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W2076018512 abstract "Compartmentalization of protein kinases and phosphatases with substrates is a means to increase the efficacy of signal transduction events. The A-kinaseanchoring protein, AKAP79, is a multivalent anchoring protein that maintains the cAMP-dependent protein kinase, protein kinase C, and protein phosphatase-2B (PP2B/calcineurin) at the postsynaptic membrane of excitatory synapses where it is recruited into complexes withN-methyl-d-aspartic acid or α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. We have used cellular targeting of AKAP79 truncation and deletion mutants as an assay to map the PP2B-binding site on AKAP79. We demonstrate that residues 315–360 are necessary and sufficient for AKAP79-PP2B anchoring in cells. Multiple determinants contained within this region bind directly to the A subunit of PP2B and inhibit phosphatase activity. Peptides spanning the 315–360 region of AKAP79 can antagonize PP2B anchoring in vitro and targeting in transfected cells. Electrophysiological experiments further emphasize this point by demonstrating that a peptide encompassing residues 330–357 of AKAP79 attenuates PP2B-dependent down-regulation of GluR1 receptor currents when perfused into HEK293 cells. We propose that the structural features of this AKAP79-PP2B-binding domain may share similarities with other proteins that serve to coordinate PP2B localization and activity. Compartmentalization of protein kinases and phosphatases with substrates is a means to increase the efficacy of signal transduction events. The A-kinaseanchoring protein, AKAP79, is a multivalent anchoring protein that maintains the cAMP-dependent protein kinase, protein kinase C, and protein phosphatase-2B (PP2B/calcineurin) at the postsynaptic membrane of excitatory synapses where it is recruited into complexes withN-methyl-d-aspartic acid or α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. We have used cellular targeting of AKAP79 truncation and deletion mutants as an assay to map the PP2B-binding site on AKAP79. We demonstrate that residues 315–360 are necessary and sufficient for AKAP79-PP2B anchoring in cells. Multiple determinants contained within this region bind directly to the A subunit of PP2B and inhibit phosphatase activity. Peptides spanning the 315–360 region of AKAP79 can antagonize PP2B anchoring in vitro and targeting in transfected cells. Electrophysiological experiments further emphasize this point by demonstrating that a peptide encompassing residues 330–357 of AKAP79 attenuates PP2B-dependent down-regulation of GluR1 receptor currents when perfused into HEK293 cells. We propose that the structural features of this AKAP79-PP2B-binding domain may share similarities with other proteins that serve to coordinate PP2B localization and activity. The efficient transmission of cellular signals often involves the orientation of signaling proteins in relation to their upstream activators and downstream targets. This is often achieved through association with anchoring and scaffolding proteins that compartmentalize signaling enzymes in distinct subcellular environments (1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Google Scholar, 2Jordan J.D. Landau E.M. Iyengar R. Cell. 2000; 103: 193-200Google Scholar, 3Hunter T. Cell. 2000; 100: 113-127Google Scholar). For example, A-kinaseanchoring proteins (AKAPs) 1The abbreviations used are: AKAP, A-kinase anchoring proteins; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PP2B, protein phosphatase-2B; AMPA, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GFP, green fluorescent protein; EGFP, enhanced GFP. 1The abbreviations used are: AKAP, A-kinase anchoring proteins; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PP2B, protein phosphatase-2B; AMPA, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GFP, green fluorescent protein; EGFP, enhanced GFP. bind the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA) to localize this broad specificity enzyme to discrete subcellular environments (4Colledge M. Scott J.D. Trends Cell Biol. 1999; 9: 216-221Google Scholar, 5Feliciello A. Gottesman M.E. Avvedimento E.V. J. Mol. Biol. 2001; 308: 99-114Google Scholar). Each AKAP contains a conserved amphipathic helix that binds to the R subunit dimer with high affinity and targeting domains that direct the PKA-AKAP complex to specific subcellular compartments (6Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D. J. Biol. Chem. 1991; 266: 14188-14192Google Scholar, 7Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Google Scholar). A likely consequence of these protein-protein interactions is that AKAP-PKA complexes are maintained in the vicinity of selected phosphoproteins and substrates for the kinase. Another important role of AKAPs is to serve as scaffolds for the assembly of multiprotein complexes that include PKA, other protein kinases, phosphodiesterases, and a variety of protein phosphatases (8Smith F.D. Scott J.D. Curr. Biol. 2002; 12: R32-R40Google Scholar). The simultaneous anchoring of kinases and phosphatases provides an efficient means to confer bi-directional control on the phosphorylation status of substrate proteins (9Westphal R.S. Tavalin S.J. Lin J.W. Alto N.M. Fraser I.D. Langeberg L.K. Sheng M. Scott J.D. Science. 1999; 285: 93-96Google Scholar, 10Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Google Scholar).A number of studies (11Fraser I.D. Scott J.D. Neuron. 