- Email: [email protected]
Subcellular Targeting of PKA through AKAPs
Chapter 165
Subcellular Targeting of PKA through AKAPs: Conserved Anchoring and Unique Targeting Domains Matthew D. Pink and Mark L. Dell’Acqua Department of Pharmacology, University of Colorado Denver School of Medicine, Aurora, Colorado
Introduction Regulation of the opposing actions of adenylyl cyclases (AC) and phosphodiesterases (PDE) controlling levels of the diffusible intracellular second messenger cAMP is central to many signaling responses. A major effector of cAMP in eukaryotic cells is the cAMP-dependent protein kinase (PKA). PKA is a heterotetrameric enzyme consisting of two regulatory subunits (R) that dimerize and each bind and inhibit a single catalytic subunit (C) to form an R2C2 holoenzyme (Figure 165.1a) [1]. Activation of the inactive PKA holoenzyme by cAMP occurs when two molecules of cAMP binding to each R subunit resulting in a conformational change that releases the active C-subunits from the inhibitory R2 dimer (Figure 165.1a). The released
(a)
active C-subunits phosphorylate serine and threonine residues in the optimal sequence contexts of RRXS/T or KRXXS/T to regulate target protein function. PKA-C subunits can phosphorylate target proteins rapidly near the site of release, and in time diffuse to more distant locations such as the nucleus, where additional targets are phosphorylated [2]. Remarkably, this ubiquitous signaling pathway is used in different cell types to efficiently transduce signals to a myriad of different yet very specific target proteins in a wide variety of cellular compartments. Thus, elucidating how this PKA signaling versatility is achieved without compromising specificity and efficiency is fundamental to understanding cAMP signal transduction pathways. Over the past two decades we have learned that both diversity and specificity in cAMP signaling is in large part
(b)
Figure 165.1 Anchoring and subcellular targeting of PKA by AKAPs. (a) Structure of the PKA holoenzyme and regulation of catalytic activity by cAMP. (b) AKAP anchoring of the PKA holoenzyme near specific cellular substrates. Handbook of Cell Signaling, Three-Volume Set 2 ed. Copyright © 2010 Elsevier Inc. All rights reserved.
1329
1330
achieved by targeting the PKA holoenzyme to discrete subcellular locations such that, upon release of the C subunit, phosphorylation of co-localized target substrates is greatly enhanced (Figure 165.1b) [2]. This subcellular targeting of PKA is mediated by a class of anchoring/scaffolding proteins called AKAPs (A-Kinase Anchoring Proteins) [3–5]. AKAPs anchor PKA by binding the R-subunit dimer through a structurally conserved anchoring domain, and then target the holoenzyme to specific locations within the cell through unique targeting domains (Figure 165.1b). AKAPs frequently contain additional protein–protein interaction motifs that serve as tethering sites for the target substrates, as well as additional signaling proteins such as other kinases, phosphatases, and scaffold and adaptor proteins. Thus, many AKAPs functions as signal-integrating scaffolds that coordinate both subcellular localization and complex assembly of multi-protein signal transduction machines. This review chapter will focus on the structurally conserved R subunit anchoring domain and examples of unique subcellular targeting domains that localize anchored-PKA for regulation of co-localized target substrates. The following chapter will deal more with the issue of AKAP multi-enzyme complexes integrating different signaling pathways.
Structurally conserved PKA anchoring determinants There are three PKA C subunit isoforms (C, C, C) and four R subunit isoforms (RI, RI, RII, RII) [1]. While the C subunit isoforms share very similar properties, the RI, and RII, subunit groups show significant differences in both cAMP binding affinity [1] and AKAP anchoring [6]. Thus, due to these differences in RI and RII, PKA is divided into type I and type II holoenzymes based on the identity of the R subunit. While it is clear that both type I and type II PKA holoenzymes can bind to AKAPs and thus be targeted to specific subcellular domains, the majority of AKAP proteins bind RII with 100- to 1000fold higher affinity than RI [6–10]. This difference in AKAP binding affinity is reflected in observations that in many, but not all, cell types PKA-RII is more discretely localized to specific cellular structures, while PKA-RI is diffusely distributed in the cytoplasm. The differences in cAMP binding affinity between RI and RII fit well with these differing distributions. Type I holoenzymes have higher cAMP binding affinities, and thus may be adapted to generate more prolonged responses in response to lower cAMP signals encountered in the bulk cytoplasm. In contrast, type II holoenzymes bind cAMP with lower affinity, a possible adaptation to being targeted by AKAPs to local environments where cAMP can be much higher due to compartmentalized regulation of either AC or PDE [11, 12]. One important function of reduced cAMP sensitivity might
PART | II Transmission: Effectors and Cytosolic Events
be to maintain low activity of type II holoenzyme anchored in local environments where even basal cAMP levels would fully activate the type I enzyme, leading to complete loss of C subunits. RI and RII also differ in that the RII, but not RI, autoinhibitory C-subunit binding domain is a PKA substrate; a recent mutagenesis study demonstrates that phosphorylation of RII increases binding to AKAPs, thus suggesting additional levels of complexity and positive feedback in AKAP-anchored PKA signaling [13]. A number of previous mutagenesis studies have shown that AKAPs bind hydrophobic residues in the N-terminus of the R subunits in a manner that depends on formation of the R–R dimer [14–16]. Indeed, NMR structural studies show that both the RI and RII N-terminal dimerization domains form anti-parallel four-helix bundles in which dimerization creates an extended hydrophobic surface that binds the AKAP (Figure 165.2c) [17–19]. This hydrophobic surface is somewhat less extensive and more sterically hinderd in RI compared to RII dimmers, possibly explaining why in general most AKAPs bind RII with higher affinity than RI. Accordingly, the AKAPs all bind to the R subunit N-terminal dimerization domain through hydrophobic interactions [18, 20–22]. The PKA anchoring domains from different AKAPs have very little primary amino acid sequence similarity, yet share a conserved hydrophobic character and secondary structure (Figure 165.2a, b) [18, 20, 21]. Thus, AKAPs are a family of functionallyrelated proteins arising from convergent evolution, as opposed to diverging from a common ancestral AKAP protein. The common hydrophobic and secondary structure in AKAP PKA anchoring domains is seen as a conserved spacing of hydrophobic residues which map to one side of an amphipathic -helix of about 18 residues in length (Figure 165.2a, b) [18, 21]. One exception to this conservation of an amphipathic-helix anchoring domain is seen in pericentrin, which anchors PKA-R subunits through a unique, more extended hydrophobic motif of unknown structure [23]. For the conserved anchoring motif found in all other AKAPs, mutagenesis studies have lent support to the amphipathic-helix structural model by showing that substitution of hydrophobic residues with either hydrophilic residues to change polarity or proline residues to break helical structure cause inhibition of R subunit binding [21, 22]. NMR structures have also been obtained showing clearly that two AKAP anchoring domain peptides, Ht31 (493–515) and AKAP79(392–413), with divergent primary sequences both bind to RII in similar helical conformations with extensive contacts made between the hydrophobic face of the helices and the large hydrophobic surface on the RII dimer (Figure 165.2c) [18]. More recent X-ray crystal structures using two additional AKAP anchoring domain peptides, D-AKAP2 and AKAP-IS, confirm the NMR structures by showing that the non-palindromic AKAP helix sits diagonally across the face of the symmetrical RII dimer, creating an asymmetrical RII–AKAP complex [24, 25].
Chapter | 165 Subcellular Targeting of PKA through AKAPs: Conserved Anchoring and Unique Targeting Domains
1331
(a)
(b)
(c)
Figure 165.2 Structurally conserved AKAP–PKA anchoring domains. (a) Divergent primary structures for PKA-anchoring in AKAP family members. Hydrophobic residues are shaded. (b) Conserved amphipathic alpha-helical secondary structures for AKAP PKA anchoring domains. Hydrophobic residues are shaded on helical wheel diagrams. (c) Conserved mechanisms of PKA anchoring revealed by NMR solution structures of AKAP–RII complexes. The structures show AKAP anchoring domain peptides from Ht31(493–515) and AKAP79(392–413) (top) bound to an RII(1–45) N-terminal domain dimer (bottom). Figure adapted from [18] (Newlon et al., 2002, EMBO J. 20: 1651–1662), with permission from P. A. Jennings and Oxford University Press.
At the interface, a series of non-polar aliphatic residues on the AKAP create a hydrophobic ridge, which interacts with a preformed cluster of hydrophobic residues from each RII protomer [24, 25]. Amino acid residues critical for RII binding to D-AKAP2 and AKAP-IS, while not completely conserved, are found in the same structural space. Of particular importance is the non-polar aliphatic amino acid Valine, found in the center of the hydrophobic ridge [24, 25]. Valine at this position is conserved amongst all RIIspecific AKAPs, and most AKAPs able to bind both RI and RII isoforms. Further, Asparagine and Glutamine at two other positions, respectively, are conserved in RII-specific AKAPs [24, 25], while acidic residues at one of these positions predominate in RI-specific and dual-specificity AKAPs. This knowledge was used to create an AKAP peptide, SuperAKAP-IS, with abolished binding for RI and increased specificity for RII. Creation of SuperAKAPIS, along with recently developed RI-specific anchoring inhibitors [26, 27], should further allow for the function of RII versus RI anchoring to be examined in various cellular functions in different cell types.
Unique subcellular targeting domains The variety and specificity that is made possible by AKAP– PKA anchoring is in large part a function of unique targeting domains in different AKAP molecules (Figure 165.1b).
