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

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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).

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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 domains (Canaves & Taylor 2002). In inside the cell by reaching and activating a subset of contrast, the linker regions are highly variable in both PKA–AKAP complex to mediate a biological effect length and sequence (Vigil et al. 2004). In addition, the (Zaccolo et al. 2002). PDEs contribute to the establish- R subunits are differentially expressed in different cells. ment of local gradients of cAMP by being localized to Whereas RIa and RIIa subunits are ubiquitously subcellular compartments and by being recruited into expressed (Lee et al. 1983, Scott et al. 1987), RIIb is multiprotein-signaling complexes organized by primarily expressed in endocrine tissues, brain, fat, and AKAPs and other scaffold proteins (Dodge et al. 2001, reproductive tissues, and RIb is mainly found in the Tasken et al. 2001). brain ( Jahnsen et al. 1986, Clegg et al. 1988, Cadd & McKnight 1989). These differences in cAMP affinities and localization between PKA isoforms are proposed to cAMP-dependent protein kinase contribute to specificity in the cAMP-signaling pathway (Skalhegg & Tasken 2000). Subcellular local- ization of PKA is mainly due to anchoring of the The PKA holoenzyme is a heterotetramer composed of R subunits by AKAPs. two catalytic (C) subunits held in an inactive state by association with a regulatory (R) subunit dimer (Corbin et al. 1973, Corbin & Keely 1977, Potter & Taylor 1979). Two classes of PKA holoenzymes, type I and II, have A-kinase-anchoring proteins been identified, which differ in their R subunits (RI and RII; Reimann et al. 1971, Corbin et al. 1975). Both The intracellular targeting and compartmentalization R subunits (RI and RII) and the C subunit exist as of PKA are controlled through association with AKAPs. multiple isoforms (RIa,RIb, RIIa, RIIb,Ca,Cb,Cg, and AKAPs are a structurally diverse family of functionally PRKX). Although there are major differences in tissue related proteins that now include more than 50 distribution and biochemical and physical properties of members when splice variants are included (Table 1). the R subunit isoforms, the holoenzyme contains two C They are defined on the basis of their ability to bind to subunits bound to RI homo- or heterodimers or RII PKA and co-precipitate catalytic activity. However, the homodimers (Scott 1991, Tasken et al. 1993). cAMP functional importance further involves targeting the binds cooperatively to two sites termed A and B on each enzyme to specific subcellular compartments thereby R subunit. In the inactive holoenzyme, only the B site is providing spatial and temporal regulation of the exposed and available for cAMP binding. When PKA-signaling events. All anchoring proteins contain a occupied, this enhances the binding of cAMP to the A PKA-binding domain and a unique targeting domain site by an intramolecular steric change. Binding of four directing the PKA–AKAP complex to defined subcel- cAMP molecules, two to each R subunit, leads to a lular structures, membranes, or organelles (Fig. 2). In conformational change and dissociation into an R addition to these two domains, several AKAPs are also subunit dimer with four cAMP molecules bound and able to form multivalent signal transduction complexes two C monomers (Kopperud et al. 2002). The C by interaction with phosphoprotein phosphatases, subunits become catalytically active and phosphorylate other kinases, PDEs, and other proteins involved in nearby target substrates on serine or threonine residues signal transduction (Coghlan et al. 1995, Schillace & presented in a sequence context of Arg-Arg-X-Ser/Thr, Scott 1999, Feliciello et al. 2001, Tasken et al. 2001). Arg-Lys-X-Ser/Thr, Lys-Arg-X-Ser/Thr, or Lys-Lys-X- Through this essential role in the spatial and temporal Ser/Thr (reviewed in Shabb (2001) and Ruppelt et al. integration of effectors and substrates, AKAPs provide a (2009)). Type I PKA is more sensitive to cAMP with an high level of specificity and temporal regulation to the activation constant (Kact) of 50–100 nM of cAMP and cAMP–PKA-signaling pathway. Type II PKA is typically was classically know to be mainly cytosolic; while about particulate and confined to subcellular structures and 75% of type II PKA is associated with organelles and compartments anchored by cell- and tissue-specific specific cellular structures and it presents a Kact of AKAPs, which explain why the majority of the AKAPs 200–400 nM of cAMP (Cadd et al. 1990, Dostmann & identified to date bind type II PKA (Colledge & Scott Taylor 1991, Gamm et al. 1996). Each R subunit of PKA 1999, Dodge & Scott 2000, Diviani & Scott 2001). contains an N-terminal docking and dimerization However, several RI-specific AKAPs have also been (D/D) domain, a PKA inhibitor site, and two tandem characterized, although the type I PKA, which is cAMP-binding domains (Heller et al. 2004). The D/D classically known to be biochemically soluble, has domain is connected to cAMP-binding domain A by an been assumed to be mainly cytoplasmic. In addition, www.endocrinology-journals.org Journal of Molecular Endocrinology (2010) 44, 271–284