1999; 23: 423-426Google Scholar) have demonstrated that anchoring of kinases and phosphatases ensures the efficient regulation of ion channels and neurotransmitter receptors. One prominent mediator of this process is the multivalent anchoring protein AKAP79 that anchors PKA, protein kinase C (PKC), and protein phosphatase-2B (PP2B/calcineurin) (12Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 24: 16816-16823Google Scholar, 13Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-112Google Scholar, 14Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Google Scholar). Precise orientation of the AKAP79 signaling complex toward substrates such as ionotropic glutamate receptors at the postsynaptic densities of neurons involves additional protein-protein interactions between the anchoring protein and membrane-associated guanylate kinase proteins. This intricate molecular architecture facilitates PKA phosphorylation at a site in the cytoplasmic tail of the GluR1 glutamate receptor channel as well as its efficient dephosphorylation by PP2B (10Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Google Scholar,15Colledge M. Dean R.A. Scott G.K. Langeberg L.K. Huganir R.L. Scott J.D. Neuron. 2000; 27: 107-119Google Scholar).Previous studies (13Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-112Google Scholar, 16Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Google Scholar, 17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar) have shown that the catalytic subunit of PP2B directly binds to sites in the C-terminal two-thirds of AKAP79 and that interaction with the anchoring protein inhibits phosphatase activity toward peptide substrates. In this report we describe experiments that map the PP2B-binding site to a region of ∼45 amino acids that contains multiple binding sites and inhibitory determinants. Furthermore, we show that a peptide from this region disrupts phosphatase targeting and attenuates PP2B-dependent down-regulation of homomeric GluR1 glutamate receptor currents inside cells.DISCUSSIONWe have mapped a region in the neuronally expressed anchoring protein AKAP79 that is necessary for binding and inhibiting the calcium/calmodulin-dependant phosphatase PP2B/calcineurin. A combination of biochemical and enzymatic approaches has identified multiple binding and inhibitory determinants that are located between residues 318 and 357 of the anchoring protein. Our characterization of this PP2B-binding region is the latest in a series of studies that have dissected functional regions within AKAP79 that are responsible for the interaction with distinct signaling enzymes. Residues 391–408 form an amphipathic helix that binds to the R subunit dimer of the PKA holoenzyme with nanomolar affinity (7Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Google Scholar, 12Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 24: 16816-16823Google Scholar, 19Hausken Z.E. Dell'Acqua M.L. Coghlan V.M. Scott J.D. J. Biol. Chem. 1996; 271: 29016-29022Google Scholar). Peptides spanning this region have been used by many investigators to disrupt a variety of cAMP-responsive events by uncoupling PKA location inside cells (20Rosenmund C. Carr D.W. Bergeson S.E. Nilaver G. Scott J.D. Westbrook G.L. Nature. 1994; 368: 853-856Google Scholar, 21Lester L.B. Langeberg L.K. Scott J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14942-14947Google Scholar, 22Fink M.A. Zakhary D.R. Mackey J.A. Desnoyer R.W. Apperson-Hansen C. Damron D.S. Bond M. Circ. Res. 2001; 88: 291-297Google Scholar, 23Feliciello A. Li Y. Avvedimento E.V. Gottesman M.E. Rubin C.S. Curr. Biol. 1997; 7: 1011-1014Google Scholar, 24Sun F. Hug M.J. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 14360-14366Google Scholar, 25Dodge K.L. Carr D.W. Sanborn B.M. Endocrinology. 1999; 140: 5165-5170Google Scholar). A PKC-binding site is located between residues 31 and 52 (14). Peptides spanning this region bind to the kinase with micromolar affinities and inhibit the bound enzyme. This effect is reversed by calcium/calmodulin, which competes for interaction with the 31–52 region and releases the active kinase from the signaling complex at the postsynaptic densities of neurons (26Faux M.C. Scott J.D. J. Biol. Chem. 1997; 272: 17038-17044Google Scholar, 27Faux M.C. Rollins E.N. Edwards A.S. Langeberg L.K. Newton A.C. Scott J.D. Biochem. J. 1999; 343: 443-452Google Scholar).In this report, we demonstrate that a family of peptides synthesized to regions of the anchoring protein between residues 318 and 357 disrupt the subcellular location of the phosphatase and inhibit PP2B-dependent phosphorylation events. Another region of AKAP79 within the membrane targeting domain (residues 88–102) may also participate in PP2B anchoring (13Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-112Google Scholar, 16Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Google Scholar, 17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar), although the inhibitory potency of peptides to this site is ∼50-fold less than the AKAP79-(330–357) peptide, and they cannot displace the phosphatase from its sites of anchoring. Therefore, we propose that the region between residues 318 and 357 is likely to contain most of the high affinity determinants for PP2B anchoring. This postulate is consistent with recent reports (17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar, 28Taigen T. De Windt L.J. Lim H.W. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1196-1201Google Scholar, 29De Windt L.J. Lim H.W. Bueno O.F. Liang Q. Delling U. Braz J.C. Glascock B.J. Kimball T.F. del Monte F. Hajjar R.J. