In neonatal rat cardiomyocytes, the phosphomimic RIIS96D increased AKAP15/18 pull-down and significantly increased phosphorylation of phospholamban and ryanodine receptor compared to RIIS96A.
Subcellular compartmentalization of the cAMP-dependent protein kinase (PKA) by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how PKA targeting to AKAPs is regulated in the intact cell. PKA binds to an amphipathic helical region of AKAPs via an N-terminal domain of the regulatory subunit. In vitro studies showed that autophosphorylation of type II regulatory subunit (RII) can alter its affinity for AKAPs and the catalytic subunit (PKAcat). We now investigate whether phosphorylation of serine 96 on RII regulates PKA targeting to AKAPs, downstream substrate phosphorylation and calcium cycling in primary cultured cardiomyocytes. We demonstrated that, whereas there is basal phosphorylation of RII subunits, persistent maximal activation of PKA results in a phosphatase-dependent loss of RII phosphorylation. To investigate the functional effects of RII phosphorylation, we constructed adenoviral vectors incorporating mutants which mimic phosphorylated (RIIS96D), nonphosphorylated (RIIS96A) RII, or wild-type (WT) RII and performed adenoviral infection of neonatal rat cardiomyocytes. Coimmunoprecipitation showed that more AKAP15/18 was pulled down by the phosphomimic, RIIS96D, than RIIS96A. Phosphorylation of phospholamban and ryanodine receptor was significantly increased in cells expressing RIIS96D versus RIIS96A. Expression of recombinant RII constructs showed significant effects on cytosolic calcium transients. We propose a model illustrating a central role of RII phosphorylation in the regulation of local PKA activity. We conclude that RII phosphorylation regulates PKA-dependent substrate phosphorylation and may have significant implications for modulation of cardiac function. Subcellular compartmentalization of the cAMP-dependent protein kinase (PKA) by protein kinase A-anchoring proteins (AKAPs) facilitates local protein phosphorylation. However, little is known about how PKA targeting to AKAPs is regulated in the intact cell. PKA binds to an amphipathic helical region of AKAPs via an N-terminal domain of the regulatory subunit. In vitro studies showed that autophosphorylation of type II regulatory subunit (RII) can alter its affinity for AKAPs and the catalytic subunit (PKAcat). We now investigate whether phosphorylation of serine 96 on RII regulates PKA targeting to AKAPs, downstream substrate phosphorylation and calcium cycling in primary cultured cardiomyocytes. We demonstrated that, whereas there is basal phosphorylation of RII subunits, persistent maximal activation of PKA results in a phosphatase-dependent loss of RII phosphorylation. To investigate the functional effects of RII phosphorylation, we constructed adenoviral vectors incorporating mutants which mimic phosphorylated (RIIS96D), nonphosphorylated (RIIS96A) RII, or wild-type (WT) RII and performed adenoviral infection of neonatal rat cardiomyocytes. Coimmunoprecipitation showed that more AKAP15/18 was pulled down by the phosphomimic, RIIS96D, than RIIS96A. Phosphorylation of phospholamban and ryanodine receptor was significantly increased in cells expressing RIIS96D versus RIIS96A. Expression of recombinant RII constructs showed significant effects on cytosolic calcium transients. We propose a model illustrating a central role of RII phosphorylation in the regulation of local PKA activity. We conclude that RII phosphorylation regulates PKA-dependent substrate phosphorylation and may have significant implications for modulation of cardiac function. The cAMP-dependent protein kinase (PKA) 2The abbreviations used are:PKAcAMP-dependent protein kinaseAKAPA-kinase-anchoring proteinsRIIregulatory subunit of PKA type IIPKAcatcatalytic subunit of PKAIBMX3-isobutyl-1-methylxanthine8-Br-cAMP8-bromo-cAMP monophosphate sodiumNCMneonatal cardiomyocytesACMadult cardiomyocytesRyRryanodine receptorPLNphospholambanSERCAsarco-endoplasmic reticulum Ca2+ ATPaseCSQcalsequestrinNCXNa+/Ca2+ exchangerWTwild typePBSphosphate-buffered salineGFPgreen fluorescent proteinGAPDHglyceraldehyde-3-phosphate dehydrogenase. 