Targeting of Protein Kinase A by Muscle A Kinase-anchoring Protein (mAKAP) Regulates Phosphorylation and Function of the Skeletal Muscle Ryanodine Receptor

Protein kinase A anchoring proteins (AKAPs) tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The muscle AKAP, mAKAP, co-localizes with the sarcoplasmic reticulum Ca2+ release channel or ryanodine receptor (RyR). The purpose of this study was to determine whether anchoring of PKA by mAKAP regulates RyR function. Either mAKAP or mAKAP-P, which is unable to anchor PKA, was expressed in CHO cells stably expressing the skeletal muscle isoform of RyR (CHO-RyR1). Immunoelectron microscopy showed that mAKAP co-localized with RyR1 in disrupted skeletal muscle. Following the addition of 10 μm forskolin to activate adenylyl cyclase, RyR1 phosphorylation in CHO-RyR1 cells expressing mAKAP increased by 42.4 ± 6.6% (n = 4) compared with cells expressing mAKAP-P. Forskolin treatment alone did not increase the amplitude of the cytosolic Ca2+ transient in CHO-RyR1 cells expressing mAKAP or mAKAP-P; however, forskolin plus 10 mm caffeine elicited a cytosolic Ca2+ transient, the amplitude of which increased by 22% (p < 0.05) in RyR1/mAKAP-expressing cells compared with RyR1/mAKAP-P-expressing cells. Therefore, localization of PKA by mAKAP at RyR1 increases both PKA-dependent RyR phosphorylation as well as efflux of Ca2+ through the RyR. Therefore, RyR1 function is regulated by mAKAP targeting of PKA, implying an important functional role for PKA phosphorylation of RyR in skeletal muscle. Protein kinase A anchoring proteins (AKAPs) tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The muscle AKAP, mAKAP, co-localizes with the sarcoplasmic reticulum Ca2+ release channel or ryanodine receptor (RyR). The purpose of this study was to determine whether anchoring of PKA by mAKAP regulates RyR function. Either mAKAP or mAKAP-P, which is unable to anchor PKA, was expressed in CHO cells stably expressing the skeletal muscle isoform of RyR (CHO-RyR1). Immunoelectron microscopy showed that mAKAP co-localized with RyR1 in disrupted skeletal muscle. Following the addition of 10 μm forskolin to activate adenylyl cyclase, RyR1 phosphorylation in CHO-RyR1 cells expressing mAKAP increased by 42.4 ± 6.6% (n = 4) compared with cells expressing mAKAP-P. Forskolin treatment alone did not increase the amplitude of the cytosolic Ca2+ transient in CHO-RyR1 cells expressing mAKAP or mAKAP-P; however, forskolin plus 10 mm caffeine elicited a cytosolic Ca2+ transient, the amplitude of which increased by 22% (p < 0.05) in RyR1/mAKAP-expressing cells compared with RyR1/mAKAP-P-expressing cells. Therefore, localization of PKA by mAKAP at RyR1 increases both PKA-dependent RyR phosphorylation as well as efflux of Ca2+ through the RyR. Therefore, RyR1 function is regulated by mAKAP targeting of PKA, implying an important functional role for PKA phosphorylation of RyR in skeletal muscle. Cyclic AMP-dependent protein kinase (PKA) 1The abbreviations used are: PKA, protein kinase A; AKAP, protein kinase A anchoring proteins; mAKAP, muscle AKAP; β2-AR, β2-adrenergic receptor; jSR, junctional sarcoplasmic reticulum; DHPR, dihydropyridine receptor (l-type voltage sensitive Ca2+ receptor); RyR1, ryanodine receptor type 1; PDE4D3, phosphodiesterase 4D3; AKAP15/18, A kinase-anchoring protein 15/18; RII, PKA regulatory subunit type II; C, PKA catalytic subunit; CHO, Chinese hamster ovary; TBS, Tris-buffered saline; ER, endoplasmic reticulum; ECFP, enhanced cyan fluorescent protein; EGFP, enhanced green fluorescent protein. 