Stoichiometric Phosphorylation of Cardiac Ryanodine Receptor on Serine 2809 by Calmodulin-dependent Kinase II and Protein Kinase A

The ryanodine receptor of cardiac muscle performs a central role in excitation-contraction coupling. Phosphorylation of the channel on serine 2809 (in rabbit or the corresponding serine 2808 in man) alters function in vitro, although the impact of this in vivo has not been established. We have produced a pair of antisera to the serine 2809 phosphorylation site to aid description of the incidence and consequence of phosphorylation of this receptor. One of these antisera is specific for the serine 2809 phosphorylated form of the cardiac ryanodine receptor; the other antiserum is specific for the serine 2809 dephosphorylated receptor. These antibodies have been used to demonstrate that both protein kinase A and calmodulin-dependent kinase II are capable of phosphorylating serine 2809 in vitro. Both kinases phosphorylate serine 2809 to full stoichiometry, but this is accompanied by the incorporation of more (radioactive) phosphate into the receptor by calmodulin-dependent kinase II than by protein kinase A. These data suggest that calmodulin-dependent kinase II phosphorylates at least four sites in addition to serine 2809 in vitro. The ryanodine receptor of cardiac muscle performs a central role in excitation-contraction coupling. Phosphorylation of the channel on serine 2809 (in rabbit or the corresponding serine 2808 in man) alters function in vitro, although the impact of this in vivo has not been established. We have produced a pair of antisera to the serine 2809 phosphorylation site to aid description of the incidence and consequence of phosphorylation of this receptor. One of these antisera is specific for the serine 2809 phosphorylated form of the cardiac ryanodine receptor; the other antiserum is specific for the serine 2809 dephosphorylated receptor. These antibodies have been used to demonstrate that both protein kinase A and calmodulin-dependent kinase II are capable of phosphorylating serine 2809 in vitro. Both kinases phosphorylate serine 2809 to full stoichiometry, but this is accompanied by the incorporation of more (radioactive) phosphate into the receptor by calmodulin-dependent kinase II than by protein kinase A. These data suggest that calmodulin-dependent kinase II phosphorylates at least four sites in addition to serine 2809 in vitro. The ryanodine receptor (RYR2) of cardiac muscle plays a central role in the coupling of electrical excitation of the muscle to mechanical contraction. It is a Ca2+ channel, which resides primarily in the sarcoplasmic reticulum (SR) 1The abbreviations used are: SR, sarcoplasmic reticulum; CaMKII, calmodulin-dependent kinase II; PKA, protein kinase A; PKI, protein kinase inhibitor; PVDF, polyvinylidene difluoride; ELISA, enzyme-linked immunosorbent assay.1The abbreviations used are: SR, sarcoplasmic reticulum; CaMKII, calmodulin-dependent kinase II; PKA, protein kinase A; PKI, protein kinase inhibitor; PVDF, polyvinylidene difluoride; ELISA, enzyme-linked immunosorbent assay. at junctions between this organelle and the t-tubular system (a specialized invaginated domain of the plasma membrane). Upon depolarization of the plasma membrane, Ca2+ enters the cell across the t-tubule membrane and interacts with the RYR2. Ca2+ binding to RYR2 opens the channel, and Ca2+ stored in the SR moves through the channel into the cytosol to initiate contraction (1Bers D.M. Nature. 2002; 415: 198-205Crossref PubMed Scopus (3118) Google Scholar). A variety of strategies are used by the cell to regulate RYR2 channel activity. It is anticipated that these regulatory strategies facilitate the fine control of E-C coupling, although evidence for this in live cells is rather limited. In vitro studies have shown that the binding of Ca2+ (2Xu L. Meissner G. Biophys. J. 1998; 75: 2302-2312Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), Mg2+ (3Hain J. Onoue H. Mayrleitner M. Fleischer S. Schindler H. J. Biol. Chem. 1995; 270: 2074-2081Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), ATP (4Xu L. Mann G. Meissner G. Circ. Res. 1996; 79: 1100-1109Crossref PubMed Scopus (166) Google Scholar), cADP ribose (5Sitsapesan R. McGarry S.J. Williams A.J. Trends Pharmacol. Sci. 1995; 16: 386-391Abstract Full Text PDF PubMed Scopus (144) Google Scholar), calmodulin (6Wagenknecht T. Berkowitz J. Grassucci R. Timerman A.P. Fleischer S. Biophys. J. 1994; 67: 2286-2295Abstract Full Text PDF PubMed Scopus (55) Google Scholar), and FKBP12.6 (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar) affect channel activity, as does the binding of pharmacological agents such as the plant alkaloid ryanodine (which was originally used to identify the channel protein; Ref. 8Meissner G. J. Biol. Chem. 1986; 261: 6300-6306Abstract Full Text PDF PubMed Google Scholar). In addition the channel is phosphorylated on at least a single residue, and this phosphorylation alters channel behavior in vitro (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar, 9Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar). In an effort to understand the regulatory role of RYR2 phosphorylation, research has focused on three aspects of the process. First, the identity of sites of phosphorylation in the receptor and kinases capable of using these sites; second, the functional consequence of site-specific phosphorylation in vitro; and third, the incidence and functional consequence of site-specific phosphorylation of the receptor in living cells. To date Ser-2809 (in the rabbit sequence (10Otsu K. Willard H.F. Khanna V.K. Zorzato F. Green N.M. MacLennan D.H. J. Biol. Chem. 1990; 265: 13472-13483Abstract Full Text PDF PubMed Google Scholar) or the corresponding Ser-2808 in man (11Tunwell R.E.A. Wickender C. Bertrand B.M.A. Shevchenko V.I. Walsh M.B. Allen P.D. Lai F.A. Biochem. J. 1996; 318: 477-487Crossref PubMed Scopus (126) Google Scholar)) has been identified as a site of phosphorylation on RYR2, which is used in vitro (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar, 9Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar). It has a counterpart in the skeletal muscle ryanodine receptor, Ser-2843, which is also phosphorylated in vitro (12Suko J. Maurer-Fogy I. Plank B. Bertel O. Wyskovsky W. Hohenegger M. Hellmann G. Biochim. Biophys. Acta. 1993; 1175: 193-206Crossref PubMed Scopus (102) Google Scholar). Other phosphorylation sites may exist in RYR2, but the identity of these await description. CaMKII was first identified as the kinase responsible for Ser-2809 phosphorylation (9Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar), and more recently phosphorylation of this site by PKA has been described (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar). It is likely that both of these observations are accurate, although this has not been demonstrated in a single study to date. Resolution of this issue is one of the objectives of the present study. Phosphorylation of RYR2 by CaMKII or PKA is accompanied by significant changes in channel function in vitro. These changes include an increased open probability (P o) (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar, 9Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar), the abrogation of the inhibitory effects of CaM (9Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar) and Mg2+ (3Hain J. Onoue H. Mayrleitner M. Fleischer S. Schindler H. J. Biol. Chem. 1995; 270: 2074-2081Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), heightened channel activity (Po) in response to step changes in Ca2+ (13Valdivia H.H. Kaplan J.H. Ellis-Davies G.C. Lederer W.J. Science. 1995; 267: 1997-2000Crossref PubMed Scopus (313) Google Scholar), dissociation of regulatory factors (e.g. FKBP12.6), expression of subconductance states, and the expression of channel activity at diastolic Ca2+ concentrations (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar). Furthermore, Ser-2809 phosphorylation appears elevated in clinical situations such as heart failure (14Reiken S. Gaburjakova M. Guatimosim S. Gomez A.M. D'Armiento J. Burkhoff D. Wang J. Vassort G. Lederer W.J. Marks A.R. J. Biol. Chem. 2003; 278: 444-453Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), and may contribute to the abnormal Ca2+ handling characteristics of cardiac muscle in these conditions. Despite good evidence of increased channel activity upon RYR2 phosphorylation in vitro, the anticipated manifestation of this is not observed in single cells (15Li Y. Kranias E.G. Mignery G.A. Bers D.M. Circ. Res. 2002; 90: 309-316Crossref PubMed Scopus (227) Google Scholar). Ca2+ spark frequency might be expected