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3322-3327Google Scholar) that recombinant fragments of AKAP79 that include this region inhibit PP2B signaling events in transfected cells and transgenic mice. Interestingly, the AKAP79-(330–357) and AKAP79-(338–357) peptides are slightly more potent inhibitors of the phosphatase than the full-length anchoring protein. Similarly we have observed that protein fragments of AKAP79 sometimes work more efficiently than the full-length protein. For example, residues 108–427 inhibit PP2B more effectively than the full-length protein (16Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Google Scholar). Likewise, the first 75 amino acids of the anchoring protein is a more potent inhibitor of PKC activity than the full-length protein (14Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Google Scholar).The AKAP79-(318–357) sequence exhibits some similarity to the binding sites of other PP2B-interacting proteins. For example, the organization of serines, threonines, and the clustering of basic amino acids in this region is reminiscent of the 38-residue PP2B-binding region in Cain, (calcineurin inhibitor)/cabin (30Lai M.M. Burnett P.E. Wolosker H. Blackshaw S. Snyder S.H. J. Biol. Chem. 1998; 273: 18325-18331Google Scholar, 31Luo S. Youn H.-D. Loh C. Stolow M. He W. Liu J.O. Immunity. 1998; 8: 703-711Google Scholar). Both proteins are non-competitive inhibitors of PP2B activity that inhibit the phosphatase in the low micromolar range and bind PP2B at a site distinct from the immunosuppressant drugs FK506 and cyclosporin A (13Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-112Google Scholar,17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar, 32Lai M.M. Luo H.R. Burnett P.E. Hong J.J. Snyder S.H. J. Biol. Chem. 2000; 275: 34017-34020Google Scholar). Another common characteristic is the presence of a loosely conserved “PIXIXIT” motif (where Xrepresents any amino acid). This sequence was first identified in the transcription factor NFATc and has been found in the docking sites of other PP2B interacting partners including Cain/cabin and the muscle-specific protein MCIP1,2 (33Aramburu J. Garcia-Cozar F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Google Scholar, 34Aramburu J. Yaffe M.B. Lopez-Rodriguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Google Scholar, 35Rothermel B. Vega R.B. Yang J. Wu H. Bassel-Duby R. Williams R.S. J. Biol. Chem. 2000; 275: 8719-8725Google Scholar). Residues 337–343 of AKAP79 ( PIA II IT) form the PIXIXIT motif, and it is totally conserved in the murine and bovine orthologs, AKAP150 and AKAP75 (36Bregman D.B. Bhattacharyya N. Rubin C.S. J. Biol. Chem. 1989; 264: 4648-4656Google Scholar, 37Bregman D.B. Hirsch A.H. Rubin C.S. J. Biol. Chem. 1991; 266: 7207-7213Google Scholar, 38Hirsch A.H. Glantz S.B. Li Y. You Y. Rubin C.S. J. Biol. Chem. 1992; 267: 2131-2134Google Scholar). Although not formally proven, it is likely that this sequence represents a principle site of contact with the phosphatase, because the AKAP79-(330–357) peptide, which includes this region, inhibits phosphatase activity at micromolar concentrations and competes for binding with the intact anchoring protein. However, it is clearly evident that other regions of AKAP79 contribute to PP2B interactions as peptides upstream of the PIXIXIT motif also bind and inhibit the phosphatase. These findings are certainly consistent with the notion that multiple determinants located in a central segment of the AKAP79 linear sequence act in tandem to anchor PP2B and inhibit phosphatase activity inside cells. Apparently multisite contact between phosphatases and their binding partners is a common theme in the subcellular targeting of this enzyme class, as a second PP2B-binding site has been defined within the regulatory domain of NFATc which may act synergistically with the PIXIXIT motif to permit a high affinity interaction with the phosphatase (39Park S. Uesugi M. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7130-7135Google Scholar). Similarly, there are examples of type 1 phosphatase targeting subunits that interact with the catalytic subunit (PP1c) through multiple sites (40Allen P.B. Kwon Y.G. Nairn A.C. Greengard P. J. Biol. Chem. 1998; 273: 4089-4095Google Scholar, 41Schillace R.V. Voltz J.W. Sim A.T. Shenolikar S. Scott J.D. J. Biol. Chem. 2001; 276: 12128-12134Google Scholar).One goal of our study was to generate peptide-based antagonists of PP2B function inside cells. Accordingly, we have shown that perfusion of the AKAP79-(330–357) peptide prevents the attenuation of GluR1 type AMPA channels by the AKAP79 signaling complex in HEK293 cells. We have shown previously that AKAP79-mediated anchoring of PP2B confers a calcium-dependent rundown of the channel through the dephosphorylation of serine 845, a site in the cytoplasmic tail of the GluR1 subunit (10Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Google Scholar). Basal phosphorylation of Ser-845 is maintained by anchored PKA, suggesting that AKAP79 contributes to the regulation of this channel by positioning kinases and phosphates in close proximity to a subset of their substrates (10Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Google Scholar, 11Fraser I.D. Scott J.D. Neuron. 1999; 23: 423-426Google Scholar, 15Colledge M. Dean R.A. Scott G.K. Langeberg L.K. Huganir R.L. Scott J.D. Neuron. 2000; 27: 107-119Google Scholar, 42Swope S.L. Moss S.I. Raymond L.A. Huganir R.L. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 49-78Google Scholar, 43Kameyama K. Lee H.K. Bear M.F. Huganir R.L. Neuron. 1998; 21: 1163-1175Google Scholar, 44Lee H.K. Kameyama K. Huganir R.L. Bear M.F. Neuron. 1998; 21: 1151-1162Google Scholar, 45Lee H.K. Barbarosie M. Kameyama K. Bear M.F. Huganir R.L. Nature. 