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Table 1 A kinase anchoring proteins

Tissue Subcellular localization Properties/function

AKAP (gene nomenclature committee name) S-AKAP84/D-AKAP1/ Testis, thyroid, heart, lung, liver, Outer mitochondrial Dual-specific AKAP. Binds lamin AKAP121/AKAP149 (AKAP1) skeletal muscle, and kidney membrane/endoplasmic B and PP1 reticulum/nuclear envelope/ Multiple splice variants. Binds sperm midpiece PDE7A AKAP-KL (AKAP2 ) Kidney, lung, thymus, and Actin cytoskeleton/apical Multiple splice variants cerebellum membrane of epithelial cells AKAP110 (AKAP3 ) Testis Axoneme Binds Ga13 AKAP82/FSC1 (AKAP4 ) Testis Fibrous sheath of sperm tail Potential role in sperm motility and capacitation Multiple splice variants. Binds both RI and RII AKAP75/79/150 (AKAP5 ) Bovine/human/rat orthologs Plasma membrane/ Polybasic domains target to Brain post-synaptic density plasma membrane and den- drites. Binds PKC, calcineurin (PP2B), b-AR, SAP97, and PSD-95 mAKAP (AKAP6 ) Heart (mAKAPb, short), skeletal Nuclear membrane Binds PDE4D3. Spectrin repeat muscle, and brain (mAKAPa, domains involved in subcellular long) targeting. Organizes signal complex with PKA, PDE4D3, Epac, and a MEKK/MEK5/ERK5 module. Longer mAKAPa also anchors PDK1 AKAP15/18 a, b, g, d (AKAP7 ) Brain, skeletal muscle, Basolateral (a) and apical (b) Targeted to plasma membrane pancreas, and heart plasma membrane, cytoplasm via fatty acid modifications (g), secretory vesicles (d) Modulation of NaC and L-type C Ca2 channels (a). ADH- mediated translocation of AQP2 from vesicles to apical mem- brane in distal kidney tubules AKAP95 (AKAP8 ) Heart, liver, skeletal muscle, Nuclear matrix Involved in the initiation of kidney, and pancreas chromosome condensation Binds Eg7/condensin Zinc-finger motif. Binds PDE7A AKAP450/AKAP350/Yotiao/ Brain, pancreas, kidney, heart, Post-synaptic density/ Binds PDE4D3, PP1, PP2A, CG-NAP/Hyperion (AKAP9 ) skeletal muscle, thymus, spleen, neuromuscular junction/ PKN, and PKC3 placenta, lung, and liver centrosomes/Golgi Targets PKA and PP1 to the NMDA receptor Multiple splice variants D-AKAP2 (AKAP10 ) Liver, lung, spleen, and brain Dual-specific AKAP AKAP220/hAKAP220 Testis and brain Vesicles/peroxisomes/ Binds PP1. Dual-specific AKAP (AKAP11 ) centrosome Gravin (AKAP12 ) Endothelium Actin cytoskeleton/cytoplasm Binds PKC and b-AR Xgravin-like (Xgl) is also a putative AKAP AKAP-Lbc/Ht31/Rt31 (AKAP13 ) Ubiquitous Cytoplasm Ht31 RII-binding site used in peptides to disrupt PKA anchoring. Rho-GEF that couples Ga12 to Rho MAP2B Ubiquitous Microtubules Binds tubulin. Modulation of L-type Ca2C channels Ezrin/AKAP78 Secretory epithelia Actin cytoskeleton Linked to CFTR via EBP50/NHERF. Dual specific AKAP. Pieters RI due to RISR domain. Scaffolds PKA-Csk immunomodulating pathway. ERM homolog Radixon also probably AKAP T-AKAP80 Testis Fibrous sheath of sperm tail (continued)

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Table 1 Continued Tissue Subcellular localization Properties/function