to increase with increased P o and with the expression of channel activity at diastolic Ca2+ concentrations (which accompany stoichiometric phosphorylation of Ser-2809; Ref. 7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar). However, Li et al. (15Li Y. Kranias E.G. Mignery G.A. Bers D.M. Circ. Res. 2002; 90: 309-316Crossref PubMed Scopus (227) Google Scholar) found that spark frequency did not increase upon cAMP generation in animals lacking the regulatory protein phospholamban. These data suggest that spark frequency was not dependent upon ryanodine receptor phosphorylation status (which would be expected to rise upon generation of cAMP) but dependent upon SR Ca2+ load, which was most heavily influenced by the phosphorylation status of phospholamban. In this study we have sought to develop tools for the study of ryanodine receptor phosphorylation in vitro and in living cells. Phosphorylation site-specific antibodies have proven to be invaluable tools in research to establish the role of phosphorylation of an individual protein in cell we the and of a pair of antisera to the Ser-2809 phosphorylation site of RYR2. One of these antisera is specific for the phosphorylated form of the Ser-2809 the other is specific for the dephosphorylated form of the These antibodies have to establish that both CaMKII and PKA phosphorylate Ser-2809 to full in vitro. The study has also that CaMKII phosphorylates RYR2 to a than PKA, with the of phosphorylation sites on RYR2 by was by the of and R. Biochem. Biophys. Res. PubMed Scopus Google Scholar). CaMKII and PKA CaMKII was also Schulman and dephosphorylated and to rabbit and and and was was cAMP was A and was and was of and dephosphorylated RYR2 to by It be that Ser-2809 in rabbit to Ser-2808 in the RYR2 but that the sequence is in these to using in the of phosphorylated Scholar), or in the of dephosphorylated G.A. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), and with of and at and and was and stored at antibodies are described to the phosphorylated and to the dephosphorylated cardiac sarcoplasmic reticulum as described C. Wang J.H. J. Biochem. 1990; PubMed Scopus Google Scholar). SR at in and for M. O. R. Vassort G. Circ. Res. 1990; 67: PubMed Scopus Google Scholar). SR by at for in and in and was by the assay. Phosphorylation at in of of dephosphorylated SR and and A. phosphorylation by CaMKII the of and PKA or (in the control Ca2+ calmodulin and CaMKII the PKA phosphorylation was in the of and CaMKII cAMP and PKA in the for the control The phosphorylation by the addition of of the by of to the by using and as described by Nature. PubMed Scopus Google Scholar). to by and binding sites for at using and at with antibodies or or and for the phosphorylated of G.A. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). A was used in with an system to the using a for both and of the of these of at in and for in and for at and at with and of incorporation into the cardiac muscle ryanodine receptor was by to for or and of the data was using for by L. Scholar) using as the for antibodies with RYR2 at and and RYR2 used as agents as described in the the are as for and was using the for of to an effort to understand the role of RYR2 phosphorylation at antibodies specific for the dephosphorylated and the phosphorylated Ser-2809 site antibodies to other have proven invaluable in the of the incidence and role of site-specific phosphorylation of protein G.A. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, D. J. Biochem. 1998; PubMed Scopus Google Scholar). A pair of RYR2 sequence used as One of the was with a at the which to Ser-2809 by the other was a dephosphorylated The and of sequence was to the of the phosphorylation status of Ser-2809 (in the between and RYR2 sequence to that the antibodies specific for RYR2 using antisera the are this study. antibodies to between phosphorylated and dephosphorylated RYR2 with produced with the and concentrations of or in used to the binding characteristics of the A by the is a of the of with the on the a not by the is to affect with as used to a which is likely to These a of binding for RYR2 as the of this with was by concentrations of concentrations of on binding to but concentrations inhibitory The for of these described a for phosphorylated RYR2 in the of the phosphorylation site in by the of