2000; 405: 955-959Google Scholar). Our electrophysiological measurements infer that perfusion of the AKAP79-(330–357) peptide shifts the equilibrium in favor of the phosphorylated state of GluR1. Importantly, our control experiments demonstrate that this peptide is only active in the presence of AKAP79, thereby indicating that PP2B targeting ensures efficient modulation of GluR1 currents. Yet our experiments are unable to delineate the mechanism of action of this peptide. Our biochemical analyses would suggest that it must function by displacing PP2B from its site of action and/or as an inhibitor of the anchored phosphatase. Nonetheless, our experiments highlight the utility of this reagent as a cell-based modulator of PP2B. Likewise, Cain has also been used to perturb PP2B function inside cells, as it binds the endocytic machinery through amphiphysin 1 to negatively regulate PP2B/dynamin-dependent synaptic vesicle endocytosis (30Lai M.M. Burnett P.E. Wolosker H. Blackshaw S. Snyder S.H. J. Biol. Chem. 1998; 273: 18325-18331Google Scholar, 32Lai M.M. Luo H.R. Burnett P.E. Hong J.J. Snyder S.H. J. Biol. Chem. 2000; 275: 34017-34020Google Scholar). Thus it is tempting to speculate that AKAP79 anchoring and inhibition of PP2B might function in a concerted manner to modulate not only AMPA receptor currents but also to attenuate the phosphorylation events that control receptor endocytosis. The efficient transmission of cellular signals often involves the orientation of signaling proteins in relation to their upstream activators and downstream targets. This is often achieved through association with anchoring and scaffolding proteins that compartmentalize signaling enzymes in distinct subcellular environments (1Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Google Scholar, 2Jordan J.D. Landau E.M. Iyengar R. Cell. 2000; 103: 193-200Google Scholar, 3Hunter T. Cell. 2000; 100: 113-127Google Scholar). For example, A-kinaseanchoring proteins (AKAPs) 1The abbreviations used are: AKAP, A-kinase anchoring proteins; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PP2B, protein phosphatase-2B; AMPA, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GFP, green fluorescent protein; EGFP, enhanced GFP. 1The abbreviations used are: AKAP, A-kinase anchoring proteins; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PP2B, protein phosphatase-2B; AMPA, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; GFP, green fluorescent protein; EGFP, enhanced GFP. bind the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA) to localize this broad specificity enzyme to discrete subcellular environments (4Colledge M. Scott J.D. Trends Cell Biol. 1999; 9: 216-221Google Scholar, 5Feliciello A. Gottesman M.E. Avvedimento E.V. J. Mol. Biol. 2001; 308: 99-114Google Scholar). Each AKAP contains a conserved amphipathic helix that binds to the R subunit dimer with high affinity and targeting domains that direct the PKA-AKAP complex to specific subcellular compartments (6Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D. J. Biol. Chem. 1991; 266: 14188-14192Google Scholar, 7Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Google Scholar). A likely consequence of these protein-protein interactions is that AKAP-PKA complexes are maintained in the vicinity of selected phosphoproteins and substrates for the kinase. Another important role of AKAPs is to serve as scaffolds for the assembly of multiprotein complexes that include PKA, other protein kinases, phosphodiesterases, and a variety of protein phosphatases (8Smith F.D. Scott J.D. Curr. Biol. 2002; 12: R32-R40Google Scholar). The simultaneous anchoring of kinases and phosphatases provides an efficient means to confer bi-directional control on the phosphorylation status of substrate proteins (9Westphal R.S. Tavalin S.J. Lin J.W. Alto N.M. Fraser I.D. Langeberg L.K. Sheng M. Scott J.D. Science. 1999; 285: 93-96Google Scholar, 10Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Google Scholar). A number of studies (11Fraser I.D. Scott J.D. Neuron. 1999; 23: 423-426Google Scholar) have demonstrated that anchoring of kinases and phosphatases ensures the efficient regulation of ion channels and neurotransmitter receptors. One prominent mediator of this process is the multivalent anchoring protein AKAP79 that anchors PKA, protein kinase C (PKC), and protein phosphatase-2B (PP2B/calcineurin) (12Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 24: 16816-16823Google Scholar, 13Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-112Google Scholar, 14Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Google Scholar). Precise orientation of the AKAP79 signaling complex toward substrates such as ionotropic glutamate receptors at the postsynaptic densities of neurons involves additional protein-protein interactions between the anchoring protein and membrane-associated guanylate kinase proteins. This intricate molecular architecture facilitates PKA phosphorylation at a site in the cytoplasmic tail of the GluR1 glutamate receptor channel as well as its efficient dephosphorylation by PP2B (10Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Google Scholar,15Colledge M. Dean R.A. Scott G.K. Langeberg L.K. Huganir R.L. Scott J.D. Neuron. 