AKAP molecules have been characterized at a myriad of distinct subcellular locations (Figure 165.3a, b) including the plasma membrane, intracellular vesicles [28, 29], actin and microtubule cytoskeletons, mitochondria, endoplasmic reticulum (ER), Golgi, and centrosomes [30, 31]. For example, the AKAP protein MAP2 is targeted to dendritic microtubules in neurons by direct binding to tubulin [32–34]. Scar/Wave1, an AKAP that also anchors the abl-Tyrosine kinase, binds to actin both in focal adhesions (Figure 165.3b) and membrane ruffles in fibroblasts where it regulates the actin polymerization activity of the Arp2/3 complex (see Chapter 166) [35]. The skeletal and cardiac muscle-enriched mAKAP protein, which also anchors PDE4D3 (see Chapter 166) [11], is targeted by a series of spectrin repeats to perinuclear ER/SR membranes including, most prominently, the nuclear membrane (Figure 165.3b) [36, 37]. dAKAP1/sAKAP84/AKAP121 can be targeted either to the ER (in the liver) or outer membrane of mitochondria (in most other cells) by two different N-terminal targeting sequences produced by alternate mRNA splicing [38–40]. AKAP95, has even been shown to associate with the nuclear matrix and chromatin, despite the fact that PKA holoenzyme/R-subunits are excluded from the nucleus during interphase (Figure 165.3a) [41, 42]. However, interactions of AKAP95 with PKA and chromatin could have important functions in regulating chromosome condensation during mitosis when the nuclear envelope is absent [43, 44]. The AKAP18 splice variant is also found in the nucleus as well as the cytoplasm, and has a conserved
1332
PART | II Transmission: Effectors and Cytosolic Events
(a)
(b)
Figure 165.3 Unique AKAP targeting domains and subcellular localizations. (a) Subcellular compartmentalization of different AKAP family members. References for AKAPLbc/Ht31 [88], MTG [89], My-RIP [90], Gravin [91], and Rab 32 [92] localization. For other references, see the text. (b) Specific subcellular localizations observed for selected AKAPs by fluorescence microscopy.
nuclear localization sequence near its N-terminus that targets it to the nuclei of kidney cells and oocytes [45]. In contrast, the related AKAP18 splice variant is found at the sarcoplasmic reticulum in cardiac myocytes and on secretory vesicles in renal collecting duct epithelial cells [46, 47]. Curiously, the central domain of AKAP18 is able to bind directly to the small molecule adenosine 5 monophosphate (5AMP), a product of cAMP and ATP hydrolysis [48]. The exact functional role of this interaction is not yet known, however, it is hypothesized that AKAP18 could somehow link cellular AMP levels to regulation of PKA phosphorylation events [48]. In certain cell types specific localization to discrete plasma membrane domains is seen, such as localization to apical or basolateral membrane domains in polarized epithelial cells and synaptic membrane specializations in neurons. AKAP75/79/150, an AKAP scaffold protein that also binds protein kinase C and calcineurin-protein phosphatase 2B(CaN-PP2B) (see Chapter 166) is targeted to the plasma membrane/cortical cytoskeleton (Figure 165.3a) by three N-terminal polybasic domains that bind to the acidic phospholipid phosphatidylinositol-4,5-bisphosphate, F-actin, and cadherin adhesion molecules [49–52]. This same N-terminal basic domain mediates specific targeting to excitatory postsynaptic membrane specializations located on actin-rich dendritic spines in neurons and cadherin-mediated adherens junctions on basolateral membranes in epithelial cells (Figure 165.3b) [50, 52]. Interestingly, membrane localization of AKAP79/150-PKA complexes is not static, and is subject to positive regulation by cadherin engagement in epithelial cells and negative regulation by NMDA-type glutamate receptor Ca2 signaling (Figure 165.4) through activation of CaN-PP2B-mediated actin reorganization and
Figure 165.4 Dynamic regulation of AKAP targeting. Punctate membrane localization of AKAP79/150 to dendritic spines in hippocampal neurons is negatively regulated by activation of NMDA receptors (agonist NMDA; antagonist APV). AKAP79/150 assumes a diffuse cytoplasmic localization in both dendrites and the cell body in NMDA-treated neurons.
phospholipase C-mediated cleavage of phosphatidylinositol4,5-bisphosphate in neurons [50, 52–54]. Thus, dynamic regulation of AKAP targeting is likely to be another important mechanism for controlling the spatio-temporal specificity of cAMP signaling in different cell types [55]. The low molecular weight AKAP15/18 splice variant is also plasma-membrane localized; however, its membrane targeting is determined by N-terminal lipid modifications of myristoylation of Gly-1 and dual palmitoylation of Cys-4, Cys-5 (Figure 165.3b) [56]. In MDCK epithelial cells this AKAP15/18 isoform selectively targets to the basolateral membrane. However, another alternate splice variant, AKAP18, that contains an additional exon coding for a 24 amino acid insert localizes to the apical membrane [57]. Interestingly, two additional AKAP families, the AKAPKL and ERM proteins (Ezrin/Radixin/Moesin), are also specifically targeted to apical membranes, where they bind to cortical actin (Figure 165.3a) [58, 59].
Chapter | 165 Subcellular Targeting of PKA through AKAPs: Conserved Anchoring and Unique Targeting Domains
Probing cellular functions of AKAP – PKA anchoring The importance of AKAP-targeted pools of PKA has been implicated in numerous cellular responses, including transcription [60, 61], secretion [56, 62, 63], cell cycle regulation [44, 64, 65], and neuronal synaptic plasticity [66–72]. Functionally, the role of AKAP–PKA targeting has been studied in the greatest detail for cAMP regulation of membrane ion channels that control muscle cell contraction and neuronal synaptic plasticity [66–74]. These studies of ion channel regulation have traditionally either used anchoring inhibitor peptides, such as the peptide Ht31, to displace PKA from endogenous AKAPs, or heterologous co-expression of an AKAP with the ion channel of interest. More recent studies have also employed expression of mutated AKAPs in primary cell culture in conjunction with RNAi-mediated suppression of endogenous AKAP expression or use of AKAP knockout, mutant knock-in, or transgenic mice. Use of anchoring inhibitor peptides to disrupt PKA-R subunit anchoring was first shown to block cAMP regulation of endogenous plasma membrane channel activity for neuronal AMPA-type glutamate receptors and cardiac and skeletal muscle L-type voltage-gated calcium channels similar to inhibition of PKA-C subunit catalytic activity [74–78]. In the case of AMPA receptors, the effects of anchoring disruption on channel currents is at least in part due to changes in channel trafficking and membrane stability [71]. Together, these studies suggested that endogenous AKAPs were important for targeting PKA to the plasma membrane in proximity to the regulated channels. Subsequent studies in a heterologous HEK-293 cell expression system confirmed the previous results by showing that co-expression of these channels with an appropriate membrane targeted AKAP partner, such as AKAP15/18 or AKAP79, could reconstitute cAMP-PKA regulation of channel activity [75, 79, 80]. Recent development of transgenic mice expressing an inducible PKA-anchoring inhibitor revealed impairments in learning and memory and PKA regulation of neuronal synaptic plasticity that is likely to involve more than one AKAP in both the pre- and postsynaptic membrane compartments [70]. Use of RNAi technology has allowed for the functions of postsynaptic AKAP79/150 in particular to be studied more directly in neurons. RNAi experiments where endogenous rat AKAP150 was knocked down and rescued with human AKAP79 unable to anchor PKA or CaN-PP2B have confirmed roles for this AKAP in neuronal AMPA receptor as well as L-type calcium channel regulation [67, 77]. Complementary studies in knockout and knock-in mice where endogenous AKAP150 was genetically deleted or mutated so that it can no longer anchor PKA has allowed for further exploration of these cellular functions in neurons. Recent mouse studies all indicate that
1333
AKAP79/150-anchored PKA is integral in regulation of AMPA receptors during certain forms of synaptic plasticity [68, 69, 72]. However, there are some differing observations regarding whether or not basal AMPA receptor phosphorylation is decreased in these different transgenic mice, and how that relates to the negative regulation of AMPA receptor activity by acute inhibition of PKA activity or disruption of PKA anchoring observed in prior studies [67, 71, 78, 80, 81]. In the case of L-type calcium channels, it is apparent from both RNAi and transgenic mice that AKAP150-anchored PKA is clearly necessary to phosphorylate Cav1.2 and regulate neuronal channel activity [66, 77]. Collectively, it has been revealed that AKAP79/150 is integral in maintaining elevated postsynaptic PKA levels, where it aids in the regulation of membrane ion channels. As an additional level of targeting specificity even beyond AKAP co-localization with ion channels in the membrane, modified leucine zipper coiled-coiled motifs that promote direct association with channel substrates have been identified on several AKAPs. While this topic is discussed more in Chapter 166, we will briefly mention two examples here. AKAP79/150 contains a modified leucine zipper coiled-coiled motif located on its C-terminus [77]. This leucine zipper allows AKAP79/150 to directly interact with neuronal L-type CaV1.2 calcium channels to promote AKAP79/150-anchored PKA and CaN-PP2B bidirectional regulation Cav1.2 activity, as discussed above, as well as signaling through CaN-PP2B to control downstream transcription factor activation [77]. A C-terminal leucine zipper motif in the AKAP Yotiao, along with its N-terminal domain, mediates its association with the cardiac potassium channel KCNQ1 [82, 83]. In response to increased heart rate stimulated by the sympathetic nervous system, this direct interaction, along with Yotiao-anchored PKA phosphorylation of KCNQ1, decreases action potential duration, thereby maintaining sufficient diastolic filling between heartbeats [82–86]. Importantly, recent research has identified a mutation in the C-terminal domain of Yotiao present in the inheritable human disease long-QT syndrome [82]. This mutant has reduced interaction with the KCNQ1 channel and decreased cAMP-dependent phosphorylation of KCNQ1 [82]. These concomitant events likely extend action potential duration and elevate the risk of death following increased sympathetic nervous system stimulation that is common in long-QT syndrome.
