the levels of the second messenger cAMP. Next, this specific target cells and tissues. Intracellularly, the leads to activation of effector molecules such as cAMP- systems for cellular communication transmit infor- dependent protein kinase A (PKA; Tasken & Aandahl mation based on well-controlled post-translational 2004, Beene & Scott 2007, Taylor et al. 2008; Fig. 1). modifications. Protein phosphorylation is one of the Transmembrane and intracellular signaling pathways most common post-translational modifications and is require a high level of spatial and temporal regulation controlled by the opposing actions of various specific to convey the appropriate inputs. The temporal control protein kinases and phosphatases (PPs). The phos- of the cAMP-signaling pathway is not only due to signal phorylation status of a protein may affect its enzymatic generation by the AC, but also depends on the action activity, cellular localization, and/or association with of cAMP–phosphodiesterases (PDEs), and different other proteins. Kinases and phosphatases have a central cell types may have high or low constitutive turnover role in signal transduction pathways and differ in of cAMP (Baillie et al. 2005, Conti & Beavo 2007). substrate specificity, acting on serine, threonine, or Moreover, spatial control is achieved through associ- tyrosine residues. Since protein phosphorylation affects ation with anchoring proteins that ensure specificity in many cellular processes, its regulation must be specific signal transduction by placing enzymes close to their and act on a defined subset of cellular targets to cognate effectors and substrates. A-kinase-anchoring ensure signal fidelity. Specificity is achieved through proteins (AKAPs) provide the means to achieve both assembly of distinct complexes of enzymes at subcel- spatial control of PKA signaling by regulating proximity lular localizations by anchoring and scaffolding as well as facilitating good temporal control by placing proteins (for review, see Scott & Pawson (2009)). PKA optimally versus small pools of cAMP that are Thus, organization of anchoring provides means for discretely temporally regulated. Indeed, AKAPs target Journal of Molecular Endocrinology (2010) 44, 271–284 DOI: 10.1677/JME-10-0010 0952–5041/10/044–271 q 2010 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/27/2021 09:45:18AM via free access 272 G PIDOUX and K TASKE´ N . Specificity and spatial dynamics of PKA signaling Adenylyl cyclase cAMP-gated ion cAMP signaling and compartmentalization channels Heterotrimeric G proteins are comprised of a, b, and g subunits (Ga,Gb, and Gg). In the resting state, the a Gβy subunit is bound to GDP and is associated with bg Gαs G-protein coupled 4 subunits. The GDP-bound heterotrimer is coupled to receptor GPCRs, which upon binding of an extracellular ligand 2 Epac Local pool of activates the trimeric G protein by increasing the rate of 3 cAMP GDP–GTP exchange on the Ga subunit. The GTP- PDEs bound, active, Ga subunit dissociates from the Gbg CCAnchored PDEs subunits; these subunits then activate their respective Anchored PKA AKAP 1 effectors. The ubiquitously expressed G protein Gas mediate receptor-dependent activation of one of several isoforms of AC resulting in increase in Figure 1 cAMP signal pathways. Ligand binding to various G protein-coupled receptors (GPCRs) activates adenylyl cyclase intracellular cAMP synthesized from ATP. cAMP in their proximity and generates pools of cAMP. The local mediates its effects through several effector molecules concentration and distribution of the cAMP gradient are limited by including PKA (Walsh et al. 1968), exchange proteins phosphodiesterases (PDEs). Particular GPCRs are confined to activated by cAMP (Epac; De Rooij et al. 1998, Kawasaki specific domains of the cell membrane in association with intracellular organelles or cytoskeletal constituents. The sub- et al. 1998), and the cyclic nucleotide-gated ion cellular structures may harbor specific isozymes of PKA that, channels (Nakamura & Gold 1987). For review and through anchoring via AKAPs, are localized in the vicinity of the references, see Tasken & Aandahl (2004), Bos (2006), receptor and the cyclase. PDEs are also anchored and serve to Beene & Scott (2007), Taylor et al. (2008) and Biel limit the extension and duration of cAMP gradients. These mechanisms serve to localize and limit the assembly and (2009) and Fig. 1. triggering of specific pathways to a defined area of the cell close Live-cell imaging and fluorescence resonance energy to the substrate. cAMP (red filled circles) has effects on a range transfer (FRET) have allowed for the visualization of of effector molecules encompassing 1) PKA, 2) PDEs, 3) guanine microdomains with a high concentration cAMP, indi- nucleotide exchange factors (GEFs) known as exchange proteins activated by cAMP (Epacs), and 4) cyclic nucleotide-gated cating that cAMP accumulation is restricted to specific ion channels. locations within the cell (Zaccolo & Pozzan 2002). The compartmentalized pools of cAMP are controlled in space and time by ACs and PDEs inside the cell. PKA to their specific substrates and assemble multi- While a family of nine membrane-bound AC isoforms is protein signal complexes that also include phospho- responsible for the production of cAMP (Hanoune & protein phosphatases and PDEs, which control the Defer 2001, Cooper 2003), the termination of cAMP temporal aspect of the regulation of the cAMP-signaling signaling is conferred by a large superfamily of enzymes pathway (Michel & Scott 2002, Tasken & Aandahl including over 40 different PDE isozyme variants that 2004, Baillie et al. 2005; Fig. 2). The role of AKAPs in the catalyze the degradation of cAMP into AMP (Baillie specificity of cAMP signal transduction is discussed in et al. 2005, Lugnier 2006). The intracellular concen- this review. trations of cAMP are therefore regulated by the AB AKAP amphipathic helix peptide N C PKA holoenzyme N N' CC Helix A' Helix A 1 3 AKAP Antiparallel RII 1–45 dimer 2 Subcellular targeting domains Helix B' Helix B Organelle C' C Figure 2 Illustration of AKAP properties. (A) AKAPs share three common properties: 1) AKAPs bind to the regulatory subunit of PKA through a conserved anchoring domain; 2) a unique subcellular targeting domain directs AKAP-signaling complexes to discrete locations inside a cell; and 3) additional binding sites for other signaling proteins such as kinases, phosphatases, or potential substrates. (B) Ribbon diagram of the NMR structure of RIIa (1–43) dimer (yellow, blue, and red) and the AKAP amphipathic helix peptide (green; Newlon et al. 2001) depicted using Accelrys Discovery Studio 2.5.1 based on the coordinates from PDB (http://www.rcsb.org/pdb/explore/explore.do?structureIdZ2DRN). Journal of Molecular Endocrinology (2010) 44, 271–284 www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/27/2021 09:45:18AM via free access Specificity and spatial dynamics of PKA signaling . G PIDOUX and K TASKE´ N 273 counterbalancing activities of ACs and PDEs that extended, highly disordered linker that contains an establish local pools of cAMP close to the effector autoinhibitory sequence and several putative phos- molecules (Conti & Jin 1999, Houslay & Adams 2003). phorylation sites. All PKA isoforms display high A local pool of cAMP can be generated by a distinct sequence homology in the D/D domain and the ligand and lead the signal to follow a specific route cAMP-binding