2000; 27: 107-119Google Scholar). Previous studies (13Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-112Google Scholar, 16Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Google Scholar, 17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar) have shown that the catalytic subunit of PP2B directly binds to sites in the C-terminal two-thirds of AKAP79 and that interaction with the anchoring protein inhibits phosphatase activity toward peptide substrates. In this report we describe experiments that map the PP2B-binding site to a region of ∼45 amino acids that contains multiple binding sites and inhibitory determinants. Furthermore, we show that a peptide from this region disrupts phosphatase targeting and attenuates PP2B-dependent down-regulation of homomeric GluR1 glutamate receptor currents inside cells. DISCUSSIONWe have mapped a region in the neuronally expressed anchoring protein AKAP79 that is necessary for binding and inhibiting the calcium/calmodulin-dependant phosphatase PP2B/calcineurin. A combination of biochemical and enzymatic approaches has identified multiple binding and inhibitory determinants that are located between residues 318 and 357 of the anchoring protein. Our characterization of this PP2B-binding region is the latest in a series of studies that have dissected functional regions within AKAP79 that are responsible for the interaction with distinct signaling enzymes. Residues 391–408 form an amphipathic helix that binds to the R subunit dimer of the PKA holoenzyme with nanomolar affinity (7Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Google Scholar, 12Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 24: 16816-16823Google Scholar, 19Hausken Z.E. Dell'Acqua M.L. Coghlan V.M. Scott J.D. J. Biol. Chem. 1996; 271: 29016-29022Google Scholar). Peptides spanning this region have been used by many investigators to disrupt a variety of cAMP-responsive events by uncoupling PKA location inside cells (20Rosenmund C. Carr D.W. Bergeson S.E. Nilaver G. Scott J.D. Westbrook G.L. Nature. 1994; 368: 853-856Google Scholar, 21Lester L.B. Langeberg L.K. Scott J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14942-14947Google Scholar, 22Fink M.A. Zakhary D.R. Mackey J.A. Desnoyer R.W. Apperson-Hansen C. Damron D.S. Bond M. Circ. Res. 2001; 88: 291-297Google Scholar, 23Feliciello A. Li Y. Avvedimento E.V. Gottesman M.E. Rubin C.S. Curr. Biol. 1997; 7: 1011-1014Google Scholar, 24Sun F. Hug M.J. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 14360-14366Google Scholar, 25Dodge K.L. Carr D.W. Sanborn B.M. Endocrinology. 1999; 140: 5165-5170Google Scholar). A PKC-binding site is located between residues 31 and 52 (14). Peptides spanning this region bind to the kinase with micromolar affinities and inhibit the bound enzyme. This effect is reversed by calcium/calmodulin, which competes for interaction with the 31–52 region and releases the active kinase from the signaling complex at the postsynaptic densities of neurons (26Faux M.C. Scott J.D. J. Biol. Chem. 1997; 272: 17038-17044Google Scholar, 27Faux M.C. Rollins E.N. Edwards A.S. Langeberg L.K. Newton A.C. Scott J.D. Biochem. J. 1999; 343: 443-452Google Scholar).In this report, we demonstrate that a family of peptides synthesized to regions of the anchoring protein between residues 318 and 357 disrupt the subcellular location of the phosphatase and inhibit PP2B-dependent phosphorylation events. Another region of AKAP79 within the membrane targeting domain (residues 88–102) may also participate in PP2B anchoring (13Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-112Google Scholar, 16Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Google Scholar, 17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar), although the inhibitory potency of peptides to this site is ∼50-fold less than the AKAP79-(330–357) peptide, and they cannot displace the phosphatase from its sites of anchoring. Therefore, we propose that the region between residues 318 and 357 is likely to contain most of the high affinity determinants for PP2B anchoring. This postulate is consistent with recent reports (17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar, 28Taigen T. De Windt L.J. Lim H.W. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1196-1201Google Scholar, 29De Windt L.J. Lim H.W. Bueno O.F. Liang Q. Delling U. Braz J.C. Glascock B.J. Kimball T.F. del Monte F. Hajjar R.J. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3322-3327Google Scholar) that recombinant fragments of AKAP79 that include this region inhibit PP2B signaling events in transfected cells and transgenic mice. Interestingly, the AKAP79-(330–357) and AKAP79-(338–357) peptides are slightly more potent inhibitors of the phosphatase than the full-length anchoring protein. Similarly we have observed that protein fragments of AKAP79 sometimes work more efficiently than the full-length protein. For example, residues 108–427 inhibit PP2B more effectively than the full-length protein (16Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Google Scholar). Likewise, the first 75 amino acids of the anchoring protein is a more potent inhibitor of PKC activity than the full-length protein (14Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Google Scholar).The AKAP79-(318–357) sequence exhibits some similarity to the binding sites of other PP2B-interacting proteins. For example, the organization of serines, threonines, and the clustering of basic amino acids in this region is reminiscent of the 38-residue PP2B-binding region in Cain, (calcineurin inhibitor)/cabin (30Lai M.M. Burnett P.E. Wolosker H. Blackshaw S. Snyder S.H. J. Biol. Chem. 1998; 273: 18325-18331Google Scholar, 31Luo S. Youn H.-D. Loh C. Stolow M. He W. Liu J.O. Immunity. 