Conclusions and future directions In summary, subcellular targeting of PKA by AKAPs is a very efficient mechanism for adapting the versatile cAMP signal transduction pathway for highly selective local regulatory phosphorylation events. The key factors in maintaining both the diversity and specificity of this system are the
1334
unique subcellular targeting domains present in different AKAP family members. Thus, it is these targeting interactions that might serve in the future as targets for the development of novel therapeutics. The attractiveness of this approach is already supported by studies described above and in the next chapter showing that peptide reagents and disease-linked mutations that disrupt the interactions between AKAPs and ion channel substrates can have the same functional effects as disrupting PKA anchoring to the AKAPs with the non-selective anchoring inhibitor peptides [82, 87]. Furthermore, recent studies of AKAP79/150 targeting to excitatory synapses suggest that regulation of AKAP targeting domains by cellular pathways may serve as important endogenous mechanisms controlling PKA signaling [50, 54]. Thus, further dissection of the mechanisms of AKAP targeting as well as the substrate binding and scaffolding interactions discussed in following chapter will continue to be active and important areas of AKAP research in the years to come.
References 1. Taylor SS, Buechler JA, Yonemoto W. cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Ann Rev Biochem 1990;59:971–1005. 2. Zhang J, Ma Y, Taylor SS, Tsien RY. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci USA 2001;98:14,997–15,002. 3. Colledge M, Scott JD. AKAPs: from structure to function. Trends Cell Biol 1999;9:216–21. 4. Feliciello A, Gottesman ME, Avvedimento EV. The biological functions of A-kinase anchor proteins. J Mol Biol 2001;308:99–114. 5. Michel JJ, Scott JD. AKAP mediated signal transduction. Annu Rev Pharmacol Toxicol 2002;42:235–57. 6. Burton KA, Johnson BD, Hausken ZE, Westenbroek RE, Idzerda RL, et al. Type II regulatory subunits are not required for the anchoringdependent modulation of Ca2 channel activity by cAMP-dependent protein kinase. Proc Natl Acad Sci USA 1997;94:11,067–11,072. 7. Angelo RG, Rubin CS. Characterization of structural features that mediate the tethering of Caenorhabditis elegans protein kinase A to a novel A kinase anchor protein. Insights into the anchoring of PKAI isoforms. J Biol Chem 2000;275:4351–62. 8. Herberg FW, Maleszka A, Eide T, Vossebein L, Tasken K. Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding. J Mol Biol 2000;298:329–39. 9. Huang LJ, Durick K, Weiner JA, Chun J, Taylor SS. Identification of a novel dual specificity protein kinase A anchoring protein, D-AKAP1. J Biol Chem 1997;272:8057–64. 10. Miki K, Eddy EM. Single amino acids determine specificity of binding of protein kinase A regulatory subunits by protein kinase A anchoring proteins. J Biol Chem 1999;274:29,057–29,062. 11. Dodge KL, Khouangsathiene S, Kapiloff MS, Mouton R, Hill EV, et al. mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J 2001;20:1921–30. 12. Tasken KA, Collas P, Kemmner WA, Witczak O, Conti M, Tasken K. Phosphodiesterase 4D and protein kinase a type II constitute a signaling unit in the centrosomal area. J Biol Chem 2001;276:21,999–22,002.
PART | II Transmission: Effectors and Cytosolic Events
13. Manni S, Mauban JH, Ward CW, Bond M. Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells. J Biol Chem 2008;283:24,145–24,154. 14. Hausken ZE, Coghlan VM, Hasting CAS, Reimann EM, Scott JD. Type II regulatory subunit (RII) of the cAMP dependent protein kinase interaction with A-kinase anchor proteins requires isoleucines 3 and 5. J Biol Chem 1994;269:24,245–24,251. 15. Hausken ZE, Dell’Acqua ML, Coghlan VM, Scott JD. Mutational analysis of the A-kinase anchoring protein (AKAP)-binding site on RII. J Biol Chem 1996;271:29,016–29,022. 16. Li Y, Rubin CS. Mutagenesis of the regulatory subunit (RIIb) of cAMP-dependent protein kinase IIb reveals hydrophobic amino acids that are essential for RIIb dimerization and/or anchoring RIIb to the cytoskeleton. J Biol Chem 1995;270:1935–44. 17. Banky P, Newlon MG, Roy M, Garrod S, Taylor SS, Jennings PA. Isoform-specific differences between the type Ialpha and IIalpha cyclic AMP-dependent protein kinase anchoring domains revealed by solution NMR. J Biol Chem 2000;275:35,146–35,152. 18. Newlon MG, Roy M, Morikis D, Carr DW, Westphal R, et al. A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes. EMBO J 2001;20:1651–62. 19. Newlon MG, Roy M, Morikis D, Hausken ZE, Coghlan V, et al. The molecular basis for protein kinase A anchoring revealed by solution NMR. Nat Struct Biol 1999;6:222–7. 20. Carr DW, Hausken ZE, Fraser ID, Stofko-Hahn RE, Scott JD. Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain. J Biol Chem 1992;267:13,376–13,382. 21. Carr DW, Stofko-Hahn RE, Fraser IDC, Bishop SM, Acott TS, et al. Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif. J Biol Chem 1991;266:14,188–14,192. 22. Glantz SB, Li Y, Rubin CS. Characterization of distinct tethering and intracellular targeting domains in AKAP75, a protein that links cAMP-dependent protein kinase II beta to the cytoskeleton. J Biol Chem 1993;268:12,796–12,804. 23. Diviani D, Langeberg LK, Doxsey SJ, Scott JD. Pericentrin anchors protein kinase A at the centrosome through a newly identified RIIbinding domain. Curr Biol 2000;10:417–20. 24. Gold MG, Lygren B, Dokurno P, Hoshi N, McConnachie G, et al. Molecular basis of AKAP specificity for PKA regulatory subunits. Mol Cell 2006;24:383–95. 25. Kinderman FS, Kim C, von Daake S, Ma Y, Pham BQ, et al. A dynamic mechanism for AKAP binding to RII isoforms of cAMPdependent protein kinase. Mol Cell 2006;24:397–408. 26. Burns-Hamuro LL, Ma Y, Kammerer S, Reineke U, Self C, et al. Designing isoform-specific peptide disruptors of protein kinase A localization. Proc Natl Acad Sci USA 2003;100:4072–7. 27. Carlson CR, Lygren B, Berge T, Hoshi N, Wong W, et al. Delineation of type I protein kinase A-selective signaling events using an RI anchoring disruptor. J Biol Chem 2006;281:21,535–21,545. 28. Lester LB, Coghlan VM, Nauert B, Scott JD. Cloning and characterization of a novel A-kinase anchoring protein: AKAP220, association with testicular peroxisomes. J Biol Chem 1996;272:9460–5. 29. Schillace RV, Scott JD. Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220. Curr Biol 1999;9:321–4. 30. Schmidt PH, Dransfield DT, Claudio JO, Hawley RG, Trotter KW, et al. AKAP350: a multiply spliced A-kinase anchoring protein associated with centrosomes. J Biol Chem 1999;274:3055–66.
Chapter | 165 Subcellular Targeting of PKA through AKAPs: Conserved Anchoring and Unique Targeting Domains
31. Witczak O, Skalhegg BS, Keryer G, Bornens M, Tasken K, et al. Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome, AKAP450. EMBO J 1999;18:1858–68. 32. Lewis SA, Wang D, Cowan NJ. Microtubule-associated protein MAP2 shares a microtubule binding motif with tau protein. Science 1988;242:936–9. 33. Luo Z, Shafit-Zagardo B, Erlichman J. Identification of the MAP2- and P75- binding domain in the regulatory subunit (RIIb) of Type II cAMP-dependent protein kinase. J Biol Chem 1990;265:21,804–21,810. 34. Rubino HM, Dammerman M, Shafit-Sagardo B, Erlichman J. Localization and characterization of the binding site for the regulatory subunit of type II cAMP-dependent protein kinase on MAP2.. Neuron 1989;3:631–8. 35. Westphal RS, Soderling SH, Alto NM, Langeberg LK, Scott JD. Scar/ WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actinassociated multi-kinase scaffold. EMBO J 2000;19:4589–600. 36. Kapiloff MS, Jackson N, Airhart N. mAKAP and the ryanodine receptor are part of a multi-component signaling complex on the cardiomyocyte nuclear envelope. J Cell Sci 2001;114:3167–76. 37. Kapiloff MS, Schillace RV, Westphal AM, Scott JD. mAKAP: an Akinase anchoring protein targeted to the nuclear membrane of differentiated myocytes. J Cell Sci 1999;112:2725–36. 38. Chen Q, Reigh-Yi L, Rubin C. Organelle-specific targeting of protein kinase AII (PKA). J Biol Chem 1997;272:15,247–15,257. 39. Huang LJ, Wang L, Ma Y, Durick K, Perkins G, et al. NH2-Terminal targeting motifs direct dual specificity A-kinase- anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum. J Cell Biol 1999;145:951–9. 40. Lin R-Y, Moss SB, Rubin CS. Characterization of S-AKAP84, a novel developmentally regulated A kinase anchor protein of male germ cells. J Biol Chem 1995;270(27):804–11. 41. Coghlan VM, Langeberg LK, Fernandez A, Lamb NJC, Scott JD. Cloning and characterization of AKAP95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase. J Biol Chem 1994;269:7658–65. 42. Eide T, Coghlan V, Orstavik S, Holsve C, Solberg R, et al. Molecular cloning, chromosomal localization and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95. Exp Cell Res 1997;238:305–16. 43. Collas P, Le Guellec K, Tasken K. The A-kinase-anchoring protein AKAP95 is a multivalent protein with a key role in chromatin condensation at mitosis. J Cell Biol 1999;147:1167–80. 44. Landsverk HB, Carlson CR, Steen RL, Vossebein L, Herberg FW, et al. Regulation of anchoring of the RIIalpha regulatory subunit of PKA to AKAP95 by threonine phosphorylation of RIIalpha: implications for chromosome dynamics at mitosis. J Cell Sci 2001;114:3255–64. 45. Brown RL, August SL, Williams CJ, Moss SB. AKAP7gamma is a nuclear RI-binding AKAP. Biochem Biophys Res Commun 2003;306:394–401. 46. Lygren B, Carlson CR, Santamaria K, Lissandron V, McSorley T, et al. AKAP complex regulates Ca2 re-uptake into heart sarcoplasmic reticulum. EMBO Rep 2007;8:1061–7. 47. Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, et al. Identification of a novel A-kinase anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin-2 shuttle in renal principal cells. J Biol Chem 2004;279:26,654–26,665. 48. Gold MG, Smith FD, Scott JD, Barford D. AKAP18 contains a phosphoesterase domain that binds AMP. J Mol Biol 2008;375: 1329–43.