2The abbreviations used are:PKAcAMP-dependent protein kinaseAKAPA-kinase-anchoring proteinsRIIregulatory subunit of PKA type IIPKAcatcatalytic subunit of PKAIBMX3-isobutyl-1-methylxanthine8-Br-cAMP8-bromo-cAMP monophosphate sodiumNCMneonatal cardiomyocytesACMadult cardiomyocytesRyRryanodine receptorPLNphospholambanSERCAsarco-endoplasmic reticulum Ca2+ ATPaseCSQcalsequestrinNCXNa+/Ca2+ exchangerWTwild typePBSphosphate-buffered salineGFPgreen fluorescent proteinGAPDHglyceraldehyde-3-phosphate dehydrogenase. is a serine/threonine kinase with a prominent role in the regulation of cardiac function. The inactive holoenzyme is composed of a regulatory subunit (R) dimer and two catalytic subunits (PKAcat). The active site of PKAcat reversibly associates with the inhibitory domain of the R subunit. Cooperative binding of cAMP molecules to the four nucleotide binding sites on the R subunit dimer causes dissociation of the PKAcat and, thus, activation of the enzyme (1Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (949) Google Scholar). However, dissociation of the R and PKAcat subunits may not always occur during activation (2Prinz A. Diskar M. Erlbruch A. Herberg F.W. Cell Signal. 2006; 18: 1616-1625Crossref PubMed Scopus (59) Google Scholar, 3Yang S. Fletcher W.H. Johnson D.A. Biochemistry. 1995; 34: 6267-6271Crossref PubMed Scopus (52) Google Scholar). It is widely known that perturbations of PKA activity, including altered PKA phosphorylation of ryanodine receptor, phospholamban, myosin-binding protein C, and troponin I, occur in the failing heart and may contribute to impaired contractile function (4Wehrens X.H. Marks A.R. Ann. Med. 2004; 36: 70-80Crossref PubMed Scopus (24) Google Scholar, 5Sipido K.R. Eisner D. Cardiovasc. Res. 2005; 68: 167-174Crossref PubMed Scopus (33) Google Scholar, 6Hasenfuss G. Cardiovasc. Res. 1998; 37: 279-289Crossref PubMed Google Scholar, 7Bodor G.S. Oakeley A.E. Allen P.D. Crimmins D.L. Ladenson J.H. Anderson P.A. Circulation. 1997; 96: 1495-1500Crossref PubMed Scopus (190) Google Scholar, 8Waggoner J.R. Kranias E.G. Heart Fail. Clin. 2005; 1: 207-218Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Decreased phosphorylation of RII is observed during heart failure; however, the mechanisms involved are poorly understood (9Zakhary D.R. Moravec C.S. Bond M. Circulation. 2000; 101: 1459-1464Crossref PubMed Scopus (69) Google Scholar). This study will focus on the functional significance of alterations in the phosphorylation state of the RII subunit. cAMP-dependent protein kinase A-kinase-anchoring proteins regulatory subunit of PKA type II catalytic subunit of PKA 3-isobutyl-1-methylxanthine 8-bromo-cAMP monophosphate sodium neonatal cardiomyocytes adult cardiomyocytes ryanodine receptor phospholamban sarco-endoplasmic reticulum Ca2+ ATPase calsequestrin Na+/Ca2+ exchanger wild type phosphate-buffered saline green fluorescent protein glyceraldehyde-3-phosphate dehydrogenase. cAMP-dependent protein kinase A-kinase-anchoring proteins regulatory subunit of PKA type II catalytic subunit of PKA 3-isobutyl-1-methylxanthine 8-bromo-cAMP monophosphate sodium neonatal cardiomyocytes adult cardiomyocytes ryanodine receptor phospholamban sarco-endoplasmic reticulum Ca2+ ATPase calsequestrin Na+/Ca2+ exchanger wild type phosphate-buffered saline green fluorescent protein glyceraldehyde-3-phosphate dehydrogenase. A-kinase-anchoring proteins (AKAPs) bind RII dimers and sequester inactive PKA holoenzyme near its substrates, facilitating phosphorylation of local PKA substrates. Sequence differences among AKAPs result in the assembly of a varied range of signaling molecules targeted to each AKAP (10Ruehr M.L. Russell M.A. Bond M. J. Mol. Cell Cardiol. 2004; 37: 653-665Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). For example, mAKAP, which targets PKA to the junctional sarcoplasmic reticulum, binds RII, protein phosphatase 2A, phosphodiesterase 4D3, and ryanodine receptor (11Kapiloff M.S. Jackson N. Airhart N. J. Cell Sci. 2001; 114: 3167-3176Crossref PubMed Google Scholar, 12Dodge-Kafka K.L. Soughayer J. Pare G.C. Carlisle Michel J.J. Langeberg L.K. Kapiloff M.S. Scott J.D. Nature. 