1998; 8: 703-711Google Scholar). Both proteins are non-competitive inhibitors of PP2B activity that inhibit the phosphatase in the low micromolar range and bind PP2B at a site distinct from the immunosuppressant drugs FK506 and cyclosporin A (13Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-112Google Scholar,17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar, 32Lai M.M. Luo H.R. Burnett P.E. Hong J.J. Snyder S.H. J. Biol. Chem. 2000; 275: 34017-34020Google Scholar). Another common characteristic is the presence of a loosely conserved “PIXIXIT” motif (where Xrepresents any amino acid). This sequence was first identified in the transcription factor NFATc and has been found in the docking sites of other PP2B interacting partners including Cain/cabin and the muscle-specific protein MCIP1,2 (33Aramburu J. Garcia-Cozar F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Google Scholar, 34Aramburu J. Yaffe M.B. Lopez-Rodriguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Google Scholar, 35Rothermel B. Vega R.B. Yang J. Wu H. Bassel-Duby R. Williams R.S. J. Biol. Chem. 2000; 275: 8719-8725Google Scholar). Residues 337–343 of AKAP79 ( PIA II IT) form the PIXIXIT motif, and it is totally conserved in the murine and bovine orthologs, AKAP150 and AKAP75 (36Bregman D.B. Bhattacharyya N. Rubin C.S. J. Biol. Chem. 1989; 264: 4648-4656Google Scholar, 37Bregman D.B. Hirsch A.H. Rubin C.S. J. Biol. Chem. 1991; 266: 7207-7213Google Scholar, 38Hirsch A.H. Glantz S.B. Li Y. You Y. Rubin C.S. J. Biol. Chem. 1992; 267: 2131-2134Google Scholar). Although not formally proven, it is likely that this sequence represents a principle site of contact with the phosphatase, because the AKAP79-(330–357) peptide, which includes this region, inhibits phosphatase activity at micromolar concentrations and competes for binding with the intact anchoring protein. However, it is clearly evident that other regions of AKAP79 contribute to PP2B interactions as peptides upstream of the PIXIXIT motif also bind and inhibit the phosphatase. These findings are certainly consistent with the notion that multiple determinants located in a central segment of the AKAP79 linear sequence act in tandem to anchor PP2B and inhibit phosphatase activity inside cells. Apparently multisite contact between phosphatases and their binding partners is a common theme in the subcellular targeting of this enzyme class, as a second PP2B-binding site has been defined within the regulatory domain of NFATc which may act synergistically with the PIXIXIT motif to permit a high affinity interaction with the phosphatase (39Park S. Uesugi M. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7130-7135Google Scholar). Similarly, there are examples of type 1 phosphatase targeting subunits that interact with the catalytic subunit (PP1c) through multiple sites (40Allen P.B. Kwon Y.G. Nairn A.C. Greengard P. J. Biol. Chem. 1998; 273: 4089-4095Google Scholar, 41Schillace R.V. Voltz J.W. Sim A.T. Shenolikar S. Scott J.D. J. Biol. Chem. 2001; 276: 12128-12134Google Scholar).One goal of our study was to generate peptide-based antagonists of PP2B function inside cells. Accordingly, we have shown that perfusion of the AKAP79-(330–357) peptide prevents the attenuation of GluR1 type AMPA channels by the AKAP79 signaling complex in HEK293 cells. We have shown previously that AKAP79-mediated anchoring of PP2B confers a calcium-dependent rundown of the channel through the dephosphorylation of serine 845, a site in the cytoplasmic tail of the GluR1 subunit (10Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Google Scholar). Basal phosphorylation of Ser-845 is maintained by anchored PKA, suggesting that AKAP79 contributes to the regulation of this channel by positioning kinases and phosphates in close proximity to a subset of their substrates (10Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Google Scholar, 11Fraser I.D. Scott J.D. Neuron. 1999; 23: 423-426Google Scholar, 15Colledge M. Dean R.A. Scott G.K. Langeberg L.K. Huganir R.L. Scott J.D. Neuron. 2000; 27: 107-119Google Scholar, 42Swope S.L. Moss S.I. Raymond L.A. Huganir R.L. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 49-78Google Scholar, 43Kameyama K. Lee H.K. Bear M.F. Huganir R.L. Neuron. 1998; 21: 1163-1175Google Scholar, 44Lee H.K. Kameyama K. Huganir R.L. Bear M.F. Neuron. 1998; 21: 1151-1162Google Scholar, 45Lee H.K. Barbarosie M. Kameyama K. Bear M.F. Huganir R.L. Nature. 2000; 405: 955-959Google Scholar). Our electrophysiological measurements infer that perfusion of the AKAP79-(330–357) peptide shifts the equilibrium in favor of the phosphorylated state of GluR1. Importantly, our control experiments demonstrate that this peptide is only active in the presence of AKAP79, thereby indicating that PP2B targeting ensures efficient modulation of GluR1 currents. Yet our experiments are unable to delineate the mechanism of action of this peptide. Our biochemical analyses would suggest that it must function by displacing PP2B from its site of action and/or as an inhibitor of the anchored phosphatase. Nonetheless, our experiments highlight the utility of this reagent as a cell-based modulator of PP2B. Likewise, Cain has also been used to perturb PP2B function inside cells, as it binds the endocytic machinery through amphiphysin 1 to negatively regulate PP2B/dynamin-dependent synaptic vesicle endocytosis (30Lai M.M. Burnett P.E. Wolosker H. Blackshaw S. Snyder S.H. J. Biol. Chem. 1998; 273: 18325-18331Google Scholar, 32Lai M.M. Luo H.R. Burnett P.E. Hong J.J. Snyder S.H. J. Biol. Chem. 2000; 275: 34017-34020Google Scholar). Thus it is tempting to speculate that AKAP79 anchoring and inhibition of PP2B might function in a concerted manner to modulate not only AMPA receptor currents but also to attenuate the phosphorylation events that control receptor endocytosis. We have mapped a region in the neuronally expressed anchoring protein