1The abbreviations used are: PKA, protein kinase A; AKAP, protein kinase A anchoring proteins; mAKAP, muscle AKAP; β2-AR, β2-adrenergic receptor; jSR, junctional sarcoplasmic reticulum; DHPR, dihydropyridine receptor (l-type voltage sensitive Ca2+ receptor); RyR1, ryanodine receptor type 1; PDE4D3, phosphodiesterase 4D3; AKAP15/18, A kinase-anchoring protein 15/18; RII, PKA regulatory subunit type II; C, PKA catalytic subunit; CHO, Chinese hamster ovary; TBS, Tris-buffered saline; ER, endoplasmic reticulum; ECFP, enhanced cyan fluorescent protein; EGFP, enhanced green fluorescent protein. has a wide range of substrates and elicits a variety of cellular responses. Protein kinase A anchoring proteins (AKAPs) modulate PKA-dependent phosphorylation by tethering this kinase to a specific subcellular location to regulate an otherwise ubiquitous signal and allow specific intracellular changes to occur. In some cases, these substrates may be bound to or are located near an AKAP. For example, the AKAP yotiao binds to its substrate, the N-methyl-d-aspartate receptor. Co-expression of these two proteins increases PKA-dependent potentiation of channel currents (1Westphal 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). Another AKAP, AKAP79, co-immunoprecipitates with the β2-adrenergic receptor (β2-AR) together with PKA, protein kinase C, and protein phosphatase 2B (calcineurin) (2Fraser I.D. Cong M. Kim J. Rollins E.N. Daaka Y. Lefkowitz R.J. Scott J.D. Curr. Biol. 2000; 10: 409-412Google Scholar). In HEK293 cells expressing AKAP79 and β2-AR, phosphorylation of the β2-AR was decreased when the interaction between AKAP79 and the receptor was disrupted (2Fraser I.D. Cong M. Kim J. Rollins E.N. Daaka Y. Lefkowitz R.J. Scott J.D. Curr. Biol. 2000; 10: 409-412Google Scholar). In other cases, AKAP anchoring of PKA can influence the phosphorylation state of substrates not in close proximity to the AKAP. For example, forskolin stimulation of HEK293 cells expressing AKAP75, which targets PKA to the cytoskeleton, resulted in a 5–10-fold higher phosphorylation of cAMP response element-binding protein compared with control cells (3Feliciello A. Li Y. Avvedimento E.V. Gottesman M.E. Rubin C.S. Curr. Biol. 1997; 7: 1011-1014Google Scholar). We previously demonstrated that upon isoproterenol stimulation, PKA-dependent phosphorylation of two myofibrillar proteins, troponin I and myosin-binding protein C, was significantly reduced in cardiac myocytes expressing Ht31 (4Fink M. Zakhary D. Mackey J. Desnoyer R. Hansen C. Damron D. Bond M. Circ. Res. 2001; 88: 291-297Google Scholar), a peptide that binds the regulatory subunit (RII) of type II PKA and prevents AKAP-PKA interactions (5Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Google Scholar). To date, no AKAP has been found to co-localize with the myofibrils in cardiac myocytes. Thus, the phosphorylation state of these two substrates may be regulated by an AKAP not in close proximity to troponin I or myosin-binding protein C. In some cases AKAPs may anchor PKA within a region of the cell subject to local elevated levels of cAMP (6Fraser I.D. Scott J.D. Neuron. 