2005; 437: 574-578Crossref PubMed Scopus (430) Google Scholar). AKAP15/18α localizes PKA to L-type Ca2+ channels. The δ isoform of AKAP15/18 was recently shown to target PKA to phospholamban (13Lygren B. Carlson C.R. Santamaria K. Lissandron V. McSorley T. Litzenberg J. Lorenz D. Wiesner B. Rosenthal W. Zaccolo M. Tasken K. Klussmann E. EMBO Rep. 2007; 8: 1061-1067Crossref PubMed Scopus (140) Google Scholar), and phosphodiesterase 3 associates with sarcoplasmic reticulum membranes (14Fischmeister R. Castro L.R. Abi-Gerges A. Rochais F. Jurevicius J. Leroy J. Vandecasteele G. Circ. Res. 2006; 99: 816-828Crossref PubMed Scopus (298) Google Scholar). Thus each AKAP and its associated signaling molecules constitute a unique multifunctional signaling complex. Ser-96 of RII is located within the RII inhibitory domain and interacts with the catalytic cleft of PKAcat (1Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (949) Google Scholar). PKAcat phosphorylates RII at Ser-96 (15Tasken K. Aandahl E.M. Physiol. Rev. 2004; 84: 137-167Crossref PubMed Scopus (610) Google Scholar). In vitro studies have shown that RII phosphorylation at Ser-96 not only regulates interaction with PKAcat (16Rangel-Aldao R. Kupiec J.W. Rosen O.M. J. Biol. Chem. 1979; 254: 2499-2508Abstract Full Text PDF PubMed Google Scholar, 17Granot J. Mildvan A.S. Hiyama K. Kondo H. Kaiser E.T. J. Biol. Chem. 1980; 255: 4569-4573Abstract Full Text PDF PubMed Google Scholar), but also affects interaction with AKAPs (18Zakhary D.R. Fink M.A. Ruehr M.L. Bond M. J. Biol. Chem. 2000; 275: 41389-41395Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Studies using PKA purified from bovine cardiac muscle showed that RII phosphorylation increases cAMP-induced dissociation of the PKA holoenzyme, leading to activation of the kinase (19Erlichman J. Rosenfeld R. Rosen O.M. J. Biol. Chem. 1974; 249: 5000-5003Abstract Full Text PDF PubMed Google Scholar). Our surface plasmon resonance spectroscopy studies showed that phosphorylation of the RII subunit alters the affinity of RII for peptides encompassing the RII binding domains of AKAP15/18, mAKAP, and AKAP-Lbc, with the greatest increase observed for AKAP15/18 (18Zakhary D.R. Fink M.A. Ruehr M.L. Bond M. J. Biol. Chem. 2000; 275: 41389-41395Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Because phosphorylation of RII can potentially affect both holoenzyme formation and subcellular localization via AKAPs, we whether the phosphorylation state of RII regulates PKA signaling in cardiomyocytes. We RII proteins that mimic phosphorylated and nonphosphorylated RII and the that RII phosphorylation holoenzyme and downstream substrate phosphorylation in intact phosphorylation of PKA affects Ca2+ that RII phosphorylation is during heart (9Zakhary D.R. Moravec C.S. Bond M. Circulation. 2000; 101: 1459-1464Crossref PubMed Scopus (69) Google Scholar), subcellular localization of PKA in of failing which may contribute to PKA protein phosphorylation and impaired in heart RII constructs and to The for RII was M.L. D.R. Bond M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To the or the in the RII the was by RII, and RIIS96D the The was by S. M. T. M.L. J. 1997; PubMed Google with by the at the of Cell and and adult cardiomyocytes A. M.S. J. Cardiovasc. PubMed Scopus Google Scholar, J. Cardiovasc. Res. PubMed Scopus Google Scholar). cultured in with bovine at cardiomyocytes at a of cells in a or cells in a infection was using of adenoviral in was to the cells The of recombinant proteins was rat cardiomyocytes on and cultured for in with with bovine with in The cells in in for and in in from and PKAcat from and for at or was used a for the cells on a using using a fluorescent with a and was using an with a for with and in with for an The with cells was on the of an and with with with for and with in and at at using to basal and The of the was by the of the