AKAP (gene nomenclature committee name) AKAP80/MAP2D Ovarian granulosa cells FSH-regulated protein, identified as MAP2D SSeCKS (Src-supressed C Testis and elongating Actin remodelling Gravin-like kinase substrate) spermatids Pericentrin Ubiquitous Centrosome Binds dynein and g-tubulin Unique RII-binding domain WAVE-1/Scar Brain Actin cytoskeleton Binds Abl and Wrp. Involved in sensorimotor and cognitive function Myosin VIIA Ubiquitous Cytoskeleton Binds RI PAP7 Steroid-producing cells (adrenal Mitochondria Hormonal regulation of gland and gonads) cholesterol transport into mitochondria. Binds RI in vivo, contains RISR Neurobeachin Brain Golgi AKAP28 Primary airway cells Ciliary axonemes Modulation of ciliary beat frequency Myeloid translocation gene Lymphocytes Golgi Binds PDE7A (MTG) 8 and 16b AKAP140 Granulosa cells and meiotic Upregulated by FSH oocytes in granulosa cells Phosphorylated by CDK1 in oocytes. Not cloned AKAP85 Lymphocytes Golgi Not cloned BIG2 (Brefeldin A-inhibited Cytosol and Golgi GEF for ADP-ribosylation factor guanine nucleotide-exchange GTPases. Binds RIa/RIb and protein 2) RIIa/RIIb through three separate PKA-binding domains. cAMP regulated translocation of BIG from cytosol to Golgi Rab32 Mitochondria Regulation of mitochondrial dynamics and fission AKAPCE Caenorhabditis elegans Binds to RI-like subunit RING-finger protein with FYVE- and TGF-b receptor- binding domain DAKAP550 Drosophila Plasma membrane/cytoplasm Contains two RII-binding sites DAKAP200 Drosophila Plasma membrane Binds F-actin and Ca–calmodulin Nervy Drosophila Axons MTG family member. Regulates repulsive axon guidance by clustering PKA and Semaphorin 1a (Sema-1a) receptor Plexin A (PlexA) AKAP97/radial spoke protein 3 Chlamydomonas Flagellar axonemes Located near inner arm dyneins (RSP3) and possibly regulates flagellar motility Synemin Heart Intercalated discs, sarcolemma, PKA–RII-binding AKAP IFs, and Z-lines Is increased significantly in failing hearts Serves dual functions in adult and neonatal cardiac myocytes as both a cytoskeletal protein and as an AKAP

(continued)

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Table 1 Continued Tissue Subcellular localization Properties/function

AKAP (gene nomenclature committee name) Myospryn Muscle-specific The costamere of striated Belongs to the tripartite motif muscle (TRIM) superfamily of proteins Binds dysbindin Closest homolog is AKAP12 Harbors three bona fide PKA-anchoring domains that bind to RIIa Alpha4 integrins Plasma membrane Binds PKA type I Involved in cell migration Sphingosine kinase type Heart Ventricular tissue Shows a partly similar sequence 1-interacting protein (SKIP) to AKAP11 Mediates the levels of two sphingolipids; sphingosine, and sphingosine-1-phosphate (S1P) MyRIP (myosin Perinuclear region Homolog to zebrafish Ze-AKAP2 Va–Rab27a-interacting protein) PKA–RII-binding AKAP Involved in protein trafficking and exocytosis SFRS17A (splicing factor, Nucleus and sliceosome Dual-specificity AKAP arginine–serine-rich 17A) Targets PKA to splicing factor compartments to confer PKA-mediated regulation of pre-mRNA splicing Binds RIb Merlin (neurofibromatosis 2) CNS, hippocampal neurons Soma, dentrites

We apologize for not being able to cite the original work identifying all the listed AKAPs due to space limitations. For review and references, see Tasken & Aandahl (2004) and Jarnaess & Tasken (2007).