1999; 23: 423-426Google Scholar). Thus, upon the appropriate stimulus, local transient increases in cAMP concentration could activate anchored PKA (7Rich T.C. Fagan K.A. Tse T.E. Schaack J. Cooper D.M.F. Karpen J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13049-13054Google Scholar). Other groups have reported changes in cell function when the interaction between AKAP and PKA is disrupted. For example, PKA anchoring was required in order to maintain AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid) responsive glutamate receptor currents in cultured hippocampal neurons (8Rosenmund C. Carr D.W. Bergeson S.E. Nilaver G. Scott J.D. Westbrook G.L. Nature. 1994; 368: 853-856Google Scholar). Both insulin secretion and increased intracellular Ca2+ are inhibited in pancreatic islet cells following treatment with Ht31, and cell-permeant forms of Ht31 inhibit PKA-dependent sperm motility (9Lester L.B. Langeberg L.K. Scott J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14942-14947Google Scholar, 10Vijayarghavan S. Goueli S.A. Davey M.P. Carr D.W. Biol. Chem. 1997; 272: 4747-4752Google Scholar). In cardiac myocytes, Ht31 reduces isoproterenol-dependent potentiation of Ca2+ current through the l-type Ca2+ channel. Disruption of the interaction between AKAP79 and the β2-AR attenuates downstream activation of the mitogen-activated protein kinase pathway (2Fraser I.D. Cong M. Kim J. Rollins E.N. Daaka Y. Lefkowitz R.J. Scott J.D. Curr. Biol. 2000; 10: 409-412Google Scholar, 11Gao T. Yatani A. Dell'Acqua M.L. Sako H. Green S.A. Dascal N. Scott J.D. Hosey M.M. Neuron. 1997; 19: 185-196Google Scholar). In both cardiac and skeletal muscle, mAKAP (formerly AKAP100) is highly expressed. In cardiac muscle, this AKAP has been immunolocalized to the perinuclear membrane and to the junctional sarcoplasmic reticulum (jSR) (12McCartney S. Little B.M. Langeberg L.K. Scott J.D. Biol. Chem. 1995; 270: 9327-9333Google Scholar, 13Yang J. Drazba J. Ferguson D. Bond M. J. Cell Biol. 1998; 142: 511-522Google Scholar, 14Kapiloff M. Schilace R.V. Westphal A. Scott J. J. Cell Sci. 1999; 112: 2725-2736Google Scholar). In both skeletal and cardiac muscle, there are several PKA substrates in this highly specialized region of the jSR/transverse tubule, notably the dihydropyridine receptor (DHPR) or l-type voltage-sensitive Ca2+ channel and the ryanodine-sensitive Ca2+ release channel (RyR). Jones et al. (15Jones L.R. Maddock S.W. Hathaway D.R. Biochim. Biophys. Acta. 1981; 641: 242-253Google Scholar) found no significant PKA II activity associated with highly purified cardiac junctional SR membrane preparation, whereas Marx et al. (16Marx S. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A. Cell. 2000; 101: 365-376Google Scholar) showed that in cardiac muscle mAKAP co-immunoprecipitates with the cardiac-specific isoform of the RyR (RyR2). These investigators also recently showed that PKA and mAKAP co-immunoprecipitated with the skeletal-specific type I isoform of the RyR (RyR1) (17Reiken S. Lacampagne A. Zhou H. Kherani A. Lehnart S.E. Ward C. Huang F. Gaburjakova M. Gaburjakova J. Yang Y.M. Rosemblit N. Warren M.S. He K. Yi G. Wang J. Burkhoff D. Vassort G. Marks A.R. J. Cell Biol. 2003; 160: 919-928Google Scholar). It is well established that PKA can regulate RyR2 function. Activation of β-adrenergic receptors results in a time-dependent increase in RyR2 phosphorylation (18Yoshida A. Takahashi M. Imagawa T. Shigekawa M. Takisawa H. Nakamura T. J. Biochem. 1992; 111: 186-190Google Scholar). PKA-dependent phosphorylation of single RyR2 channels increases mean open probability, open frequency, and open time (19Uehara A. Yasukochi M. Mejia-Alvarez R. Fill M. Imanaga I. Pfluegers Arch. Eur. J. Physiol. 