using the PKA in and neonatal with or a of in with and with or with in for at PKA was by with or with in or for from cells and in the with for In with phosphatase was with the cells for and at the during the cardiomyocytes with or a of in The cells in with and proteins via cardiomyocytes in and from the in to of from and for on at for 3 the was and with of RII for at of and for at The in in and in a for of proteins was in using the by using or to for with of the was performed by using the The used the a from of and receptor and receptor, and calsequestrin exchanger or used The was used a in the to the are was using of the not to a the on was was of on RII phosphorylated of RII in vitro in purified cardiac and muscle It that RII in the phosphorylated in (16Rangel-Aldao R. Kupiec J.W. Rosen O.M. J. Biol. Chem. 1979; 254: 2499-2508Abstract Full Text PDF PubMed Google Scholar, D.R. Fink M.A. Ruehr M.L. Bond M. J. Biol. Chem. 2000; 275: 41389-41395Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). To investigate whether the of phosphorylation of RII in intact cardiomyocytes we the phosphorylation of RII at and in primary rat neonatal and adult cardiomyocytes. In neonatal phosphorylation of RII was observed with or significant in RII phosphorylation versus was observed with or in with the phosphodiesterase we observed loss of the phosphorylated RII with the cAMP results adult cardiomyocytes in the that are not to neonatal was of adult cardiomyocytes with or and however, the of cells with or and was in of adult cells with and was in the two In neonatal we observed a for at The was also to This may a regulated of RII not in adult The RII was to the of but of neonatal with not affect the of the of neonatal with the of and not affect the RII that RII is phosphorylated in cardiomyocytes The results are with in muscle J. Biol. Chem. Full Text PDF PubMed Google and that of of Ser-96 is from the of In of by of cAMP or the of may result in dissociation of RII and PKAcat and of the RII subunit by Expression and of RII in widely used to mimic phosphorylated or of a protein is of the to or A. M. N. N. S. N. Mol. Cell Biol. 2005; PubMed Scopus Google Scholar, X.H. S. J.A. A. Marks A.R. Sci. S. A. 2006; PubMed Scopus Google Scholar). To the functional effects of altered RII phosphorylation in cardiac we adenoviral wild-type RII and two mutants that mimic phosphorylated Ser-96 to and Ser-96 to a of the RII proteins using the a of to the of recombinant with only in cells the wild-type RII The observed phosphorylation of recombinant wild-type RII that RII is phosphorylated basal The not and RIIS96D of the in neonatal with to RII and The two PKA the and and performed to whether of constructs altered localization of PKAcat and In the of the was used to neonatal cells expressing the was proteins the The PKAcat in the The of and of and differences in localization of PKAcat from neonatal expressing recombinant proteins for PKAcat by cells with an significant in the of with or to cells expressing observed a significant increase in a result of of recombinant PKAcat in vitro studies showed that phosphorylation of RII RII affinity for PKAcat (16Rangel-Aldao R. Kupiec J.W. Rosen O.M. J. Biol. Chem. 1979; 254: 2499-2508Abstract Full Text PDF PubMed Google Scholar, 17Granot J. Mildvan A.S. Hiyama K. Kondo H. Kaiser E.T. J. Biol. Chem. 1980; 255: 4569-4573Abstract Full Text PDF PubMed Google Scholar). RIIS96D and are of phosphorylated and RII, we binding of PKAcat to RIIS96D versus RIIS96A. We the of PKAcat which with RIIS96D versus RIIS96A. PKAcat RIIS96D with thus, that constructs are of phosphorylated and RII of RII on in is a prominent AKAP in cardiomyocytes Langeberg L.K. Scott J.D. EMBO J. 1998; PubMed Scopus Google Scholar, T. Sci. S. A. PubMed Scopus Google Scholar). also of in cardiomyocytes (13Lygren B. Carlson C.R. Santamaria K. Lissandron V. McSorley T. Litzenberg J. Lorenz D. Wiesner B. Rosenthal W. Zaccolo M. Tasken K. Klussmann E. EMBO Rep. 2007; 8: 1061-1067Crossref PubMed Scopus (140) Google Scholar). plasmon resonance have shown that the affinity