1335
49. Dell’Acqua ML, Faux MC, Thorburn J, Thorburn A, Scott JD. Membrane-targeting sequences on AKAP79 bind phosphatidylinositol4, 5- bisphosphate. EMBO J 1998;17:2246–60. 50. Gomez LL, Alam S, Smith KE, Horne E, Dell’Acqua ML. Regulation of A-kinase anchoring protein 79/150-cAMP-dependent protein kinase postsynaptic targeting by NMDA receptor activation of calcineurin and remodeling of dendritic actin. J Neurosci 2002;22:7027–44. 51. Li Y, Ndubuka C, Rubin CS. A kinase anchor protein 75 targets regulatory (RII) subunits of cAMP-dependent protein kinase II to the cortical actin cytoskeleton in non-neuronal cells. J Biol Chem 1996;271:16,862–16,869. 52. Gorski JA, Gomez LL, Scott JD, Dell’Acqua ML. Association of an A-kinase-anchoring protein signaling scaffold with cadherin adhesion molecules in neurons and epithelial cells. Mol Biol Cell 2005;16: 3574–90. 53. Horne EA, Dell’Acqua ML. Phospholipase C is required for changes in postsynaptic structure and function associated with NMDA receptordependent long-term depression. J Neurosci 2007;27:3523–34. 54. Smith KE, Gibson ES, Dell’Acqua ML. cAMP-dependent protein kinase postsynaptic localization regulated by NMDA receptor activation through translocation of an A-kinase anchoring protein scaffold protein. J Neurosci 2006;26:2391–402. 55. Dell’acqua ML, Smith KE, Gorski JA, Horne EA, Gibson ES, Gomez LL. Regulation of neuronal PKA signaling through AKAP targeting dynamics. Eur J Cell Biol 2006;85:627–33. 56. Fraser ID, Tavalin SJ, Lester LB, Langeberg LK, Westphal AM, et al. A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMPresponsive membrane events. EMBO J 1998;17:2261–72. 57. Trotter KW, Fraser ID, Scott GK, Stutts MJ, Scott JD, Milgram SL. Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms. J Cell Biol 1999;147:1481–92. 58. Dong F, Felsmesser M, Casadevall A, Rubin CS. Molecular characterization of a cDNA that encodes six isoforms of a novel murine A kinase anchor protein. J Biol Chem 1998;273:6533–41. 59. Dransfield DT, Bradford AJ, Smith J, Martin M, Roy C, et al. Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO J 1997;16:101–9. 60. Feliciello A, Li Y, Avvedimento EV, Gottesman ME, Rubin CS. Akinase anchor protein 75 increases the rate and magnitude of cAMP signaling to the nucleus. Curr Biol 1997;7:1011–14. 61. Paolillo M, Feliciello A, Porcellini A, Garbi C, Bifulco M, et al. The type and the localization of cAMP-dependent protein kinase regulate transmission of cAMP signals to the nucleus in cortical and cerebellar granule cells. J Biol Chem 1999;274:6546–52. 62. Lester LB, Faux MC, Nauert JB, Scott JD. Targeted protein kinase A and PP-2B regulate insulin secretion through reversible phosphorylation.. Endocrinology 2001;142:1218–27. 63. Lester LB, Scott JD. Anchoring and scaffold proteins for kinases and phosphatases. Recent Progr Hormone Res 1997;52:409–30. 64. Carlson CR, Witczak O, Vossebein L, Labbe JC, Skalhegg BS, et al. CDK1-mediated phosphorylation of the RIIalpha regulatory subunit of PKA works as a molecular switch that promotes dissociation of RIIalpha from centrosomes at mitosis. J Cell Sci 2001;114:3243–54. 65. Keryer G, Yassenko M, Labbe JC, Castro A, Lohmann SM, et al. Mitosis-specific phosphorylation and subcellular redistribution of the RIIalpha regulatory subunit of cAMP-dependent protein kinase. J Biol Chem 1998;273:34,594–34,602. 66. Hall DD, Davare MA, Shi M, Allen ML, Weisenhaus M, et al. Critical role of cAMP-dependent protein kinase anchoring to the L-type calcium channel Cav1.2 via A-kinase anchor protein 150 in neurons. Biochemistry 2007;46:1635–46.
1336
67. Hoshi N, Langeberg LK, Scott JD. Distinct enzyme combinations in AKAP signalling complexes permit functional diversity. Nat Cell Biol 2005;7:1066–73. 68. Lu Y, Allen M, Halt AR, Weisenhaus M, Dallapiazza RF, et al. Agedependent requirement of AKAP150-anchored PKA and GluR2-lacking AMPA receptors in LTP. EMBO J 2007;26:4879–90. 69. Lu Y, Zhang M, Lim IA, Hall DD, Allen M, et al. AKAP150-anchored PKA activity is important for LTD during its induction phase. J Physiol 2008;586:4155–64. 70. Nie T, McDonough CB, Huang T, Nguyen PV, Abel T. Genetic disruption of protein kinase A anchoring reveals a role for compartmentalized kinase signaling in theta-burst long-term potentiation and spatial memory. J Neurosci 2007;27:10,278–10,288. 71. Snyder EM, Colledge M, Crozier RA, Chen WS, Scott JD, Bear MF. Role for A kinase-anchoring proteins (AKAPS) in glutamate receptor trafficking and long term synaptic depression. J Biol Chem 2005;280:16,962–16,968. 72. Tunquist BJ, Hoshi N, Guire ES, Zhang F, Mullendorff K, et al. Loss of AKAP150 perturbs distinct neuronal processes in mice. Proc Natl Acad Sci USA 2008;105:12,557–12,562. 73. Fraser ID, Scott JD. Modulation of ion channels: a “current” view of AKAPs. Neuron 1999;23:423–6. 74. Gray PC, Tibbs VC, Catterall WA, Murphy BJ. Identification of a 15-kDa cAMP-dependent protein kinase-anchoring protein associated with skeletal muscle L-type calcium channels. J Biol Chem 1997;272:6297–302. 75. Gao T, Yatani A, Dell’Acqua ML, Sako H, Green SA, et al. cAMPdependent regulation of cardiac L-type Ca2 channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 1997;19:185–96. 76. Johnson BD, Scheuer T, Catterall WA. Voltage-dependent potentiation of L-type Ca2 channels in skeletal muscle cells requires anchored cAMP-dependent protein kinase. Proc Natl Acad Sci USA 1994;91:11,492–11,496. 77. Oliveria SF, Dell’Acqua ML, Sather WA. AKAP79/150 anchoring of calcineurin controls neuronal L-type Ca2 channel activity and nuclear signaling.. Neuron 2007;55:261–75. 78. Rosenmund C, Carr DW, Bergeson SE, Nilaver G, Scott JD, Westbrook GL. Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons. Nature 1994;368:853–6. 79. Gray III PC, Johnson BD, Westenbroek RE, Hays LG, Yates JR, et al. Primary structure and function of an A kinase anchoring protein associated with calcium channels. Neuron 1998;20:1017–26.
PART | II Transmission: Effectors and Cytosolic Events
80. Tavalin SJ, Colledge M, Hell JW, Langeberg LK, Huganir RL, Scott JD. Regulation of GluR1 by the A-kinase anchoring protein 79 (AKAP79) signaling complex shares properties with long-term depression. J Neurosci 2002;22:3044–51. 81. Kameyama K, Lee HK, Bear MF, Huganir RL. Involvement of a postsynaptic protein kinase A substrate in the expression of homosynaptic long-term depression. Neuron 1998;21:1163–75. 82. Chen L, Marquardt ML, Tester DJ, Sampson KJ, Ackerman MJ, Kass RS. Mutation of an A-kinase-anchoring protein causes long-QT syndrome. Proc Natl Acad Sci USA 2007;104:20,990–20,995. 83. Marx SO, Kurokawa J, Reiken S, Motoike H, D’Armiento J, et al. Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel. Science 2002;295:496–9. 84. Chen L, Kass RS. Dual roles of the A kinase-anchoring protein Yotiao in the modulation of a cardiac potassium channel: a passive adaptor versus an active regulator. Eur J Cell Biol 2006;85:623–6. 85. Chen L, Kurokawa J, Kass RS. Phosphorylation of the A-kinaseanchoring protein Yotiao contributes to protein kinase A regulation of a heart potassium channel. J Biol Chem 2005;280:31,347–31,352. 86. Kurokawa J, Motoike HK, Rao J, Kass RS. Regulatory actions of the A-kinase anchoring protein Yotiao on a heart potassium channel downstream of PKA phosphorylation. Proc Natl Acad Sci USA 2004;101:16,374–16,378. 87. Hulme JT, Ahn M, Hauschka SD, Scheuer T, Catterall WA. A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2 channel and modulates its function. J Biol Chem 2002;277:4079–87. 88. Diviani D, Soderling J, Scott JD. AKAP-Lbc anchors protein kinase A and nucleates Galpha 12-selective Rho- mediated stress fiber formation. J Biol Chem 2001;276:44,247–44,257. 89. Schillace RV, Andrews SF, Liberty GA, Davey MP, Carr DW. Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes. J Immunol 2002;168:1590–9. 90. Goehring AS, Pedroja BS, Hinke SA, Langeberg LK, Scott JD. MyRIP anchors protein kinase A to the exocyst complex. J Biol Chem 2007;282:33,155–33,167. 91. Nauert JB, Klauck TM, Langeberg LK, Scott JD. Gravin, an autoantigen recognized by serum from myasthenia gravis patients, is a kinase scaffold protein. Curr Biol 1997;7:52–62. 92. Alto NM, Soderling J, Scott JD. Rab32 is an A-kinase anchoring protein and participates in mitochondrial dynamics. J Cell Biol 2002;158:659–68.
