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Architecture and Dynamics of an A-Kinase Anchoring Protein 79 (AKAP79) Signaling Complex

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Architecture and Dynamics of an A-Kinase Anchoring Protein 79 (AKAP79) Signaling Complex Architecture and dynamics of an A-kinase anchoring protein 79 (AKAP79) signaling complex Matthew G. Golda,b,1,2, Florian Stengelc,1, Patrick J. Nygrena, Chad R. Weisbrodd, James E. Bruced, Carol V. Robinsonc, David Barfordb, and John D. Scotta aHoward Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, 1959 Pacific Avenue NE, Seattle, WA 98195; bSection of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, United Kingdom; cDepartment of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QZ, United Kingdom; and dDepartment of Genome Sciences, University of Washington School of Medicine, 1959 Pacific Avenue NE, Seattle, WA 98195 Edited* by Tony Pawson, Samuel Lunenfeld Research Institute, Toronto, Canada, and approved February 16, 2011 (received for review September 28, 2010) A-kinase anchoring protein 79 (AKAP79) is a human anchoring density, where it coordinates phosphoregulation of GluR1 recep- protein that organizes cAMP-dependent protein kinase (PKA), tors. Consequently, mice deficient in AKAP150 exhibit impaired Ca2þ∕calmodulin (CaM)-dependent protein phosphatase (PP2B), synaptic plasticity and learning deficits (12). AKAP79 also targets and protein kinase C (PKC) for phosphoregulation of synaptic PKC to KCNQ2 channels for receptor-induced suppression of signaling. Quantitative biochemical analyses of selected AKAP79 the M current (13), a property that provides AKAP150 knockout complexes have determined the quaternary structure of these animals with a measure of resistance to seizures induced by the signaling complexes. We show that AKAP79 dimerizes, and we nonselective muscarinic agonist pilocarpine (12). demonstrate that, upon addition of a lysine-reactive cross-linker, Prior to this study, our understanding of the molecular basis parallel homomeric dimers are stabilized through K328–K328 and of the AKAP79 signaling complex was limited to structures of K333–K333 cross-links. An assembly of greater complexity compris- AKAP-derived peptides in complex with D∕D domains (14, 15). ing AKAP79, PP2B, a type II regulatory subunit fragment (RII 1–45) Here, we determine the stoichiometry of a core AKAP79 com- of PKA, and CaM was reconstituted in vitro. Using native MS, we plex by mass spectrometry (MS) of intact protein assemblies determined the molecular mass of this complex as 466 kDa. This (16, 17), and we map a parallel dimer interface by chemical indicates that dimeric AKAP79 coordinates two RII 1–45 homodi- cross-linking. Each protomer of AKAP79 is capable of binding BIOCHEMISTRY mers, four PP2B heterodimers, and two CaM molecules. Binding two PP2B heterodimers, a PKA regulatory homodimer and one of Ca2þ∕CaM to AKAP79 stabilizes the complex by generating a CaM. Each kinase/phosphatase pair is anchored in a symmetrical 2þ second interface for PP2B. This leads to activation of the anchored manner. Finally, we show that binding of Ca ∕CaM to AKAP79 phosphatases. Our architectural model reveals how dimeric generates an additional PP2B interaction site on the anchoring AKAP79 concentrates pockets of second messenger responsive protein to activate the tethered phosphatase. enzyme activities at the plasma membrane. Results ollowing its discovery, cyclic AMP (cAMP) was thought to AKAP79 Dimerizes. We developed a multistep purification system A be freely diffusible within the cell (1). Subsequently, it was for reconstitution of AKAP79 complexes in vitro (Fig. 1 ). A F ∕ α demonstrated that utilization of this second messenger is more GST fusion protein with the D D domain of RII (residues – ∕ sophisticated. For example, treatment of cardiac myocytes with 1 45, hereafter abbreviated D D) was used to purify AKAP79, Sf21 norepinephrine, but not prostaglandin E1, stimulates contraction which was expressed with an N-terminal His6 tag in insect and glycogen metabolism, despite the fact that both hormones cells. AKAP79 and associated proteins were released from glu- elevate cAMP to activate cAMP-dependent protein kinase tathione sepharose resin by incubation with PreScission protease ∕ (PKA) (2). It is now recognized that intracellular cAMP levels that specifically cleaved the link between GST and the D D fluctuate in cellular microdomains where cAMP effector proteins domain. A nickel affinity step was included to further enrich such as PKA, Epac guanine nucleotide exchange factors, and His6-tagged AKAP79 and associated proteins before final puri- – ∕ cyclic nucleotide-gated ion channels reside (3–5). Likewise, other fication by gel filtration. AKAP79 D D eluted from a Superdex second messengers such as Ca2þ and certain phospholipids oper- S200 gel filtration column with an elution volume of 66.6 mL A ate in cellular microdomains where they interface with their