binding to on the was at concentrations of phosphorylated and dephosphorylated and The of was observed with as would be of identified a for dephosphorylated RYR2 in the of by the and demonstrated inhibitory characteristics at and for the dephosphorylated and phosphorylated The of the by of phosphorylated or dephosphorylated with is most in the of the of binding of to on the a with to dephosphorylated RYR2 but a with to phosphorylated RYR2 we is a function of the of the antiserum A of antibodies exist the which a of binding for the In the of the of the antibodies for dephosphorylated RYR2 has a that a inhibitory of the by this for phosphorylated RYR2 is more a inhibitory of These that antibodies have been produced which are specific for the RYR2 phosphorylation site at Ser-2809 and with this site in a of this have been described with other protein G.A. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) and have proven invaluable in the description of the role of protein phosphorylation in cardiac cells. of RYR2 observed and by these we with RYR2 protein in a phosphorylation dependent cardiac SR of RYR2 protein; this was phosphorylated by with CaMKII and to that RYR2 protein is present in the SR with A and and the does not between control and CaMKII RYR2 is by into as has been described (9Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar). also that interacts with the RYR2 as a protein of to that by the RYR2 other or in cardiac SR by this The of with RYR2 was dependent on the phosphorylation status of as phosphorylated RYR2 but not dephosphorylated RYR2 of RYR2 on the that this interacts with RYR2 Ser-2809 is we that the of with this the phosphorylation status of Ser-2809 is the for other The of RYR2 in control by this that Ser-2809 phosphorylation is present to to which be to the of phosphorylation the of SR of phosphorylation is increased upon to CaMKII as by the increase in with in the with the control and would be expected the with the dephosphorylated was the of that with the phosphorylation It the RYR2 protein and the was upon addition of but not upon addition of the was upon CaMKII of SR These data that both of these antibodies RYR2 protein in a which is dependent upon the phosphorylation status of Ser-2809; is specific for Ser-2809 phosphorylated RYR2, and is specific for Ser-2809 dephosphorylated RYR2. suggest that kinases phosphorylate RYR2 on Ser-2809 (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar, 9Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar). It is likely that both are and that both CaMKII and PKA, phosphorylate this site in vitro. A of this which be by of the of both kinases a single study. was in the First, the phosphorylation status of RYR2 was by in an effort to kinase A in by and an increase in by evidence of of Ser-2809 of RYR2 by of cardiac SR with or for A of dephosphorylated SR was produced by with for by at for and an step (in to SR and the The SR was to a protein kinase in the of of the other The site-specific phosphorylation of PKA, Ref. G.A. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) was used to that the activity of kinase in was observed The but not phosphate and and and phosphorylation of in a was not observed upon CaMKII addition at this but in increased upon PKA addition phosphorylation was to PKA addition at but increased in the of CaMKII These data that the used of the activity of individual kinases The phosphorylation status of RYR2 on Ser-2809 was by in these of Ser-2809 phosphorylation in control kinase but Ser-2809 phosphorylation was with CaMKII or PKA that a significant increase in Ser-2809 phosphorylation upon addition of CaMKII or PKA We sought to establish RYR2 was phosphorylated to the by of these was most by of the of RYR2 with is in PKA phosphorylated RYR2 to on as was protein in the CaMKII did not this of phosphorylation in this These data suggest that Ser-2809 is a phosphorylation site for PKA, as this is to the stoichiometric phosphorylation of this et al. (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar) the using a phosphorylation However, in the present the of CaMKII and PKA in the which might contribute to the of phosphorylation observed in To this cardiac SR was phosphorylated by of CaMKII of and PKA of a increase in CaMKII activity not as the specific activity of CaMKII used in the was than that used in the these Ser-2809 was phosphorylated to by kinase the was to protein kinase such we that Ser-2809 be phosphorylated to full in vitro by CaMKII or RYR2 on Ser-2809 good has been for the of a single site for PKA phosphorylation on RYR2, as of Ser-2809 in the of PKA phosphorylation (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar). et al. (9Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar) suggest that Ser-2809 is the phosphorylation site for have observed that CaMKII phosphorylates RYR2 to a than PKA M. J. Biochem. J. 1993; PubMed Scopus Google Scholar, T. T. K. T. M. J. Biochem. 