2002; 444: 202-212Google Scholar). In pancreatic β-cells, the activation of PKA by forskolin increases the caffeine-dependent Ca2+ transient through RyR2 (20Islam S. Leibiger I. Leibiger B. Rossi D. Sorrentino V. Ekstrom T. Westerblad H. Andrade F. Berggren P.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6145-6150Google Scholar). In general, PKA-dependent phosphorylation of RyR2 increases Ca2+ efflux from the SR into the cytosol. However, little information is currently available the of PKA phosphorylation of of the channel can regulate its function Scholar, J. S. M. S. H. Biophys. J. 1994; Scholar). is by that phosphatase co-localizes with RyR1 in a skeletal muscle cell implying a role for of this channel D.W. A. Kim J. Biophys. J. 2002; Scholar). by also Ca2+ implying a functional role for RyR1 phosphorylation D.W. A. Kim J. Biophys. J. 2002; Scholar). mAKAP co-immunoprecipitates with RyR2 (16Marx S. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A. Cell. 2000; 101: 365-376Google Scholar) and RyR1 (17Reiken S. Lacampagne A. Zhou H. Kherani A. Lehnart S.E. Ward C. Huang F. Gaburjakova M. Gaburjakova J. Yang Y.M. Rosemblit N. Warren M.S. He K. Yi G. Wang J. Burkhoff D. Vassort G. Marks A.R. J. Cell Biol. 2003; 160: 919-928Google Scholar), a functional of mAKAP targeting of PKA at the RyR RyR function has not been CHO cells stably expressing RyR1 M. J. S. H. J. Biophys. J. 1997; Scholar), have the of whether mAKAP anchoring of PKA PKA-dependent RyR phosphorylation in RyR function. of for the of mAKAP or of mAKAP is by et al. M. Schilace R.V. Westphal A. Scott J. J. Cell Sci. 1999; 112: 2725-2736Google Scholar). mAKAP or a of mAKAP, which not PKA, was into to green fluorescent proteins M. Schilace R.V. Westphal A. Scott J. J. Cell Sci. 1999; 112: 2725-2736Google Scholar). The of a for an the of the of mAKAP The for was from or and and with from of to or and of cells stably expressing M. J. S. H. J. Biophys. J. 1997; Scholar, J. H. J. Biophys. J. 1997; Scholar, J. H. J. J. Physiol. 1997; Scholar) and with or to the cell is used as a for RyR function M. J. S. H. J. Biophys. J. 1997; Scholar, J. H. J. Biophys. J. 1997; Scholar, J. H. J. J. Physiol. 1997; Scholar, M. J. H. J. Biophys. J. 1999; Scholar). We to the to or cells and to cell expressing of these However, these cells showed a Ca2+ response in the of Therefore, cells with or between and and these cells used For or be to as mAKAP, mAKAP-P, or RyR1, The addition of a green fluorescent protein not the function or targeting of RyR1 M. J. S. H. J. Biophys. J. 1997; Scholar, N.M. M. N. Arch. Biochem. Biophys. 2001; Scholar) or mAKAP M. Schilace R.V. Westphal A. Scott J. J. Cell Sci. 1999; 112: 2725-2736Google Scholar, M.L. Zakhary D.R. Damron Bond M. J. Biol. Chem. 1999; Scholar). of mAKAP by Immunoelectron The muscle from an was disrupted with a single through a of the changes by of the in a by of the in the appropriate for in mm and 10 with mAKAP The was with as and the with in the that to and with in with the To with and with 10 mm for microscopy and with and and in a and of RyR in CHO was used to that CHO cells expressed the appropriate proteins and also to whether the phosphorylation state of RyR is in the or of mAKAP or following the activation of to in a PKA activity in the CHO cells was by cells for to 10 μm an of adenylyl cells by with protein a and phosphatase from the cell to and with or To was to of the with for and with in in Tris-buffered with for in and in and with in in for at as and to of the was demonstrated by with phosphatase not RyR was from CHO cell and the was by to and with A was at