Subcellular Targeting of PKA through AKAPs: Conserved Anchoring and Unique Targeting Domains Matthew D. Pink and Mark L. Dell’Acqua Department of Pharmacology, University of Colorado Denver School of Medicine, Aurora, Colorado
Introduction Regulation of the opposing actions of adenylyl cyclases (AC) and phosphodiesterases (PDE) controlling levels of the diffusible intracellular second messenger cAMP is central to many signaling responses. A major effector of cAMP in eukaryotic cells is the cAMP-dependent protein kinase (PKA). PKA is a heterotetrameric enzyme consisting of two regulatory subunits (R) that dimerize and each bind and inhibit a single catalytic subunit (C) to form an R2C2 holoenzyme (Figure 165.1a) [1]. Activation of the inactive PKA holoenzyme by cAMP occurs when two molecules of cAMP binding to each R subunit resulting in a conformational change that releases the active C-subunits from the inhibitory R2 dimer (Figure 165.1a). The released
(a)
active C-subunits phosphorylate serine and threonine residues in the optimal sequence contexts of RRXS/T or KRXXS/T to regulate target protein function. PKA-C subunits can phosphorylate target proteins rapidly near the site of release, and in time diffuse to more distant locations such as the nucleus, where additional targets are phosphorylated [2]. Remarkably, this ubiquitous signaling pathway is used in different cell types to efficiently transduce signals to a myriad of different yet very specific target proteins in a wide variety of cellular compartments. Thus, elucidating how this PKA signaling versatility is achieved without compromising specificity and efficiency is fundamental to understanding cAMP signal transduction pathways. Over the past two decades we have learned that both diversity and specificity in cAMP signaling is in large part
(b)
Figure 165.1 Anchoring and subcellular targeting of PKA by AKAPs. (a) Structure of the PKA holoenzyme and regulation of catalytic activity by cAMP. (b) AKAP anchoring of the PKA holoenzyme near specific cellular substrates. Handbook of Cell Signaling, Three-Volume Set 2 ed. Copyright © 2010 Elsevier Inc. All rights reserved.
1329
1330
achieved by targeting the PKA holoenzyme to discrete subcellular locations such that, upon release of the C subunit, phosphorylation of co-localized target substrates is greatly enhanced (Figure 165.1b) [2]. This subcellular targeting of PKA is mediated by a class of anchoring/scaffolding proteins called AKAPs (A-Kinase Anchoring Proteins) [3–5]. AKAPs anchor PKA by binding the R-subunit dimer through a structurally conserved anchoring domain, and then target the holoenzyme to specific locations within the cell through unique targeting domains (Figure 165.1b). AKAPs frequently contain additional protein–protein interaction motifs that serve as tethering sites for the target substrates, as well as additional signaling proteins such as other kinases, phosphatases, and scaffold and adaptor proteins. Thus, many AKAPs functions as signal-integrating scaffolds that coordinate both subcellular localization and complex assembly of multi-protein signal transduction machines. This review chapter will focus on the structurally conserved R subunit anchoring domain and examples of unique subcellular targeting domains that localize anchored-PKA for regulation of co-localized target substrates. The following chapter will deal more with the issue of AKAP multi-enzyme complexes integrating different signaling pathways.
Structurally conserved PKA anchoring determinants There are three PKA C subunit isoforms (C, C, C) and four R subunit isoforms (RI, RI, RII, RII) [1]. While the C subunit isoforms share very similar properties, the RI, and RII, subunit groups show significant differences in both cAMP binding affinity [1] and AKAP anchoring [6]. Thus, due to these differences in RI and RII, PKA is divided into type I and type II holoenzymes based on the identity of the R subunit. While it is clear that both type I and type II PKA holoenzymes can bind to AKAPs and thus be targeted to specific subcellular domains, the majority of AKAP proteins bind RII with 100- to 1000fold higher affinity than RI [6–10]. This difference in AKAP binding affinity is reflected in observations that in many, but not all, cell types PKA-RII is more discretely localized to specific cellular structures, while PKA-RI is diffusely distributed in the cytoplasm. The differences in cAMP binding affinity between RI and RII fit well with these differing distributions. Type I holoenzymes have higher cAMP binding affinities, and thus may be adapted to generate more prolonged responses in response to lower cAMP signals encountered in the bulk cytoplasm. In contrast, type II holoenzymes bind cAMP with lower affinity, a possible adaptation to being targeted by AKAPs to local environments where cAMP can be much higher due to compartmentalized regulation of either AC or PDE [11, 12]. One important function of reduced cAMP sensitivity might
PART | II Transmission: Effectors and Cytosolic Events
be to maintain low activity of type II holoenzyme anchored in local environments where even basal cAMP levels would fully activate the type I enzyme, leading to complete loss of C subunits. RI and RII also differ in that the RII, but not RI, autoinhibitory C-subunit binding domain is a PKA substrate; a recent mutagenesis study demonstrates that phosphorylation of RII increases binding to AKAPs, thus suggesting additional levels of complexity and positive feedback in AKAP-anchored PKA signaling [13]. A number of previous mutagenesis studies have shown that AKAPs bind hydrophobic residues in the N-terminus of the R subunits in a manner that depends on formation of the R–R dimer [14–16]. Indeed, NMR structural studies show that both the RI and RII N-terminal dimerization domains form anti-parallel four-helix bundles in which dimerization creates an extended hydrophobic surface that binds the AKAP (Figure 165.2c) [17–19]. This hydrophobic surface is somewhat less extensive and more sterically hinderd in RI compared to RII dimmers, possibly explaining why in general most AKAPs bind RII with higher affinity than RI. Accordingly, the AKAPs all bind to the R subunit N-terminal dimerization domain through hydrophobic interactions [18, 20–22]. The PKA anchoring domains from different AKAPs have very little primary amino acid sequence similarity, yet share a conserved hydrophobic character and secondary structure (Figure 165.2a, b) [18, 20, 21]. Thus, AKAPs are a family of functionallyrelated proteins arising from convergent evolution, as opposed to diverging from a common ancestral AKAP protein. The common hydrophobic and secondary structure in AKAP PKA anchoring domains is seen as a conserved spacing of hydrophobic residues which map to one side of an amphipathic -helix of about 18 residues in length (Figure 165.2a, b) [18, 21]. One exception to this conservation of an amphipathic-helix anchoring domain is seen in pericentrin, which anchors PKA-R subunits through a unique, more extended hydrophobic motif of unknown structure [23]. For the conserved anchoring motif found in all other AKAPs, mutagenesis studies have lent support to the amphipathic-helix structural model by showing that substitution of hydrophobic residues with either hydrophilic residues to change polarity or proline residues to break helical structure cause inhibition of R subunit binding [21, 22]. NMR structures have also been obtained showing clearly that two AKAP anchoring domain peptides, Ht31 (493–515) and AKAP79(392–413), with divergent primary sequences both bind to RII in similar helical conformations with extensive contacts made between the hydrophobic face of the helices and the large hydrophobic surface on the RII dimer (Figure 165.2c) [18]. More recent X-ray crystal structures using two additional AKAP anchoring domain peptides, D-AKAP2 and AKAP-IS, confirm the NMR structures by showing that the non-palindromic AKAP helix sits diagonally across the face of the symmetrical RII dimer, creating an asymmetrical RII–AKAP complex [24, 25].
Chapter | 165 Subcellular Targeting of PKA through AKAPs: Conserved Anchoring and Unique Targeting Domains
1331
(a)
(b)
(c)
Figure 165.2 Structurally conserved AKAP–PKA anchoring domains. (a) Divergent primary structures for PKA-anchoring in AKAP family members. Hydrophobic residues are shaded. (b) Conserved amphipathic alpha-helical secondary structures for AKAP PKA anchoring domains. Hydrophobic residues are shaded on helical wheel diagrams. (c) Conserved mechanisms of PKA anchoring revealed by NMR solution structures of AKAP–RII complexes. The structures show AKAP anchoring domain peptides from Ht31(493–515) and AKAP79(392–413) (top) bound to an RII(1–45) N-terminal domain dimer (bottom). Figure adapted from [18] (Newlon et al., 2002, EMBO J. 20: 1651–1662), with permission from P. A. Jennings and Oxford University Press.
At the interface, a series of non-polar aliphatic residues on the AKAP create a hydrophobic ridge, which interacts with a preformed cluster of hydrophobic residues from each RII protomer [24, 25]. Amino acid residues critical for RII binding to D-AKAP2 and AKAP-IS, while not completely conserved, are found in the same structural space. Of particular importance is the non-polar aliphatic amino acid Valine, found in the center of the hydrophobic ridge [24, 25]. Valine at this position is conserved amongst all RIIspecific AKAPs, and most AKAPs able to bind both RI and RII isoforms. Further, Asparagine and Glutamine at two other positions, respectively, are conserved in RII-specific AKAPs [24, 25], while acidic residues at one of these positions predominate in RI-specific and dual-specificity AKAPs. This knowledge was used to create an AKAP peptide, SuperAKAP-IS, with abolished binding for RI and increased specificity for RII. Creation of SuperAKAPIS, along with recently developed RI-specific anchoring inhibitors [26, 27], should further allow for the function of RII versus RI anchoring to be examined in various cellular functions in different cell types.
Unique subcellular targeting domains The variety and specificity that is made possible by AKAP– PKA anchoring is in large part a function of unique targeting domains in different AKAP molecules (Figure 165.1b).