AKAP79 that is necessary for binding and inhibiting the calcium/calmodulin-dependant phosphatase PP2B/calcineurin. A combination of biochemical and enzymatic approaches has identified multiple binding and inhibitory determinants that are located between residues 318 and 357 of the anchoring protein. Our characterization of this PP2B-binding region is the latest in a series of studies that have dissected functional regions within AKAP79 that are responsible for the interaction with distinct signaling enzymes. Residues 391–408 form an amphipathic helix that binds to the R subunit dimer of the PKA holoenzyme with nanomolar affinity (7Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Google Scholar, 12Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 24: 16816-16823Google Scholar, 19Hausken Z.E. Dell'Acqua M.L. Coghlan V.M. Scott J.D. J. Biol. Chem. 1996; 271: 29016-29022Google Scholar). Peptides spanning this region have been used by many investigators to disrupt a variety of cAMP-responsive events by uncoupling PKA location inside cells (20Rosenmund C. Carr D.W. Bergeson S.E. Nilaver G. Scott J.D. Westbrook G.L. Nature. 1994; 368: 853-856Google Scholar, 21Lester L.B. Langeberg L.K. Scott J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14942-14947Google Scholar, 22Fink M.A. Zakhary D.R. Mackey J.A. Desnoyer R.W. Apperson-Hansen C. Damron D.S. Bond M. Circ. Res. 2001; 88: 291-297Google Scholar, 23Feliciello A. Li Y. Avvedimento E.V. Gottesman M.E. Rubin C.S. Curr. Biol. 1997; 7: 1011-1014Google Scholar, 24Sun F. Hug M.J. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 14360-14366Google Scholar, 25Dodge K.L. Carr D.W. Sanborn B.M. Endocrinology. 1999; 140: 5165-5170Google Scholar). A PKC-binding site is located between residues 31 and 52 (14). Peptides spanning this region bind to the kinase with micromolar affinities and inhibit the bound enzyme. This effect is reversed by calcium/calmodulin, which competes for interaction with the 31–52 region and releases the active kinase from the signaling complex at the postsynaptic densities of neurons (26Faux M.C. Scott J.D. J. Biol. Chem. 1997; 272: 17038-17044Google Scholar, 27Faux M.C. Rollins E.N. Edwards A.S. Langeberg L.K. Newton A.C. Scott J.D. Biochem. J. 1999; 343: 443-452Google Scholar). In this report, we demonstrate that a family of peptides synthesized to regions of the anchoring protein between residues 318 and 357 disrupt the subcellular location of the phosphatase and inhibit PP2B-dependent phosphorylation events. Another region of AKAP79 within the membrane targeting domain (residues 88–102) may also participate in PP2B anchoring (13Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-112Google Scholar, 16Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Google Scholar, 17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar), although the inhibitory potency of peptides to this site is ∼50-fold less than the AKAP79-(330–357) peptide, and they cannot displace the phosphatase from its sites of anchoring. Therefore, we propose that the region between residues 318 and 357 is likely to contain most of the high affinity determinants for PP2B anchoring. This postulate is consistent with recent reports (17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar, 28Taigen T. De Windt L.J. Lim H.W. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1196-1201Google Scholar, 29De Windt L.J. Lim H.W. Bueno O.F. Liang Q. Delling U. Braz J.C. Glascock B.J. Kimball T.F. del Monte F. Hajjar R.J. Molkentin J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3322-3327Google Scholar) that recombinant fragments of AKAP79 that include this region inhibit PP2B signaling events in transfected cells and transgenic mice. Interestingly, the AKAP79-(330–357) and AKAP79-(338–357) peptides are slightly more potent inhibitors of the phosphatase than the full-length anchoring protein. Similarly we have observed that protein fragments of AKAP79 sometimes work more efficiently than the full-length protein. For example, residues 108–427 inhibit PP2B more effectively than the full-length protein (16Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Google Scholar). Likewise, the first 75 amino acids of the anchoring protein is a more potent inhibitor of PKC activity than the full-length protein (14Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Google Scholar). The AKAP79-(318–357) sequence exhibits some similarity to the binding sites of other PP2B-interacting proteins. For example, the organization of serines, threonines, and the clustering of basic amino acids in this region is reminiscent of the 38-residue PP2B-binding region in Cain, (calcineurin inhibitor)/cabin (30Lai M.M. Burnett P.E. Wolosker H. Blackshaw S. Snyder S.H. J. Biol. Chem. 1998; 273: 18325-18331Google Scholar, 31Luo S. Youn H.-D. Loh C. Stolow M. He W. Liu J.O. Immunity. 