RyR. the to the RyR at a higher and was from of the other the in this was cell To that this was the a of purified RyR1 by of as a at the as the in and showed a time-dependent increase in phosphorylation levels when the RyR1 was with the catalytic subunit of PKA not The of phosphorylation was by of the of the as the RyR. that this was RyR was its in the from CHO cells. To control for in protein the of the was by the of the protein concentration was at for the of cells expressing RyR at with μm at following with mAKAP or mAKAP-P. a with the the cells at in mm mm mm 10 mm mm and mm at of and and at cells an as previously (4Fink M. Zakhary D. Mackey J. Desnoyer R. Hansen C. Damron D. Bond M. Circ. Res. 2001; 88: 291-297Google Scholar). at a of and with a as previously H. Damron S.J. D.R. Circ. Res. 1997; Scholar). intracellular Ca2+ levels as the of in cells in for with 10 μm forskolin in to activate adenylyl to 10 mm caffeine in the of in amplitude of the intracellular Ca2+ transient for cell by the of Ca2+ of from of following caffeine of RyR1 and mAKAP at the PKA to within a It is that upon activation of the PKA PKA, which is to an AKAP in the of local increases in be compared with PKA not anchored at these AKAPs may also function by PKA to a PKA Thus, that PKA is anchoring of PKA to its regulate the of phosphorylation of that Both PKA and mAKAP with RyR2 (16Marx S. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A. Cell. 2000; 101: 365-376Google Scholar) and RyR1 (17Reiken S. Lacampagne A. Zhou H. Kherani A. Lehnart S.E. Ward C. Huang F. Gaburjakova M. Gaburjakova J. Yang Y.M. Rosemblit N. Warren M.S. He K. Yi G. Wang J. Burkhoff D. Vassort G. Marks A.R. J. Cell Biol. 2003; 160: 919-928Google Scholar). Thus, at the whether mAKAP is co-localized with RyR in skeletal muscle. mAKAP is to two subcellular It is in the perinuclear region in myocytes and cardiac J. Drazba J. Ferguson D. Bond M. J. Cell Biol. 1998; 142: 511-522Google Scholar, 14Kapiloff M. Schilace R.V. Westphal A. Scott J. J. Cell Sci. 1999; 112: 2725-2736Google Scholar). by previously showed that mAKAP is found near the jSR/transverse in cardiac muscle cells J. Drazba J. Ferguson D. Bond M. J. Cell Biol. 1998; 142: 511-522Google Scholar), an in RyR. To mAKAP localization at higher and to determine whether mAKAP is located to the used microscopy to determine the location of mAKAP and RyR in disrupted skeletal muscle. mAKAP was located in close proximity to the which has been to be the RyR C. J. Cell Biol. Scholar). Thus, not mAKAP RyR in in also co-localizes with the RyR at the in cardiac To the of mAKAP the phosphorylation state of the RyR and its RyR to study these proteins in a cell CHO cells with RyR1 (CHO-RyR1). RyR1 is to the endoplasmic reticulum and Ca2+ release from the ER, the of Ca2+ release from the SR of muscle cells M. J. S. H. J. Biophys. J. 1997; Scholar, J. J. Biol. Chem. 2002; Scholar). that mAKAP and RyR expressed in CHO cells CHO cells did not mAKAP or RyR1 mAKAP PKA-dependent RyR1 determine the of of mAKAP the phosphorylation state of CHO-RyR1 cells with mAKAP or mAKAP-P. We that upon stimulation of PKA, the of RyR phosphorylation be in cells expressing mAKAP not PKA near the RyR. PKA stimulation compared with cells expressing mAKAP-P, RyR phosphorylation was increased by 42.4 ± 6.6% ± (n = 4) in cells expressing mAKAP (p < 0.05) The is for could be used to PKA-dependent phosphorylation of the the phosphorylation for PKA the RyR is at J. I. B. M. G. Biochim. Biophys. Acta. Scholar). protein