AKAP molecules have been characterized at a myriad of distinct subcellular locations (Figure 165.3a, b) including the plasma membrane, intracellular vesicles [28, 29], actin and microtubule cytoskeletons, mitochondria, endoplasmic reticulum (ER), Golgi, and centrosomes [30, 31]. For example, the AKAP protein MAP2 is targeted to dendritic microtubules in neurons by direct binding to tubulin [32–34]. Scar/Wave1, an AKAP that also anchors the abl-Tyrosine kinase, binds to actin both in focal adhesions (Figure 165.3b) and membrane ruffles in fibroblasts where it regulates the actin polymerization activity of the Arp2/3 complex (see Chapter 166) [35]. The skeletal and cardiac muscle-enriched mAKAP protein, which also anchors PDE4D3 (see Chapter 166) [11], is targeted by a series of spectrin repeats to perinuclear ER/SR membranes including, most prominently, the nuclear membrane (Figure 165.3b) [36, 37]. dAKAP1/sAKAP84/AKAP121 can be targeted either to the ER (in the liver) or outer membrane of mitochondria (in most other cells) by two different N-terminal targeting sequences produced by alternate mRNA splicing [38–40]. AKAP95, has even been shown to associate with the nuclear matrix and chromatin, despite the fact that PKA holoenzyme/R-subunits are excluded from the nucleus during interphase (Figure 165.3a) [41, 42]. However, interactions of AKAP95 with PKA and chromatin could have important functions in regulating chromosome condensation during mitosis when the nuclear envelope is absent [43, 44]. The AKAP18 splice variant is also found in the nucleus as well as the cytoplasm, and has a conserved
1332
PART | II Transmission: Effectors and Cytosolic Events
(a)
(b)
Figure 165.3 Unique AKAP targeting domains and subcellular localizations. (a) Subcellular compartmentalization of different AKAP family members. References for AKAPLbc/Ht31 [88], MTG [89], My-RIP [90], Gravin [91], and Rab 32 [92] localization. For other references, see the text. (b) Specific subcellular localizations observed for selected AKAPs by fluorescence microscopy.
nuclear localization sequence near its N-terminus that targets it to the nuclei of kidney cells and oocytes [45]. In contrast, the related AKAP18 splice variant is found at the sarcoplasmic reticulum in cardiac myocytes and on secretory vesicles in renal collecting duct epithelial cells [46, 47]. Curiously, the central domain of AKAP18 is able to bind directly to the small molecule adenosine 5 monophosphate (5AMP), a product of cAMP and ATP hydrolysis [48]. The exact functional role of this interaction is not yet known, however, it is hypothesized that AKAP18 could somehow link cellular AMP levels to regulation of PKA phosphorylation events [48]. In certain cell types specific localization to discrete plasma membrane domains is seen, such as localization to apical or basolateral membrane domains in polarized epithelial cells and synaptic membrane specializations in neurons. AKAP75/79/150, an AKAP scaffold protein that also binds protein kinase C and calcineurin-protein phosphatase 2B(CaN-PP2B) (see Chapter 166) is targeted to the plasma membrane/cortical cytoskeleton (Figure 165.3a) by three N-terminal polybasic domains that bind to the acidic phospholipid phosphatidylinositol-4,5-bisphosphate, F-actin, and cadherin adhesion molecules [49–52]. This same N-terminal basic domain mediates specific targeting to excitatory postsynaptic membrane specializations located on actin-rich dendritic spines in neurons and cadherin-mediated adherens junctions on basolateral membranes in epithelial cells (Figure 165.3b) [50, 52]. Interestingly, membrane localization of AKAP79/150-PKA complexes is not static, and is subject to positive regulation by cadherin engagement in epithelial cells and negative regulation by NMDA-type glutamate receptor Ca2 signaling (Figure 165.4) through activation of CaN-PP2B-mediated actin reorganization and
Figure 165.4 Dynamic regulation of AKAP targeting. Punctate membrane localization of AKAP79/150 to dendritic spines in hippocampal neurons is negatively regulated by activation of NMDA receptors (agonist NMDA; antagonist APV). AKAP79/150 assumes a diffuse cytoplasmic localization in both dendrites and the cell body in NMDA-treated neurons.
phospholipase C-mediated cleavage of phosphatidylinositol4,5-bisphosphate in neurons [50, 52–54]. Thus, dynamic regulation of AKAP targeting is likely to be another important mechanism for controlling the spatio-temporal specificity of cAMP signaling in different cell types [55]. The low molecular weight AKAP15/18 splice variant is also plasma-membrane localized; however, its membrane targeting is determined by N-terminal lipid modifications of myristoylation of Gly-1 and dual palmitoylation of Cys-4, Cys-5 (Figure 165.3b) [56]. In MDCK epithelial cells this AKAP15/18 isoform selectively targets to the basolateral membrane. However, another alternate splice variant, AKAP18, that contains an additional exon coding for a 24 amino acid insert localizes to the apical membrane [57]. Interestingly, two additional AKAP families, the AKAPKL and ERM proteins (Ezrin/Radixin/Moesin), are also specifically targeted to apical membranes, where they bind to cortical actin (Figure 165.3a) [58, 59].
Chapter | 165 Subcellular Targeting of PKA through AKAPs: Conserved Anchoring and Unique Targeting Domains
Probing cellular functions of AKAP – PKA anchoring The importance of AKAP-targeted pools of PKA has been implicated in numerous cellular responses, including transcription [60, 61], secretion [56, 62, 63], cell cycle regulation [44, 64, 65], and neuronal synaptic plasticity [66–72]. Functionally, the role of AKAP–PKA targeting has been studied in the greatest detail for cAMP regulation of membrane ion channels that control muscle cell contraction and neuronal synaptic plasticity [66–74]. These studies of ion channel regulation have traditionally either used anchoring inhibitor peptides, such as the peptide Ht31, to displace PKA from endogenous AKAPs, or heterologous co-expression of an AKAP with the ion channel of interest. More recent studies have also employed expression of mutated AKAPs in primary cell culture in conjunction with RNAi-mediated suppression of endogenous AKAP expression or use of AKAP knockout, mutant knock-in, or transgenic mice. Use of anchoring inhibitor peptides to disrupt PKA-R subunit anchoring was first shown to block cAMP regulation of endogenous plasma membrane channel activity for neuronal AMPA-type glutamate receptors and cardiac and skeletal muscle L-type voltage-gated calcium channels similar to inhibition of PKA-C subunit catalytic activity [74–78]. In the case of AMPA receptors, the effects of anchoring disruption on channel currents is at least in part due to changes in channel trafficking and membrane stability [71]. Together, these studies suggested that endogenous AKAPs were important for targeting PKA to the plasma membrane in proximity to the regulated channels. Subsequent studies in a heterologous HEK-293 cell expression system confirmed the previous results by showing that co-expression of these channels with an appropriate membrane targeted AKAP partner, such as AKAP15/18 or AKAP79, could reconstitute cAMP-PKA regulation of channel activity [75, 79, 80]. Recent development of transgenic mice expressing an inducible PKA-anchoring inhibitor revealed impairments in learning and memory and PKA regulation of neuronal synaptic plasticity that is likely to involve more than one AKAP in both the pre- and postsynaptic membrane compartments [70]. Use of RNAi technology has allowed for the functions of postsynaptic AKAP79/150 in particular to be studied more directly in neurons. RNAi experiments where endogenous rat AKAP150 was knocked down and rescued with human AKAP79 unable to anchor PKA or CaN-PP2B have confirmed roles for this AKAP in neuronal AMPA receptor as well as L-type calcium channel regulation [67, 77]. Complementary studies in knockout and knock-in mice where endogenous AKAP150 was genetically deleted or mutated so that it can no longer anchor PKA has allowed for further exploration of these cellular functions in neurons. Recent mouse studies all indicate that
1333
AKAP79/150-anchored PKA is integral in regulation of AMPA receptors during certain forms of synaptic plasticity [68, 69, 72]. However, there are some differing observations regarding whether or not basal AMPA receptor phosphorylation is decreased in these different transgenic mice, and how that relates to the negative regulation of AMPA receptor activity by acute inhibition of PKA activity or disruption of PKA anchoring observed in prior studies [67, 71, 78, 80, 81]. In the case of L-type calcium channels, it is apparent from both RNAi and transgenic mice that AKAP150-anchored PKA is clearly necessary to phosphorylate Cav1.2 and regulate neuronal channel activity [66, 77]. Collectively, it has been revealed that AKAP79/150 is integral in maintaining elevated postsynaptic PKA levels, where it aids in the regulation of membrane ion channels. As an additional level of targeting specificity even beyond AKAP co-localization with ion channels in the membrane, modified leucine zipper coiled-coiled motifs that promote direct association with channel substrates have been identified on several AKAPs. While this topic is discussed more in Chapter 166, we will briefly mention two examples here. AKAP79/150 contains a modified leucine zipper coiled-coiled motif located on its C-terminus [77]. This leucine zipper allows AKAP79/150 to directly interact with neuronal L-type CaV1.2 calcium channels to promote AKAP79/150-anchored PKA and CaN-PP2B bidirectional regulation Cav1.2 activity, as discussed above, as well as signaling through CaN-PP2B to control downstream transcription factor activation [77]. A C-terminal leucine zipper motif in the AKAP Yotiao, along with its N-terminal domain, mediates its association with the cardiac potassium channel KCNQ1 [82, 83]. In response to increased heart rate stimulated by the sympathetic nervous system, this direct interaction, along with Yotiao-anchored PKA phosphorylation of KCNQ1, decreases action potential duration, thereby maintaining sufficient diastolic filling between heartbeats [82–86]. Importantly, recent research has identified a mutation in the C-terminal domain of Yotiao present in the inheritable human disease long-QT syndrome [82]. This mutant has reduced interaction with the KCNQ1 channel and decreased cAMP-dependent phosphorylation of KCNQ1 [82]. These concomitant events likely extend action potential duration and elevate the risk of death following increased sympathetic nervous system stimulation that is common in long-QT syndrome.
Conclusions and future directions In summary, subcellular targeting of PKA by AKAPs is a very efficient mechanism for adapting the versatile cAMP signal transduction pathway for highly selective local regulatory phosphorylation events. The key factors in maintaining both the diversity and specificity of this system are the
1334
unique subcellular targeting domains present in different AKAP family members. Thus, it is these targeting interactions that might serve in the future as targets for the development of novel therapeutics. The attractiveness of this approach is already supported by studies described above and in the next chapter showing that peptide reagents and disease-linked mutations that disrupt the interactions between AKAPs and ion channel substrates can have the same functional effects as disrupting PKA anchoring to the AKAPs with the non-selective anchoring inhibitor peptides [82, 87]. Furthermore, recent studies of AKAP79/150 targeting to excitatory synapses suggest that regulation of AKAP targeting domains by cellular pathways may serve as important endogenous mechanisms controlling PKA signaling [50, 54]. Thus, further dissection of the mechanisms of AKAP targeting as well as the substrate binding and scaffolding interactions discussed in following chapter will continue to be active and important areas of AKAP research in the years to come.