1998; 8: 703-711Google Scholar). Both proteins are non-competitive inhibitors of PP2B activity that inhibit the phosphatase in the low micromolar range and bind PP2B at a site distinct from the immunosuppressant drugs FK506 and cyclosporin A (13Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-112Google Scholar,17Kashishian A. Howard M. Loh C. Gallatin W.M. Hoekstra M.F. Lai Y. J. Biol. Chem. 1998; 273: 27412-27419Google Scholar, 32Lai M.M. Luo H.R. Burnett P.E. Hong J.J. Snyder S.H. J. Biol. Chem. 2000; 275: 34017-34020Google Scholar). Another common characteristic is the presence of a loosely conserved “PIXIXIT” motif (where Xrepresents any amino acid). This sequence was first identified in the transcription factor NFATc and has been found in the docking sites of other PP2B interacting partners including Cain/cabin and the muscle-specific protein MCIP1,2 (33Aramburu J. Garcia-Cozar F. Raghavan A. Okamura H. Rao A. Hogan P.G. Mol. Cell. 1998; 1: 627-637Google Scholar, 34Aramburu J. Yaffe M.B. Lopez-Rodriguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Google Scholar, 35Rothermel B. Vega R.B. Yang J. Wu H. Bassel-Duby R. Williams R.S. J. Biol. Chem. 2000; 275: 8719-8725Google Scholar). Residues 337–343 of AKAP79 ( PIA II IT) form the PIXIXIT motif, and it is totally conserved in the murine and bovine orthologs, AKAP150 and AKAP75 (36Bregman D.B. Bhattacharyya N. Rubin C.S. J. Biol. Chem. 1989; 264: 4648-4656Google Scholar, 37Bregman D.B. Hirsch A.H. Rubin C.S. J. Biol. Chem. 1991; 266: 7207-7213Google Scholar, 38Hirsch A.H. Glantz S.B. Li Y. You Y. Rubin C.S. J. Biol. Chem. 1992; 267: 2131-2134Google Scholar). Although not formally proven, it is likely that this sequence represents a principle site of contact with the phosphatase, because the AKAP79-(330–357) peptide, which includes this region, inhibits phosphatase activity at micromolar concentrations and competes for binding with the intact anchoring protein. However, it is clearly evident that other regions of AKAP79 contribute to PP2B interactions as peptides upstream of the PIXIXIT motif also bind and inhibit the phosphatase. These findings are certainly consistent with the notion that multiple determinants located in a central segment of the AKAP79 linear sequence act in tandem to anchor PP2B and inhibit phosphatase activity inside cells. Apparently multisite contact between phosphatases and their binding partners is a common theme in the subcellular targeting of this enzyme class, as a second PP2B-binding site has been defined within the regulatory domain of NFATc which may act synergistically with the PIXIXIT motif to permit a high affinity interaction with the phosphatase (39Park S. Uesugi M. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7130-7135Google Scholar). Similarly, there are examples of type 1 phosphatase targeting subunits that interact with the catalytic subunit (PP1c) through multiple sites (40Allen P.B. Kwon Y.G. Nairn A.C. Greengard P. J. Biol. Chem. 1998; 273: 4089-4095Google Scholar, 41Schillace R.V. Voltz J.W. Sim A.T. Shenolikar S. Scott J.D. J. Biol. Chem. 2001; 276: 12128-12134Google Scholar). One goal of our study was to generate peptide-based antagonists of PP2B function inside cells. Accordingly, we have shown that perfusion of the AKAP79-(330–357) peptide prevents the attenuation of GluR1 type AMPA channels by the AKAP79 signaling complex in HEK293 cells. We have shown previously that AKAP79-mediated anchoring of PP2B confers a calcium-dependent rundown of the channel through the dephosphorylation of serine 845, a site in the cytoplasmic tail of the GluR1 subunit (10Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Google Scholar). Basal phosphorylation of Ser-845 is maintained by anchored PKA, suggesting that AKAP79 contributes to the regulation of this channel by positioning kinases and phosphates in close proximity to a subset of their substrates (10Tavalin S.J. Colledge M. Hell J.W. Langeberg L.K. Huganir R.L. Scott J.D. J. Neurosci. 2002; 22: 3044-3051Google Scholar, 11Fraser I.D. Scott J.D. Neuron. 1999; 23: 423-426Google Scholar, 15Colledge M. Dean R.A. Scott G.K. Langeberg L.K. Huganir R.L. Scott J.D. Neuron. 2000; 27: 107-119Google Scholar, 42Swope S.L. Moss S.I. Raymond L.A. Huganir R.L. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 49-78Google Scholar, 43Kameyama K. Lee H.K. Bear M.F. Huganir R.L. Neuron. 1998; 21: 1163-1175Google Scholar, 44Lee H.K. Kameyama K. Huganir R.L. Bear M.F. Neuron. 1998; 21: 1151-1162Google Scholar, 45Lee H.K. Barbarosie M. Kameyama K. Bear M.F. Huganir R.L. Nature. 2000; 405: 955-959Google Scholar). Our electrophysiological measurements infer that perfusion of the AKAP79-(330–357) peptide shifts the equilibrium in favor of the phosphorylated state of GluR1. Importantly, our control experiments demonstrate that this peptide is only active in the presence of AKAP79, thereby indicating that PP2B targeting ensures efficient modulation of GluR1 currents. Yet our experiments are unable to delineate the mechanism of action of this peptide. Our biochemical analyses would suggest that it must function by displacing PP2B from its site of action and/or as an inhibitor of the anchored phosphatase. Nonetheless, our experiments highlight the utility of this reagent as a cell-based modulator of PP2B. Likewise, Cain has also been used to perturb PP2B function inside cells, as it binds the endocytic machinery through amphiphysin 1 to negatively regulate PP2B/dynamin-dependent synaptic vesicle endocytosis (30Lai M.M. Burnett P.E. Wolosker H. Blackshaw S. Snyder S.H. J. Biol. Chem. 1998; 273: 18325-18331Google Scholar, 32Lai M.M. Luo H.R. Burnett P.E. Hong J.J. Snyder S.H. J. Biol. Chem. 2000; 275: 34017-34020Google Scholar). Thus it is tempting to speculate that AKAP79 anchoring and inhibition of PP2B might function in a concerted manner to modulate not only AMPA receptor currents but also to attenuate the phosphorylation events that control receptor endocytosis. We thank Brian Nauert for pCDNA3 PP2B vectors; Iain Fraser for pCDNA AKAP18-GFP; Yvonne Lai (ICOS Corp.) for anti-AKAP79 rabbit polyclonal antisera (918I); and Lisa L. Gomez and Shuvo Alam for technical assistance." @default.
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W2076018512 title "Mapping the Protein Phosphatase-2B Anchoring Site on AKAP79" @default.
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