kinase has also been reported to RyR this J. I. B. M. G. Biochim. Biophys. Acta. however, in the addition of the RyR1 phosphorylation in CHO-RyR1 cells expressing mAKAP that with forskolin In the activation of the adenylyl pathway by forskolin did not increase intracellular Ca2+ forskolin treatment not in activation of Thus, can that the increase in RyR1 phosphorylation is the of activation of of mAKAP PKA-dependent by RyR1 from to that RyR1 phosphorylation is in CHO-RyR1 cells expressing mAKAP as compared with CHO-RyR1 cells expressing mAKAP-P, to determine whether of mAKAP Ca2+ release from the PKA-dependent phosphorylation has previously been to increase Ca2+ efflux from the SR RyR2 (20Islam S. Leibiger I. Leibiger B. Rossi D. Sorrentino V. Ekstrom T. Westerblad H. Andrade F. Berggren P.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6145-6150Google Scholar). For example, in pancreatic β-cells, the caffeine-dependent Ca2+ transient of Ca2+ release from the is significantly increased in the of forskolin (20Islam S. Leibiger I. Leibiger B. Rossi D. Sorrentino V. Ekstrom T. Westerblad H. Andrade F. Berggren P.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6145-6150Google Scholar). was no significant in cytosolic Ca2+ when CHO cells with 10 μm forskolin no forskolin as in the However, treatment with 10 mm caffeine in the of 10 μm forskolin significantly increased the amplitude of the intracellular Ca2+ transient in CHO-RyR1 cells expressing mAKAP compared with cells expressing The results of this study that by anchoring PKA in close to RyR1, mAKAP regulates RyR function. PKA is to RyR1 (17Reiken S. Lacampagne A. Zhou H. Kherani A. Lehnart S.E. Ward C. Huang F. Gaburjakova M. Gaburjakova J. Yang Y.M. Rosemblit N. Warren M.S. He K. Yi G. Wang J. Burkhoff D. Vassort G. Marks A.R. J. Cell Biol. 2003; 160: 919-928Google Scholar), and mAKAP as the anchoring protein to this kinase to RyR1 (17Reiken S. Lacampagne A. Zhou H. Kherani A. Lehnart S.E. Ward C. Huang F. Gaburjakova M. Gaburjakova J. Yang Y.M. Rosemblit N. Warren M.S. He K. Yi G. Wang J. Burkhoff D. Vassort G. Marks A.R. J. Cell Biol. 2003; 160: 919-928Google Scholar). study is the to that mAKAP a role in PKA-dependent changes in RyR1 phosphorylation and Ca2+ efflux from an important regulatory role of mAKAP in RyR1 function. These are with that of the interaction by the addition or of a peptide PKA-dependent phosphorylation results in in PKA-dependent cellular function (2Fraser I.D. Cong M. Kim J. Rollins E.N. Daaka Y. Lefkowitz R.J. Scott J.D. Curr. Biol. 2000; 10: 409-412Google Scholar, M. Zakhary D. Mackey J. Desnoyer R. Hansen C. Damron D. Bond M. Circ. Res. 2001; 88: 291-297Google Scholar, L.B. Langeberg L.K. Scott J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14942-14947Google Scholar, 10Vijayarghavan S. Goueli S.A. Davey M.P. Carr D.W. Biol. Chem. 1997; 272: 4747-4752Google Scholar). these results to the role of PKA-dependent RyR phosphorylation in muscle, that in response to activation of adenylyl cyclase, increases in cAMP near the increased RyR phosphorylation as a Ca2+ efflux through this channel. In cardiac muscle, PKA-dependent activation of RyR2 could be as a of of cAMP by PDE4D3, the PKA-dependent response S. M.S. R. E.V. Langeberg L.K. Scott J.D. J. 2001; Scholar). may also be the in skeletal muscle, to be It is also that phosphorylation of RyR is also by protein phosphatase in both skeletal and cardiac and by protein phosphatase in cardiac muscle, these are to RyR by specific targeting proteins (17Reiken S. Lacampagne A. Zhou H. Kherani A. Lehnart S.E. Ward C. Huang F. Gaburjakova M. Gaburjakova J. Yang Y.M. Rosemblit N. Warren M.S. He K. Yi G. Wang J. Burkhoff D. Vassort G. Marks A.R. J. Cell Biol. 2003; 160: 919-928Google Scholar). 