References 1. Taylor SS, Buechler JA, Yonemoto W. cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Ann Rev Biochem 1990;59:971–1005. 2. Zhang J, Ma Y, Taylor SS, Tsien RY. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci USA 2001;98:14,997–15,002. 3. Colledge M, Scott JD. AKAPs: from structure to function. Trends Cell Biol 1999;9:216–21. 4. Feliciello A, Gottesman ME, Avvedimento EV. The biological functions of A-kinase anchor proteins. J Mol Biol 2001;308:99–114. 5. Michel JJ, Scott JD. AKAP mediated signal transduction. Annu Rev Pharmacol Toxicol 2002;42:235–57. 6. Burton KA, Johnson BD, Hausken ZE, Westenbroek RE, Idzerda RL, et al. Type II regulatory subunits are not required for the anchoringdependent modulation of Ca2 channel activity by cAMP-dependent protein kinase. Proc Natl Acad Sci USA 1997;94:11,067–11,072. 7. Angelo RG, Rubin CS. Characterization of structural features that mediate the tethering of Caenorhabditis elegans protein kinase A to a novel A kinase anchor protein. Insights into the anchoring of PKAI isoforms. J Biol Chem 2000;275:4351–62. 8. Herberg FW, Maleszka A, Eide T, Vossebein L, Tasken K. Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding. J Mol Biol 2000;298:329–39. 9. Huang LJ, Durick K, Weiner JA, Chun J, Taylor SS. Identification of a novel dual specificity protein kinase A anchoring protein, D-AKAP1. J Biol Chem 1997;272:8057–64. 10. Miki K, Eddy EM. Single amino acids determine specificity of binding of protein kinase A regulatory subunits by protein kinase A anchoring proteins. J Biol Chem 1999;274:29,057–29,062. 11. Dodge KL, Khouangsathiene S, Kapiloff MS, Mouton R, Hill EV, et al. mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J 2001;20:1921–30. 12. Tasken KA, Collas P, Kemmner WA, Witczak O, Conti M, Tasken K. Phosphodiesterase 4D and protein kinase a type II constitute a signaling unit in the centrosomal area. J Biol Chem 2001;276:21,999–22,002.
PART | II Transmission: Effectors and Cytosolic Events
13. Manni S, Mauban JH, Ward CW, Bond M. Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells. J Biol Chem 2008;283:24,145–24,154. 14. Hausken ZE, Coghlan VM, Hasting CAS, Reimann EM, Scott JD. Type II regulatory subunit (RII) of the cAMP dependent protein kinase interaction with A-kinase anchor proteins requires isoleucines 3 and 5. J Biol Chem 1994;269:24,245–24,251. 15. Hausken ZE, Dell’Acqua ML, Coghlan VM, Scott JD. Mutational analysis of the A-kinase anchoring protein (AKAP)-binding site on RII. J Biol Chem 1996;271:29,016–29,022. 16. Li Y, Rubin CS. Mutagenesis of the regulatory subunit (RIIb) of cAMP-dependent protein kinase IIb reveals hydrophobic amino acids that are essential for RIIb dimerization and/or anchoring RIIb to the cytoskeleton. J Biol Chem 1995;270:1935–44. 17. Banky P, Newlon MG, Roy M, Garrod S, Taylor SS, Jennings PA. Isoform-specific differences between the type Ialpha and IIalpha cyclic AMP-dependent protein kinase anchoring domains revealed by solution NMR. J Biol Chem 2000;275:35,146–35,152. 18. Newlon MG, Roy M, Morikis D, Carr DW, Westphal R, et al. A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes. EMBO J 2001;20:1651–62. 19. Newlon MG, Roy M, Morikis D, Hausken ZE, Coghlan V, et al. The molecular basis for protein kinase A anchoring revealed by solution NMR. Nat Struct Biol 1999;6:222–7. 20. Carr DW, Hausken ZE, Fraser ID, Stofko-Hahn RE, Scott JD. Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain. J Biol Chem 1992;267:13,376–13,382. 21. Carr DW, Stofko-Hahn RE, Fraser IDC, Bishop SM, Acott TS, et al. Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif. J Biol Chem 1991;266:14,188–14,192. 22. Glantz SB, Li Y, Rubin CS. Characterization of distinct tethering and intracellular targeting domains in AKAP75, a protein that links cAMP-dependent protein kinase II beta to the cytoskeleton. J Biol Chem 1993;268:12,796–12,804. 23. Diviani D, Langeberg LK, Doxsey SJ, Scott JD. Pericentrin anchors protein kinase A at the centrosome through a newly identified RIIbinding domain. Curr Biol 2000;10:417–20. 24. Gold MG, Lygren B, Dokurno P, Hoshi N, McConnachie G, et al. Molecular basis of AKAP specificity for PKA regulatory subunits. Mol Cell 2006;24:383–95. 25. Kinderman FS, Kim C, von Daake S, Ma Y, Pham BQ, et al. A dynamic mechanism for AKAP binding to RII isoforms of cAMPdependent protein kinase. Mol Cell 2006;24:397–408. 26. Burns-Hamuro LL, Ma Y, Kammerer S, Reineke U, Self C, et al. Designing isoform-specific peptide disruptors of protein kinase A localization. Proc Natl Acad Sci USA 2003;100:4072–7. 27. Carlson CR, Lygren B, Berge T, Hoshi N, Wong W, et al. Delineation of type I protein kinase A-selective signaling events using an RI anchoring disruptor. J Biol Chem 2006;281:21,535–21,545. 28. Lester LB, Coghlan VM, Nauert B, Scott JD. Cloning and characterization of a novel A-kinase anchoring protein: AKAP220, association with testicular peroxisomes. J Biol Chem 1996;272:9460–5. 29. Schillace RV, Scott JD. Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220. Curr Biol 1999;9:321–4. 30. Schmidt PH, Dransfield DT, Claudio JO, Hawley RG, Trotter KW, et al. AKAP350: a multiply spliced A-kinase anchoring protein associated with centrosomes. J Biol Chem 1999;274:3055–66.
Chapter | 165 Subcellular Targeting of PKA through AKAPs: Conserved Anchoring and Unique Targeting Domains
31. Witczak O, Skalhegg BS, Keryer G, Bornens M, Tasken K, et al. Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome, AKAP450. EMBO J 1999;18:1858–68. 32. Lewis SA, Wang D, Cowan NJ. Microtubule-associated protein MAP2 shares a microtubule binding motif with tau protein. Science 1988;242:936–9. 33. Luo Z, Shafit-Zagardo B, Erlichman J. Identification of the MAP2- and P75- binding domain in the regulatory subunit (RIIb) of Type II cAMP-dependent protein kinase. J Biol Chem 1990;265:21,804–21,810. 34. Rubino HM, Dammerman M, Shafit-Sagardo B, Erlichman J. Localization and characterization of the binding site for the regulatory subunit of type II cAMP-dependent protein kinase on MAP2.. Neuron 1989;3:631–8. 35. Westphal RS, Soderling SH, Alto NM, Langeberg LK, Scott JD. Scar/ WAVE-1, a Wiskott-Aldrich syndrome protein, assembles an actinassociated multi-kinase scaffold. EMBO J 2000;19:4589–600. 36. Kapiloff MS, Jackson N, Airhart N. mAKAP and the ryanodine receptor are part of a multi-component signaling complex on the cardiomyocyte nuclear envelope. J Cell Sci 2001;114:3167–76. 37. Kapiloff MS, Schillace RV, Westphal AM, Scott JD. mAKAP: an Akinase anchoring protein targeted to the nuclear membrane of differentiated myocytes. J Cell Sci 1999;112:2725–36. 38. Chen Q, Reigh-Yi L, Rubin C. Organelle-specific targeting of protein kinase AII (PKA). J Biol Chem 1997;272:15,247–15,257. 39. Huang LJ, Wang L, Ma Y, Durick K, Perkins G, et al. NH2-Terminal targeting motifs direct dual specificity A-kinase- anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum. J Cell Biol 1999;145:951–9. 40. Lin R-Y, Moss SB, Rubin CS. Characterization of S-AKAP84, a novel developmentally regulated A kinase anchor protein of male germ cells. J Biol Chem 1995;270(27):804–11. 41. Coghlan VM, Langeberg LK, Fernandez A, Lamb NJC, Scott JD. Cloning and characterization of AKAP95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase. J Biol Chem 1994;269:7658–65. 42. Eide T, Coghlan V, Orstavik S, Holsve C, Solberg R, et al. Molecular cloning, chromosomal localization and cell cycle-dependent subcellular distribution of the A-kinase anchoring protein, AKAP95. Exp Cell Res 1997;238:305–16. 43. Collas P, Le Guellec K, Tasken K. The A-kinase-anchoring protein AKAP95 is a multivalent protein with a key role in chromatin condensation at mitosis. J Cell Biol 1999;147:1167–80. 44. Landsverk HB, Carlson CR, Steen RL, Vossebein L, Herberg FW, et al. Regulation of anchoring of the RIIalpha regulatory subunit of PKA to AKAP95 by threonine phosphorylation of RIIalpha: implications for chromosome dynamics at mitosis. J Cell Sci 2001;114:3255–64. 45. Brown RL, August SL, Williams CJ, Moss SB. AKAP7gamma is a nuclear RI-binding AKAP. Biochem Biophys Res Commun 2003;306:394–401. 46. Lygren B, Carlson CR, Santamaria K, Lissandron V, McSorley T, et al. AKAP complex regulates Ca2 re-uptake into heart sarcoplasmic reticulum. EMBO Rep 2007;8:1061–7. 47. Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, et al. Identification of a novel A-kinase anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin-2 shuttle in renal principal cells. J Biol Chem 2004;279:26,654–26,665. 48. Gold MG, Smith FD, Scott JD, Barford D. AKAP18 contains a phosphoesterase domain that binds AMP. J Mol Biol 2008;375: 1329–43.