of the RII binding region of AKAP15/18 for RII significantly increases Ser-96 of RII is phosphorylated (18Zakhary D.R. Fink M.A. Ruehr M.L. Bond M. J. Biol. Chem. 2000; 275: 41389-41395Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We whether more AKAP15/18 is by RIIS96D with RIIS96A. We that we protein of recombinant and AKAP15/18α RII to was used to AKAP15/18 The of AKAP15/18α with each was to recombinant AKAP15/18α with the RII mimic with the phosphorylated mimic RIIS96D or wild-type RII results the recombinant with to of RII not with the results that interaction is significantly increased RII is phosphorylated at serine thus, that may a by which are regulated in of RII on PKA the that altered binding of phosphorylation regulates local PKA activity, we phosphorylation of two PKA ryanodine receptor and phospholamban Kranias E.G. Rev. Mol. Cell Biol. PubMed Scopus Google Scholar). The of ryanodine receptor and phospholamban showed significant cells expressing recombinant proteins with and basal the of phosphorylated ryanodine ryanodine receptor was significantly in neonatal cells expressing the phosphorylated mimic RIIS96D, with wild-type RII, or the of phosphorylated phospholamban was significantly in neonatal expressing RIIS96D, with and we also that basal phosphorylation of phospholamban was significantly in wild-type RII and to The increase in PKA-dependent phosphorylation of both ryanodine receptor and phospholamban, in cells expressing the phosphorylated mimic RIIS96D versus the mimic the that increased phosphorylation of RII phosphorylated results in increased PKA binding to AKAPs, PKA localization near its substrates, and PKA-dependent substrate phosphorylation. of RII on Ca2+ whether adenoviral of Ca2+ regulatory We of and Na+/Ca2+ exchanger protein The of calsequestrin and Na+/Ca2+ exchanger protein in of cells expressing with or cells with However, was significantly in of cells expressing recombinant with or cells expressing The in of was not to adenoviral infection cells with to not a in We used the Ca2+ to the of of on Ca2+ in the of of was in and neonatal cardiomyocytes of wild-type RII RIIS96D However, the of Ca2+ is regulated by Ca2+ of Ca2+ from the sarcoplasmic reticulum, Ca2+ Ca2+ the sarcoplasmic reticulum, and Ca2+ We the sarcoplasmic reticulum Ca2+ of the Ca2+ by the with Because we observed that protein was constructs was to of Ca2+ from the a result of Ca2+ a Ca2+ for each a significant in protein with the was not significantly in RIIS96D cells versus and In the was significantly in cells expressing and in cells expressing wild-type RII with from results that the Ca2+ is in neonatal expressing the wild-type with of effects of of RII RIIS96D and to in a we observed a in protein in neonatal cardiomyocytes expressing the of the Ca2+ was significantly in cells expressing wild-type RII and RIIS96D versus The and of PKA its subcellular localization by AKAPs (10Ruehr M.L. Russell M.A. Bond M. J. Mol. Cell Cardiol. 2004; 37: 653-665Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In vitro studies have that phosphorylation of RII regulates its binding to both AKAPs and We the that the phosphorylation state of RII is to of local PKA activity. We used adenoviral infection to wild-type RII and RII mutants that mimic phosphorylated or nonphosphorylated RII, in neonatal rat cardiomyocytes. of AKAP15/18 and the catalytic subunit of PKA that RIIS96D phosphorylated RII and nonphosphorylated We demonstrated that the RII and phosphorylation of PKA and Ca2+ results that the phosphorylation state of the RII subunit is a which regulates PKA substrate phosphorylation, and Ca2+ signaling in rat cardiomyocytes. RII Phosphorylation in in vitro studies that RII is phosphorylated at basal (16Rangel-Aldao R. Kupiec J.W. Rosen O.M. J. Biol. Chem. 1979; 254: 2499-2508Abstract Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, O.M. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We have now for the the of cAMP on the phosphorylation state of RII in cardiomyocytes. We that of neonatal or adult cardiomyocytes with or in with to RII phosphorylation. This result with the increase in phosphorylation observed with PKA phospholamban, ryanodine receptor, troponin which a increase in phosphorylation a result of increased are with in vitro studies of cAMP RII in purified bovine cardiac muscle or muscle O.M. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In with or and and and not a significant in RII phosphorylation, with We conclude that have to phosphorylated serine 96 cAMP is at and the holoenzyme is the phosphorylation state of RII in cardiomyocytes is a function of the of not only of PKAcat but also of and associated with each signaling (14Fischmeister R. Castro L.R. Abi-Gerges A. Rochais F. Jurevicius J. Leroy J. Vandecasteele G. Circ. Res. 2006; 99: 816-828Crossref PubMed Scopus (298) Google Scholar, M. J. Cell Biol. 2006; PubMed Scopus Google Scholar). RII Phosphorylation in surface plasmon resonance studies showed that the region encompassing the RII binding domain of AKAP15/18, showed the increase in binding affinity for RII the RII subunit is with AKAPs (18Zakhary D.R. Fink M.A. Ruehr M.L. Bond M. J. Biol. Chem. 2000; 275: 41389-41395Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We to study the effects of RII phosphorylation on with AKAP15/18 in cardiac We observed that the phosphorylated RII mimic more AKAP15/18α to the that RII phosphorylation increases interaction in cardiomyocytes. The AKAP15/18α binding of is than observed in surface plasmon resonance studies we AKAP15/18α binding to phosphorylated and recombinant This is to the that we used only the RII binding domain of the AKAP in Because the of the RII binding domain is the for AKAP15/18 we also increased binding of phosphorylated RII to which was recently shown to with phospholamban (13Lygren B. Carlson C.R. Santamaria K. Lissandron V. McSorley T. Litzenberg J. Lorenz D. Wiesner B. Rosenthal W. Zaccolo M. Tasken K. Klussmann E. EMBO Rep. 2007; 8: 1061-1067Crossref PubMed Scopus (140) Google Scholar). results that PKA binding to AKAPs is regulated by the phosphorylation state of RII in cardiomyocytes. RII Phosphorylation PKA Phosphorylation of and with of constructs not alter of PKAcat G.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Thus observed in PKA activity, by altered substrate phosphorylation, to altered of Because RII phosphorylation can increase binding of RII to AKAPs and binding of R. Rosen O.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), we increased PKA substrate phosphorylation in cells expressing the RIIS96D versus RIIS96A. In with B. G. M. D. K. H. H. Biochem. J. 2006; PubMed Scopus Google the study demonstrated phosphorylation of ryanodine receptor in cardiomyocytes PKA phosphorylation of ryanodine receptor was also observed in neonatal expressing In of ryanodine receptor phosphorylation was significantly increased in neonatal expressing RIIS96D, with RIIS96A. Our of phosphorylation of phospholamban showed a with phosphorylation of phospholamban at was significantly increased in cells to cardiomyocytes phosphorylation of ryanodine receptor and phospholamban are significantly increased in cells expressing the phosphorylated RII mimic (RIIS96D), with the mimic results the that the PKA binding to AKAPs and increased PKA localization by AKAPs, a result of increased RII phosphorylation, to increased phosphorylation of PKA substrates. the effects of the phosphorylated and RII on phosphorylation of PKA substrates. for the that PKA targeting to AKAPs, by RII phosphorylation, regulates substrate phosphorylation in cardiac phospholamban phosphorylation in neonatal expressing wild-type RII and was significantly with phospholamban phosphorylation in was not observed with ryanodine receptor phosphorylation. differences in basal phosphorylation of phospholamban and ryanodine receptor a result of of wild-type We the is now that unique signaling M. G. Lissandron V. A. Biochem. 2006; 34: PubMed Scopus Google Scholar). For example, and within subcellular are to have in PKA signaling K.L. Soughayer J. Pare G.C. Carlisle Michel J.J. Langeberg L.K. Kapiloff M.S. Scott J.D. Nature. 