dual-specificity AKAPs (D-AKAPs) are capable of divided into two regions: residues 1–23 forming the anchoring both types of R subunits (Huang et al. AKAP-binding surface and residues 24–44, which cover 1997a,b). the majority of the contacts between the two parts of One of the classical characteristics of an AKAP is that dimer. Deletion of residues 1–5 in RII abolishes it binds to the R subunit of PKA through a stretch of binding, without interfering with dimerization, since 14–18 amino acids forming an amphipathic helix. This the branched side chains at residues Ile3 and Ile5 are helix is found in almost all AKAPs characterized to date, needed for the interaction with the AKAPs in the and consists of hydrophobic residues aligned along one hydrophobic groove formed on top of the N-terminal face of the helix and charged residues along the other helices (Hausken et al. 1994, 1996). The RI subunits (Carr et al. 1991). The residues form five turns of an dimerize in a similar way by making a helix-turn-helix a-helix, and this helical shape is important to maintain motif, although the dimerization domain is shifted binding to PKA. Proline substitutions of one or more further from the N-terminus and include amino acids residues within the helix of numerous known AKAPs 12–61 (Leon et al. 1997, Banky et al. 1998, 2000). disrupt their PKA-binding capability (Carr et al. 1991). The structural basis has recently been modelled using The RII subunits normally bind to AKAPs with affinity an NMR structure of the RI D/D domain (Banky et al. in the low nanomolar range (Herberg et al. 2000), while 2003; Fig. 3). The difference in AKAP-binding speci- the affinity of RI subunits is usually high nanomolar to ficity between RI and RII may be explained by the fact submicromolar. Nuclear magnetic resonance (NMR) that the extreme N-terminus in the RI subunit is data (Newlon et al. 1999, 2001) and crystal structures believed to be helical and that this additional helix folds (Gold et al. 2006, Kinderman et al.2006)ofthe back on to the four-helix bundle. Furthermore, the RIa RII–AKAP complex have demonstrated that the RII homodimer is rather compact having a Y-like shape with subunits dimerize at their N-terminus, which is a maximum diameter of 14 nm (Heller et al. 2004), necessary for AKAP binding. The D/D domain consists while the RIIa and RIIb dimers seem to be completely of an antiparallel dimer of two polypeptide chains in a extended with a maximum diameter of nearly helix-turn-helix motif, forming an X-type four-helix 20 nm (Vigil et al. 2004). In addition, the RIa subunit bundle that provides the docking surface for the AKAP undergoes a major conformational change when it helix (Fig. 3). The N-terminal RII fragment can be associates with the catalytic subunit (Vigil et al. 2005),

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RI RII AKAP docking domain AKAP docking domain

NN' N N'

Helix A' Helix A Helix A' Helix A

Helix B' Helix B Helix B' Helix B

C' C C' C Figure 3 Comparison of the RIa and the RIIa D/D domains (Newlon et al. 2001, Banky et al. 2003). NMR structures were depicted using Accelrys Discovery Studio 2.5.1 based on the coordinates from PDB (for RI: 2EZW; http://www.rcsb.org/pdb/explore/explore.do?structureIdZ2EZW; and for RII: 2DRN, http://www. rcsb.org/pdb/explore/explore.do?structureIdZ2DRN). The extreme N-termini are shown in yellow, helices A and A0 in blue, and helices B and B0 in red respectively. while the RIIa subunits remain fully extended (Zhao PKA-binding region of D-AKAP2. The peptides were et al. 1998). In contrast, the RIIb subunits round up designated as AKB–RI (where AKB stands for A-kinase to form a globular protein upon complex formation binding) and AKB–RII (Burns-Hamuro et al. 2003). In with the catalytic subunits (Vigil et al. 2006). parallel, a third generation of peptides was developed based on AKAP-IS utilizing the same two-dimensional peptide array technology to test for both RI and RII Delineation of PKA–AKAP interaction with binding. Upon substitutions selecting for either RI or disruptor peptides RII specificity, the RI-anchoring disruptor (RIAD) and the SuperAKAP-IS were developed with three-order To establish a functional role for various AKAPs, magnitude selectivity for RI and RII respectively peptides have been developed to disrupt the (Carlson et al. 2006, Gold et al. 2006). From these interaction between PKA and AKAPs and serve as studies, it became apparent that type I PKA binds more important tools to study the relevance of PKA in cellular N-terminally in the A-kinase-binding region and processes. All peptides developed have attempted to tolerates more acidic residues on the hydrophilic face mimic the PKA-binding domains of AKAPs and are of the amphipathic helix (Carlson et al. 2006). made to be small stretches of amino acids forming Furthermore, the recent discovery of a region in amphipathic helixes. The protein originally called D-AKAPs that enhance anchoring of type I PKA, the Ht31, now known as AKAP-Lbc, was first characterized RI specifier region (RISR), has led to more insight into with respect to its PKA-binding domain and shown to how type I PKA can bind to AKAPs, although the affinity contain an 18-amino acid amphipathic helix that binds for the amphipathic helix is comparably lower PKA (Carr et al. 1991, 1992). Peptides encompassing ( Jarnaess et al. 2008). In addition, a 13-mer peptide this sequence display low nanomolar affinity for RII derived from RISR was developed and shown to disrupt Z G the ezrin–type I PKA interaction independently of (KD 2.2 0.03 nM; Newlon et al. 2001), were shown to disrupt R–AKAP interactions inside cells, and have been peptides that mimic the amphipathic helix, and this used extensively as the archetypical PKA-anchoring released cAMP-mediated inhibition of T-cell immune disruptor (Rosenmund et al. 1994). Experiments using function ( Jarnaess et al. 2008). These third generation Ht31 have demonstrated that this peptide disrupts anchoring disruptors serve as tools to delineate interactions both with type I and type II PKA with biological effects specifically mediated by the type I AKAPs (Herberg et al. 2000), and later other peptides and the type II PKA, see Fig. 4 for illustration of effect have been developed that specifically disrupt the (Carlson et al. 2006, Torheim et al. 2009). AKAP–RI or AKAP–RII interaction. The peptide AKAP-in silico (AKAP-IS) is a peptide derived from the minimal RII-binding domains of ten AKAPs and AKAPs as unique targeting vehicles optimized with regards to RII binding, yielding subnanomolar binding affinity for RII (Alto et al.2003). In addition to the PKA-binding domain, AKAPs contain By the use of two-dimensional peptide arrays, two specialized targeting domains responsible for tethering peptides with higher affinities and specificities the complex to different subcellular localizations for either RI or RII were developed based on the through protein–lipid or protein–protein interaction. www.endocrinology-journals.org Journal of Molecular Endocrinology (2010) 44, 271–284