1335
49. Dell’Acqua ML, Faux MC, Thorburn J, Thorburn A, Scott JD. Membrane-targeting sequences on AKAP79 bind phosphatidylinositol4, 5- bisphosphate. EMBO J 1998;17:2246–60. 50. Gomez LL, Alam S, Smith KE, Horne E, Dell’Acqua ML. Regulation of A-kinase anchoring protein 79/150-cAMP-dependent protein kinase postsynaptic targeting by NMDA receptor activation of calcineurin and remodeling of dendritic actin. J Neurosci 2002;22:7027–44. 51. Li Y, Ndubuka C, Rubin CS. A kinase anchor protein 75 targets regulatory (RII) subunits of cAMP-dependent protein kinase II to the cortical actin cytoskeleton in non-neuronal cells. J Biol Chem 1996;271:16,862–16,869. 52. Gorski JA, Gomez LL, Scott JD, Dell’Acqua ML. Association of an A-kinase-anchoring protein signaling scaffold with cadherin adhesion molecules in neurons and epithelial cells. Mol Biol Cell 2005;16: 3574–90. 53. Horne EA, Dell’Acqua ML. Phospholipase C is required for changes in postsynaptic structure and function associated with NMDA receptordependent long-term depression. J Neurosci 2007;27:3523–34. 54. Smith KE, Gibson ES, Dell’Acqua ML. cAMP-dependent protein kinase postsynaptic localization regulated by NMDA receptor activation through translocation of an A-kinase anchoring protein scaffold protein. J Neurosci 2006;26:2391–402. 55. Dell’acqua ML, Smith KE, Gorski JA, Horne EA, Gibson ES, Gomez LL. Regulation of neuronal PKA signaling through AKAP targeting dynamics. Eur J Cell Biol 2006;85:627–33. 56. Fraser ID, Tavalin SJ, Lester LB, Langeberg LK, Westphal AM, et al. A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMPresponsive membrane events. EMBO J 1998;17:2261–72. 57. Trotter KW, Fraser ID, Scott GK, Stutts MJ, Scott JD, Milgram SL. Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms. J Cell Biol 1999;147:1481–92. 58. Dong F, Felsmesser M, Casadevall A, Rubin CS. Molecular characterization of a cDNA that encodes six isoforms of a novel murine A kinase anchor protein. J Biol Chem 1998;273:6533–41. 59. Dransfield DT, Bradford AJ, Smith J, Martin M, Roy C, et al. Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO J 1997;16:101–9. 60. Feliciello A, Li Y, Avvedimento EV, Gottesman ME, Rubin CS. Akinase anchor protein 75 increases the rate and magnitude of cAMP signaling to the nucleus. Curr Biol 1997;7:1011–14. 61. Paolillo M, Feliciello A, Porcellini A, Garbi C, Bifulco M, et al. The type and the localization of cAMP-dependent protein kinase regulate transmission of cAMP signals to the nucleus in cortical and cerebellar granule cells. J Biol Chem 1999;274:6546–52. 62. Lester LB, Faux MC, Nauert JB, Scott JD. Targeted protein kinase A and PP-2B regulate insulin secretion through reversible phosphorylation.. Endocrinology 2001;142:1218–27. 63. Lester LB, Scott JD. Anchoring and scaffold proteins for kinases and phosphatases. Recent Progr Hormone Res 1997;52:409–30. 64. Carlson CR, Witczak O, Vossebein L, Labbe JC, Skalhegg BS, et al. CDK1-mediated phosphorylation of the RIIalpha regulatory subunit of PKA works as a molecular switch that promotes dissociation of RIIalpha from centrosomes at mitosis. J Cell Sci 2001;114:3243–54. 65. Keryer G, Yassenko M, Labbe JC, Castro A, Lohmann SM, et al. Mitosis-specific phosphorylation and subcellular redistribution of the RIIalpha regulatory subunit of cAMP-dependent protein kinase. J Biol Chem 1998;273:34,594–34,602. 66. Hall DD, Davare MA, Shi M, Allen ML, Weisenhaus M, et al. Critical role of cAMP-dependent protein kinase anchoring to the L-type calcium channel Cav1.2 via A-kinase anchor protein 150 in neurons. Biochemistry 2007;46:1635–46.
1336
67. Hoshi N, Langeberg LK, Scott JD. Distinct enzyme combinations in AKAP signalling complexes permit functional diversity. Nat Cell Biol 2005;7:1066–73. 68. Lu Y, Allen M, Halt AR, Weisenhaus M, Dallapiazza RF, et al. Agedependent requirement of AKAP150-anchored PKA and GluR2-lacking AMPA receptors in LTP. EMBO J 2007;26:4879–90. 69. Lu Y, Zhang M, Lim IA, Hall DD, Allen M, et al. AKAP150-anchored PKA activity is important for LTD during its induction phase. J Physiol 2008;586:4155–64. 70. Nie T, McDonough CB, Huang T, Nguyen PV, Abel T. Genetic disruption of protein kinase A anchoring reveals a role for compartmentalized kinase signaling in theta-burst long-term potentiation and spatial memory. J Neurosci 2007;27:10,278–10,288. 71. Snyder EM, Colledge M, Crozier RA, Chen WS, Scott JD, Bear MF. Role for A kinase-anchoring proteins (AKAPS) in glutamate receptor trafficking and long term synaptic depression. J Biol Chem 2005;280:16,962–16,968. 72. Tunquist BJ, Hoshi N, Guire ES, Zhang F, Mullendorff K, et al. Loss of AKAP150 perturbs distinct neuronal processes in mice. Proc Natl Acad Sci USA 2008;105:12,557–12,562. 73. Fraser ID, Scott JD. Modulation of ion channels: a “current” view of AKAPs. Neuron 1999;23:423–6. 74. Gray PC, Tibbs VC, Catterall WA, Murphy BJ. Identification of a 15-kDa cAMP-dependent protein kinase-anchoring protein associated with skeletal muscle L-type calcium channels. J Biol Chem 1997;272:6297–302. 75. Gao T, Yatani A, Dell’Acqua ML, Sako H, Green SA, et al. cAMPdependent regulation of cardiac L-type Ca2 channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 1997;19:185–96. 76. Johnson BD, Scheuer T, Catterall WA. Voltage-dependent potentiation of L-type Ca2 channels in skeletal muscle cells requires anchored cAMP-dependent protein kinase. Proc Natl Acad Sci USA 1994;91:11,492–11,496. 77. Oliveria SF, Dell’Acqua ML, Sather WA. AKAP79/150 anchoring of calcineurin controls neuronal L-type Ca2 channel activity and nuclear signaling.. Neuron 2007;55:261–75. 78. Rosenmund C, Carr DW, Bergeson SE, Nilaver G, Scott JD, Westbrook GL. Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons. Nature 1994;368:853–6. 79. Gray III PC, Johnson BD, Westenbroek RE, Hays LG, Yates JR, et al. Primary structure and function of an A kinase anchoring protein associated with calcium channels. Neuron 1998;20:1017–26.
PART | II Transmission: Effectors and Cytosolic Events
80. Tavalin SJ, Colledge M, Hell JW, Langeberg LK, Huganir RL, Scott JD. Regulation of GluR1 by the A-kinase anchoring protein 79 (AKAP79) signaling complex shares properties with long-term depression. J Neurosci 2002;22:3044–51. 81. Kameyama K, Lee HK, Bear MF, Huganir RL. Involvement of a postsynaptic protein kinase A substrate in the expression of homosynaptic long-term depression. Neuron 1998;21:1163–75. 82. Chen L, Marquardt ML, Tester DJ, Sampson KJ, Ackerman MJ, Kass RS. Mutation of an A-kinase-anchoring protein causes long-QT syndrome. Proc Natl Acad Sci USA 2007;104:20,990–20,995. 83. Marx SO, Kurokawa J, Reiken S, Motoike H, D’Armiento J, et al. Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel. Science 2002;295:496–9. 84. Chen L, Kass RS. Dual roles of the A kinase-anchoring protein Yotiao in the modulation of a cardiac potassium channel: a passive adaptor versus an active regulator. Eur J Cell Biol 2006;85:623–6. 85. Chen L, Kurokawa J, Kass RS. Phosphorylation of the A-kinaseanchoring protein Yotiao contributes to protein kinase A regulation of a heart potassium channel. J Biol Chem 2005;280:31,347–31,352. 86. Kurokawa J, Motoike HK, Rao J, Kass RS. Regulatory actions of the A-kinase anchoring protein Yotiao on a heart potassium channel downstream of PKA phosphorylation. Proc Natl Acad Sci USA 2004;101:16,374–16,378. 87. Hulme JT, Ahn M, Hauschka SD, Scheuer T, Catterall WA. A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2 channel and modulates its function. J Biol Chem 2002;277:4079–87. 88. Diviani D, Soderling J, Scott JD. AKAP-Lbc anchors protein kinase A and nucleates Galpha 12-selective Rho- mediated stress fiber formation. J Biol Chem 2001;276:44,247–44,257. 89. Schillace RV, Andrews SF, Liberty GA, Davey MP, Carr DW. Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes. J Immunol 2002;168:1590–9. 90. Goehring AS, Pedroja BS, Hinke SA, Langeberg LK, Scott JD. MyRIP anchors protein kinase A to the exocyst complex. J Biol Chem 2007;282:33,155–33,167. 91. Nauert JB, Klauck TM, Langeberg LK, Scott JD. Gravin, an autoantigen recognized by serum from myasthenia gravis patients, is a kinase scaffold protein. Curr Biol 1997;7:52–62. 92. Alto NM, Soderling J, Scott JD. Rab32 is an A-kinase anchoring protein and participates in mitochondrial dynamics. J Cell Biol 2002;158:659–68.