own (Fig. S2 ), which, according to gel filtration standards, corre- effector proteins (6). sponds to a protein of approximately 200 kDa. This suggests A-kinase anchoring proteins (AKAPs) organize responses that AKAP79 oligomerizes. Peak fractions (Coomassie-stained, B to these second messengers. AKAP79 is a prototypical AKAP, Fig. 1 ) were pooled and concentrated for analysis by native MS. – ∕ exemplifying the three properties that are characteristic of the MS of purified AKAP79 D D at minimal activation revealed a i α – series of AKAP79–D∕D oligomers (Fig. S3A). Fig. 1C shows the family: ( ) an amphipathic -helix (residues 387 406) that binds – ∕ to the D∕D domain (residues 1–45) of PKA RII subunits (7); (ii) tandem MS (MSMS) spectrum of AKAP79 D D where ions m∕z a subcellular localization signal, in its case, three tandem mem- were selected at 4,500 . Upon activation, this complex loses aD∕D subunit to form a stripped complex of 106,820 Da brane-binding basic regions (MBBRs) that bind to PIP2 (8); and (iii) the ability to interact with multiple signaling molecules. 2þ AKAP79 targets PKA, the Ca -dependent Ser/Thr protein Author contributions: M.G.G., F.S., C.V.R., D.B., and J.D.S. designed research; M.G.G., F.S., phosphatase PP2B (calcineurin) (9), and protein kinase C (PKC) P.J.N., and C.R.W. performed research; M.G.G., F.S., P.J.N., C.R.W., J.E.B., C.V.R., D.B., and (10) to substrates including transmembrane receptors and ion J.D.S. analyzed data; and M.G.G., F.S., D.B., and J.D.S. wrote the paper. channels. A more comprehensive list of AKAP79 binding part- The authors declare no conflict of interest. ners is shown in Fig. S1. Ancillary protein–lipid and protein– *This Direct Submission article had a prearranged editor. protein interactions serve to localize AKAP79 with particular Freely available online through the PNAS open access option. membrane substrates (8). For example, a leucine zipper-like 1M.G.G. and F.S. contributed equally to this work. motif in AKAP79 associates with the cytoplasmic tail of L-type 2To whom correspondence should be addressed. E-mail: [email protected]. 2þ Ca channels, enabling channel regulation by PKA and PP2B This article contains supporting information online at www.pnas.org/lookup/suppl/ (11). AKAP79 is also a principal AKAP in the postsynaptic doi:10.1073/pnas.1014400108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1014400108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 27, 2021 AB Fig. 1. AKAP79 purification and basic stoichiometry. (A) Purification protocol for the AKAP79–D∕D com- plex. The positions of two additional steps sufficient E for AKDPC purification are indicated. (B) Coomassie- C stained 4–12% SDS-PAGE showing 10 μg purified AKAP79–D∕D. (C) MSMS spectrum of AKAP79–D∕D. 106,820 Da [2 Ã AKAP79 þ 1 Ã D∕D] (gold) and 101,036 Da [2 Ã AKAP79] (blue) dissociation products are indicated. Spectra are shown at a collision cell voltage of 100 V. (D) Topology diagram illustrating the location of 42 lysine residues (white bars) in AKAP79 and the boundaries of three constructs. The locations of dimeric cross-linking sites (red) and F the PIAIIIT motif (green) are indicated. (E) Anti-strep immunoblot (IB) of Strep–AKAP79 pulled-down by GST, GST–AKAP79, or GST fused to the three regions D of AKAP79 as depicted in D. Relative densitometry analysis (n ¼ 3, Top) and pull-down Coomassie (Bot- tom) are shown. (F) Control immunoglobulin (IgG) or anti-FLAG immunoprecipitates from HEK293 cells co- H expressing FLAG–AKAP79 and V5–AKAP79 were im- G munoblotted as indicated. Relative densitometry analysis (n ¼ 3, Top). (G) Anti-Strep IB shows Strep– AKAP79 dimer cross-linking with 0 (lane 1), 50 (lane 2) and 500 (lane 3) μM DSS, indicated by anti-strep immunoblotting. (H) MS spectra showing peptides generated by trypsinization of cross-linked Strep– AKAP79. The spectra correspond to K328–K328 (Top) and K333–K333 (Bottom) dimeric cross-linked peptides. [2 Ã AKAP79 þ 1 Ã D∕D] (Fig. 1C, gold). Upon additional activa- lysine-specific cross-linker disuccinimidyl suberate (DSS) to pur- tion, a further D∕D subunit is lost to form a 101,036-Da complex ified Strep–AKAP79 produced a species of approximately twice consisting of the AKAP79 dimer (Fig. 1C, blue). Therefore, the apparent molecular weight, as detected by immunoblotting the original precursor ion is assigned unambiguously to the (Fig. 1G). Low levels of trimeric and higher oligomeric species 25þ charge state of a complex consisting of [2 Ã AKAP79 þ 2Ã of AKAP79 were also stabilized by addition of DSS (Fig. 1G). D∕D], supporting AKAP79 dimerization. MS of the AKAP79– Detection of the 150-kDa species was more prominent with D∕D complex was also performed under denaturing conditions. 500-μM DSS (Fig. 1G, lanes 2 and 3; Coomassie staining is shown A more precise mass of AKAP79 was calculated by this method at in Fig. S3D). For mapping studies, Strep–AKAP79 was treated with 50;461 Æ 18 B Da (Fig.
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