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AKAPs have been identified in association with a variety of cellular compartments, including centrosomes, endoplasmic reticulum, the Golgi apparatus, mito- A chondria, microtubules, cell membranes, nuclear

Target 1 Target 2 matrices, secretory granules, and the cytoskeleton (Wong & Scott 2004). Several studies suggest that protein–lipid interactions target most AKAP–PKA AKAP AKAP cAMP cAMP complexes to the correct subcellular environment, C C Type I PKA Type II PKA while protein–protein interactions precisely orient the C C kinase toward its substrate (Carnegie & Scott 2003). For example, AKAP15/18a is targeted to the mem- brane by myristoylation and palmitoylation signals in C close proximity to PKA substrates such as Ca2 and Biological effect 1 Biological effect 2 C Na channels (Fraser et al. 1998, Gray et al. 1998). C B In addition, AKAP15/18a interacts with L-type Ca2 Target 1 Target 2 channel via a modified leucine zipper motif (Hulme et al. 2003). Furthermore, AKAP79 is localized AKAP AKAP cAMP to the post-synaptic membranes in neurons through C repeated sequences that bind negatively charged RIAD Type II PKA C phospholipids, whereas this anchoring protein cAMP is selectively coupled to substrates such as a-amino- C Type I 3-hydroxy-5-methyl-4isoxazolepropionic acid-type PKA C C C glutamate receptor ion channels and Ca2 or K Biological effect 2 channel through specific protein–protein interactions C (Dodge & Scott 2000). Target 1 Target 2 Different AKAPs can also be targeted to the same subcellular organelle, indicating that within a single

AKAP AKAP cellular compartment, various AKAPs function to target cAMP PKA to distinct subdomains and to specific substrates. C Type I PKA SuperAKAP-/S Also, splice variants and isoforms of an AKAP can be C cAMP diversely targeted. Four anchoring proteins (D-AKAP1, C D-AKAP2, PAP7, and Rab32) anchor PKA to the Type II PKA C mitochondria, while ezrin, WAVE-1 (Wiskott-Aldrich Biological effect 1 syndrome protein family verprolin homologous protein), and AKAP-Lbc all tether PKA to distinct D parts of the actin cytoskeleton (Tasken & Aandahl Target 1 Target 2 2004). In addition, the AKAPs pericentrin and AKAP450 both secure precise PKA phosphorylation at AKAP AKAP the centrosomes (Tasken & Aandahl 2004). While AKAP450 is targeted to the centrosomes, its splice RIAD SuperAKAP-/S variant, Yotiao, is targeted to post-synaptic densities. cAMP cAMP Similarly, the different isoforms of AKAP18 have C C a Type I PKA Type II PKA distinct subcellular localization. The -isoform is C C located at the lateral membrane of polarized epithelial cells, the b-isoform is located at the apical membrane of E polarized epithelial cells, while the g-isoform is mainly

Target 1 Target 2 cytoplasmic (Trotter et al. 1999).