2005; 437: 574-578Crossref PubMed Scopus (430) Google Scholar, M. McSorley T. S. A. Lissandron V. A. E. A. T. Zaccolo M. Circ. Res. 2004; PubMed Scopus Google Scholar, F. Abi-Gerges A. K. F. M. R. Vandecasteele G. Circ. Res. 2006; PubMed Scopus Google Scholar). Because PKAcat and adenoviral of wild-type RII results in a of wild-type Thus wild-type RII molecules will not phosphorylated by PKAcat at the compartmentalization of signaling molecules in cardiac and differences in local and of and state RII phosphorylation will in of the cell. of phospholamban phosphorylation of wild-type RII that RII is in the RII Phosphorylation Ca2+ have shown that of RIIS96D versus regulates phosphorylation of proteins to Ca2+ We that of RII also affect of cytosolic we observed significant in protein of ryanodine receptor, phospholamban, and Na+/Ca2+ exchanger in neonatal cardiomyocytes expressing with significantly with and phospholamban phosphorylation, the of the Ca2+ was in cells expressing versus results are with in an mimic of phospholamban and calcium cycling G. J.W. W. J. Kranias E.G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In a in in versus cells expressing RIIS96D showed in of the Ca2+ and in of Ca2+ We conclude that the increased phosphorylation of ryanodine receptor, with phospholamban phosphorylation, a result of of the phosphorylated RII for and the sarcoplasmic reticulum to and Ca2+ in to neonatal expressing constructs Ca2+ than The of in cytosolic Ca2+ during cardiac is by regulated by phospholamban, and by the Na+/Ca2+ exchanger Nature. PubMed Scopus Google Scholar, Eisner D.A. PubMed Scopus Google Scholar). Our Ca2+ cycling in the of and in Ca2+ the sarcoplasmic reticulum of Ca2+ from the sarcoplasmic reticulum via the ryanodine receptor, and in the of via the Na+/Ca2+ was also by an Ca2+ Because of the Na+/Ca2+ exchanger was in altered of the exchanger for the differences The for the increased of of Ca2+ in cells to In heart and phospholamban is to result in activity, and of the Ca2+ B. S. Res. Cardiol. Google Scholar, V. J. J. Cardiovasc. Med. 2000; PubMed Scopus Google Scholar, R. Moravec C.S. Kranias E.G. J. Mol. Cell Cardiol. 2001; Full Text PDF PubMed Scopus Google Scholar). However, of a phospholamban heart in M. S. M. M. J. K.R. Med. 8: PubMed Scopus Google Scholar). studies have shown a result of increased or of proteins phospholamban, F. T. A. Sci. S. A. 2000; PubMed Scopus Google Scholar, J. G. Kranias E.G. J. Physiol. Heart Circ. Physiol. 2000; PubMed Google Scholar), or signaling K. Kranias E.G. T. G. J. Clin. 2001; PubMed Scopus Google Scholar, Mol. Full Text Full Text PDF PubMed Scopus Google Scholar). may for heart the of the RIIS96D not of phospholamban phosphorylation and Ca2+ signaling and of a RII of in cardiac is of or of phospholamban J.R. Kranias E.G. Heart Fail. Clin. 2005; 1: 207-218Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, M. M. F. Kranias E.G. PubMed Scopus Google Scholar). In results that the phosphorylation state of the RII subunit phosphorylation of PKA substrates, and Ca2+ The results can by an modulation of PKA RII subunits within the PKA are phosphorylated at basal cAMP binds RII subunits, leading to PKAcat RII, with increased of serine 96 to is of RII its affinity for AKAPs but increases its affinity for PKAcat R. Rosen O.M. J. Biol. Chem. Full Text PDF PubMed Google a result of phosphodiesterase also the of RII and cAMP to of PKA holoenzyme autophosphorylation of RII by PKAcat increases affinity for the AKAP and the In we have to the that regulation of phosphorylation of RII is of an for local activation and of PKA activity.
Manni et al. (Fri,) reported a other. Adenoviral vectors with phosphomimic RII (RIIS96D) vs. Nonphosphorylated RII mimic (RIIS96A) or wild-type RII was evaluated on PKA targeting to AKAPs, downstream substrate phosphorylation, and calcium cycling. In neonatal rat cardiomyocytes, the phosphomimic RIIS96D increased AKAP15/18 pull-down and significantly increased phosphorylation of phospholamban and ryanodine receptor compared to RIIS96A.