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J. 1994; Scholar), Ca2+ efflux through the channel. of RyR1 may be for channel in response to activation by the Y. J. Physiol. 1997; Scholar) as well as for Ca2+ release from the SR Scholar). Thus, several groups have that phosphorylation of RyR1 regulates its function by Ca2+ efflux through this receptor. proteins two AKAPs have been in the between the and membrane The of the of the skeletal located at the has also been to to a region of the RyR1 G. J. Biol. Chem. 1994; Scholar, G. J. Biol. Chem. 1995; 270: Scholar, T. T. S. Nature. Scholar). It has been that there is a between the and RyR1 that the activation of RyR1 in skeletal muscle T. H. A. V. Takahashi H. K. M. H. T. S. Nature. Scholar, C.S. Green D. D.R. Biophys. Biol. 2002; Scholar). also has been that the is associated with AKAP, J. Biol. Chem. 1997; 272: Scholar, I.D. Tavalin S.J. L.B. Langeberg L.K. Westphal Scott J.D. J. 1998; Scholar, R.E. T. Neuron. 1998; Scholar). that from these is two AKAPs are located in this between the and membrane and whether PKA to the could the RyR or showed that mAKAP has a for and that this for the phosphorylation state of D.R. M.L. Bond M. J. Biol. Chem. 2000; Scholar). may be to mAKAP or its phosphorylation a to modulate PKA-dependent signal It is not whether PKA to the could the RyR or whether PKA to RyR could the that the catalytic subunit can through the and substrates that are not in the of an AKAP (3Feliciello A. Li Y. Avvedimento E.V. Gottesman M.E. Rubin C.S. Curr. Biol. 1997; 7: 1011-1014Google Scholar), the between and the PKA phosphorylation RyR or mAKAP and the PKA phosphorylation are not The RyR is a protein with of its receptor may the of the from PKA to mAKAP to the from this receptor the of the several other proteins are bound to the RyR and also may the of the to In the has been to as a may be significantly from in the and may be Science. Scholar, M.E. J. Physiol. 1995; Scholar, G. J. Physiol. 1997; Scholar, Biophys. J. 2000; Scholar). to or mAKAP, other proteins have been found to be to both the and the RyR in skeletal muscle. For example, in skeletal muscle, is associated with R. A. J. Res. Cell 2002; Scholar) as well as with RyR1 D.W. A. Kim J. Biophys. J. 2002; Scholar). phosphatase could regulate the phosphorylation state of both the and was of the protein to with RyR1 and RyR2 G. Circ. Res. Scholar), and may activate or inhibit the channel the concentration of Ca2+ G. Circ. Res. Scholar, A. J. Biol. Chem. 2002; Scholar, S. 2002; 7: Scholar). also binds the of the and a role in the function of this receptor S. 2002; 7: Scholar). Thus, the tethering of specific proteins AKAPs to both the and the RyR may regulate in this highly specialized In current study the role of mAKAP targeting of PKA in PKA-dependent Ca2+ results that mAKAP co-localizes with RyR1 in skeletal muscle. We that anchoring of PKA increases the phosphorylation of RyR1 in the of elevated levels of when mAKAP PKA to RyR1, the activation of PKA regulates the function of the RyR by Ca2+ efflux through this receptor. Therefore, Ca2+ efflux through the RyR regulates the of muscle that of this Ca2+ channel may regulate not cardiac muscle as is well may also a role in skeletal muscle