AKAP AKAP Figure 4 Schematic illustration of the effect of specific anchoring disruptor peptides. (A) Model of type I PKA and type II PKA signal Ht31 Ht31 complexes organized by specific AKAPs and mediating biological cAMP cAMP effects 1 and 2 respectively. (B and C) Models for effect of RIAD or SuperAKAP-IS on biological effects 1 and 2. (D and E) Effect of C C Type I PKA Type II PKA RIAD and SuperAKAP-IS together or Ht31 on biological effects C C 1 and 2. cAMP, red-filled circles; phosphate, blue-filled circles.

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AKAPs as multienzyme scaffolding C complexes C cAMP AKAPs mediate high levels of specificity and complexity in the cAMP–PKA signaling pathway through the mAKAP interaction with various signaling enzymes and by PDE cAMP assembly of multiprotein complexes. Several AKAPs Epac with this property have been identified that provide precise spatiotemporal regulation of the cAMP–PKA Rap1 pathway and integration with other signaling pathways in one signal complex. Recent examples are AKAP79, AKAP450, AKAP220, AKAP-Lbc, ezrin, gravin, mAKAP, and WAVE that all have been shown to form multi- MEKK enzyme complexes, and it is likely that we are still at the very beginning of understanding the roles AKAPs play in the orchestration of intracellular signaling events in health and disease. The potential for multivalency in MEK5 AKAPs was first demonstrated for AKAP79, which in addition to PKA interacts with protein kinase C (PKC) and the calcium/calmodulin-dependent phosphatase, ERK5 PP2B (Coghlan et al. 1995, Klauck et al. 1996). More recent work on AKAP79 using meticulously designed Figure 5 Signal complex consisting of mAKAP, PKA, PDE4D3, FRET reporters and mutant versions of the protein has Epac, and a MEKK/MEK5/ERK5 module. mAKAP anchors PKA shown that distinct combinations of enzymes targeted and PDE4D3, whereas PDE4D3 scaffolds an Epac–Rap1 pathway that coordinates a MEKK/MEK5/ERK5 module. cAMP, by AKAP79 in different contexts give functional red-filled circle; phosphate, blue-filled circle. diversity with respect to what signal pathways the AKAP-targeted complex participates in (Hoshi et al. synchronized within the same AKAP complex, indicat- 2003, 2005). Furthermore, the same authors recently ing that local control of cAMP signaling by AKAP demonstrated that interaction with AKAP79 alters the proteins is more intricate than previously anticipated. regulation and pharmacological profile of the bound PKC (Hoshi et al. 2010). Subsequently, other AKAPs have been shown to work as multi-scaffolding proteins as well. AKAP220 interacts with protein phosphatases 1 Functional consequences of PKA anchoring (PP1; Schillace & Scott 1999), AKAP450 anchors PKC, polynucleotide kinase, PP2A, PPI, and PDE4D3 to the The biological relevance of anchoring is extensively centrosomes (for review, see Tasken & Aandahl studied using inhibitory peptides that disrupt PKA (2004)), and AKAP149, when located at the nuclear anchoring and expression of compartment-specific membrane, binds PP1 and PKC (Steen et al. 2000, AKAPs to redistribute PKA to defined subcellular Kuntziger et al. 2006). An interesting and more sites. Later studies have also used siRNA-mediated complex example is the muscle-specific AKAP, knockdown and reconstitution experiments expressing mAKAP, that coordinates two integrated cAMP effector a mutant AKAP that does not bind PKA or employed pathways. Both PKA and PDE4D3 are present in the engineering of mutant mice to study the function mAKAP-signaling complex (Dodge et al. 2001), creating of AKAPs. a negative feedback loop where PKA phosphorylation Early studies focused mainly on rapid cAMP-respon- of PDE4D3 increases its activity and facilitates more sive events, such as modulation of ion channels. Use of rapid signal termination. Moreover, PDE4D3 serves as the prototypical disruptor peptide Ht31 showed that an adaptor protein for Epac and the extracellular anchored PKA is involved in the regulation of cardiac C C signal-regulated kinase (ERK), ERK5 (Dodge-Kafka and skeletal muscle L-type Ca2 channels and Ca2 - C et al. 2005). ERK5 phosphorylation of PDE4D3 activated K channels in smooth muscle (Gao et al. decreases the PDE activity (Hoffmann et al. 1999), 1997). AKAP79, AKAP15/18, and AKAP-KL have all thereby favoring a local increase of cAMP and been shown to be involved in the modulation of ion successive PKA and Epac activation. Epac can then channels through directing pools of kinases and activate the small Ras-like GTPase Rap1, which inhibits phosphatases near particular channel subunits (Fraser the MAPK/ERK kinase kinase (MEKK) thereby relea- & Scott 1999). Expression of AKAP79 has been used to sing inhibition of PDE4D3 by ERK5 (Fig. 5). Thus, demonstrate an increase in cAMP-dependant modu- C two coupled cAMP-dependent feedback loops are lation of L-type Ca2 channels (Gao et al. 1997) as well www.endocrinology-journals.org Journal of Molecular Endocrinology (2010) 44, 271–284

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C as in the native K secretory channels (ROMK) in the type II PKA by several protein interaction method- kidney (Ali et al.1998). Likewise, expression of ologies, and was found to be co-localized with the AKAP15/18 stimulates the process of insulin secretion catalytic (C) subunit of PKA in splicing factor compart- from intact cells by targeting PKA to the membrane, ments in the nucleus. In addition, SFRS17A regulated while peptide-mediated disruption of PKA–RII E1A alternative splicing as shown in E1A minigene interaction leads to a decrease. This might be a result experiments, indicating that SFRS17A targets PKA to C of enhanced Ca2 channels (Fraser et al. 1998). splicing factor compartments to confer PKA-mediated PKA was identified as the kinase involved in the regulation of pre-mRNA splicing ( Jarnaess et al. 2009). recycling of b1-adrenergic receptor with the use of the To study the function of the WAVE-1 signal complex peptide Ht31. However, it was through the use of siRNA in vivo, targeted disruption was used to knock out interference that the AKAP responsible for anchoring the WAVE-1 gene showing that loss of this AKAP PKA, AKAP79, was identified (Gardner et al. 2006). causes sensorimotor retardation and reduced learning Moreover, while the use of Ht31 demonstrated the and memory in mice, phenotypes analogous to involvement of an AKAP in cAMP-mediated immuno- patients with 3p-syndrome mental retardation modulation, siRNA-mediated knockdown of the AKAP (Soderling et al. 2003). Later, null mutant mice have ezrin eliminated type I PKA from T-cell lipid rafts and been generated for AKAP149 (Newhall et al. 2006), disrupted cAMP-mediated inhibition of T-cell immune AKAP79 (Hall et al. 2007, Navedo et al. 2008, Tunquist function (Ruppelt et al. 2007), whereas reconstitution et al. 2008), AKAP95 (Yang et al. 2006) and ezrin experiments reversed the effects seen with siRNA alone (Saotome et al. 2004) demonstrating distinct roles on ( Jarnaess et al. 2008). differentiation, development and hormonal regulation The AKAP18d is involved in the recruitment of PKA of physiological function. to a supramolecular complex containing phospholam- C ban (PLB) and sarcoplasmic reticulum Ca2 -ATPase 2 (SERCA2), and is important to discretely regulate PKA AKAPs in disease phosphorylation of PLB and thereby the PLB-inhibitory C effect on SERCA2 and Ca2 reuptake into heart A genetic polymorphism in D-AKAP2 (Ile646Val), sarcoplasmic reticulum. Displacement of PLB from which decreases the affinity for type I PKA R subunit, AKAP18d and siRNA knockdown experiments revealed has been shown to be associated with familial breast that scaffolding of the complex by AKAP18d to allow cancer. Likewise, another study has shown that the precise spatiotemporal control of PKA phosphorylation AKAP-Lbc Lys526Gln polymorphism is associated with C of PLB is necessary for adrenergic effect on Ca2 familial breast cancer. This amino acid substitution reuptake (Lygren et al. 2007; Fig. 6). alters the secondary structure of AKAP-Lbc and affects A recent work has demonstrated that the RISR is the function of the protein. Carriers of both mutations, found in D-AKAPs, and this sequence was utilized in a D-AKAP2 Ile646Val and AKAP-Lbc Lys526Gln, in bioinformatic search to identify a novel anchoring combination have an even greater risk of developing protein, the splicing factor arginine–serine-rich 17A breast cancer (Wirtenberger et al. 2007). Furthermore, (SFRS17A). SFRS17A was found to bind both type I and the Ile646Val substitution in D-AKAP2 has been

ABCa2+ Ca2+ Ca2+ Ca2+

SR SR S S E E R R C C A A 2 2 PLB CytosolPLB Cytosol AKAP18δ Ca2+ AKAP18δ cAMP 2+ Ca2+ Ca C C Ca2+ C C 2+ Ca Ca2+ Figure 6 PKA–AKAP18d–PLB–SERCA2 complex. (A) Resting situation, no adrenergic drive, low heart rate, SERCA2 inhibited by PLB with low ATP and energy consumption. (B) Adrenergic stimulation paces the heart and increased heart rate. SERCA2 released from PLB inhibition by PKA phosphorylation leading to fast Ca2C reabsorption and high ATP and energy consumption.

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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 281 associated with increased P–R interval in electrocardiog- Declaration of interest raphy, whereas a Ser1570Lys substitution in Yotiao, which delocalizes the AKAP itself, has been linked to The authors declare that there is no conflict of interest that could be long QT syndrome and cardiac arrhythmias (Kammerer perceived as prejudicing the impartiality of the research reported. et al. 2003, Chen et al. 2007). Funding

Concluding remarks Our work is supported by the Functional Genomics Program, Research Council of Norway, The Norwegian Cancer Society, and Although a number of early studies indicated possibil- Novo Nordic Research Foundation. ities of compartmentalization of cAMP, the predominat- ing view just over a decade ago was still that cAMP would be elevated throughout the cell in response to many Acknowledgements ligands. Detailed studies of compartmentalization of specific receptors and ACs to distinct membrane We are grateful to Dr Arnaud David for his help with preparing figures subdomains, as well as live-cell imaging of cAMP and with illustrations of structures. unravelling of the subcellular targeting of PDEs have now made clear that physiological increases in cAMP occur in discrete microdomains. Similarly, although References PKA type II was well known to be biochemically particulate and AKAPs were starting to be identified Ali S, Chen X, Lu M, Xu JZ, Lerea KM, Hebert SC & Wang WH 1998 20 years ago, the prevailing view was still for a long time The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels. PNAS 95 that many effects of cAMP would be mediated by en bloc 10274–10278. activation of PKA over large areas of the cell and/or Alto NM, Soderling SH, Hoshi N, Langeberg LK, Fayos R, Jennings PA that the C subunit would be released from a PKA & Scott JD 2003 Bioinformatic design of A-kinase anchoring holoenzyme complex and travel some distance to find protein-in silico: a potent and selective peptide antagonist of type II protein kinase A anchoring. PNAS 100 4445–4450. its substrate. However, since then it has become clear Baillie GS, Scott JD & Houslay MD 2005 Compartmentalisation of that a large spectrum of AKAP proteins is available phosphodiesterases and protein kinase A: opposites attract. FEBS (more than 50 AKAPs per date when differentially Letters 579 3264–3270. targeted splice variants are included, Table 1). Further- Banky P, Huang LJ & Taylor SS 1998 Dimerization/docking domain of more, new AKAPs for PKA type I, long thought to be the type Ia regulatory subunit of cAMP-dependent protein kinase. Requirements for dimerization and docking are distinct but primarily cytoplasmic and freely diffusible, are now overlapping. Journal of Biological Chemistry 273 35048–35055. increasingly reported. In addition, the requirement for Banky P, Newlon MG, Roy M, Garrod S, Taylor SS & Jennings PA 2000 anchoring of PKA in order to regulate specific Isoform-specific differences between the type Ia and IIalpha substrates as well as to mediate a number of physiologi- cyclic AMP-dependent protein kinase anchoring domains revealed by solution NMR. Journal of Biological Chemistry 275 cal effects has been extensively studied over the past 35146–35152. decade. With few exceptions, it has been shown that Banky P, Roy M, Newlon MG, Morikis D, Haste NM, Taylor SS & most cAMP–PKA-regulated physiological processes Jennings PA 2003 Related protein–protein interaction modules require an anchored kinase. It has been made clear present drastically different surface topographies despite a from studies discussed here that the localized cAMP conserved helical platform. 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