1991; PubMed Scopus Google Scholar), which phosphorylation sites used by CaMKII or a of Ser-2809 phosphorylation CaMKII with in these To we the of phosphorylation of RYR2 of stoichiometric phosphorylation of Ser-2809 by PKA and that stoichiometric phosphorylation of Ser-2809 by PKA was accompanied by the incorporation of of phosphate into the RYR2 Phosphorylation by CaMKII in the incorporation of more into RYR2. was used to the incorporation of phosphate into RYR2 by PKA or CaMKII the incorporation of of for by PKA we that PKA phosphorylates a single site on RYR2 (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar) and that stoichiometric phosphorylation of this site was in this incorporation into RYR2 a single site is phosphorylated is by in CaMKII the incorporation of this into RYR2, which be in of stoichiometric phosphorylation of Ser-2809 stoichiometric phosphorylation of a four sites or stoichiometric phosphorylation of Ser-2809 phosphorylation of more than four of the sequence of RYR2 has identified CaMKII phosphorylation sites and a single PKA site A of sites are observed in the rabbit RYR2 phosphorylation sites in RYR2. phosphorylation sites in the RYR2 sequence identified using using the sequence for CaMKII or for PKA is and at a single The of phosphorylation site for CaMKII is shown the the of PKA phosphorylation sites is shown the It be that Ser-2809 in rabbit RYR2 to Ser-2808 in In this study we have produced and a pair of antibodies that with the cardiac ryanodine receptor in a dependent upon the phosphorylation status of One of the interacts with RYR2, Ser-2809 is and the binding of this is the is The other interacts with RYR2 Ser-2809 is and the binding is upon of this These antibodies are specific for the dephosphorylated and phosphorylated The antibodies used to RYR2 phosphorylation at Ser-2809 in cardiac SR These studies data that an in the CaMKII PKA phosphorylate Both CaMKII and PKA to phosphorylate Ser-2809 to CaMKII appears to phosphorylate at least four sites on RYR2. of site-specific antibodies are to be a in with in of antibodies in The data of this study description G.A. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) that specific be produced by the of with to and to a The of does not to be as used in this study and and both These antibodies on the phosphorylation status of Ser-2809 of RYR2 and are by the phosphorylation status of other sites in the receptor the between and in which the phosphorylation status of Ser-2809 is but not the of The of other the was by of the phosphorylation site in The sequence of this site (12Suko J. Maurer-Fogy I. Plank B. Bertel O. Wyskovsky W. Hohenegger M. Hellmann G. Biochim. Biophys. Acta. 1993; 1175: 193-206Crossref PubMed Scopus (102) Google Scholar) is to the site the antibodies described in this study between these The binding of to the corresponding sequence or as is than binding to RYR2 sequence this we that both antibodies with in the sequence and that the in sequence RYR2 and the of RYR2 does not other phosphorylation site which the Ser-2809 more than does the sequence we that the antibodies used in this study would to with other RYR2 phosphorylation sites and that observed with these antibodies is dependent upon Ser-2809 phosphorylation status The pair of one to the dephosphorylated receptor and the other to the phosphorylated receptor, in this as was the which of the of phosphorylation was to be in and a the pair of antibodies has proven an of as the observed with one is the of of the other was used in the first description of phosphorylation which to Nature. PubMed Scopus Google Scholar). Furthermore, the antibodies to the of phosphorylation, as the of dephosphorylated protein was to by PKA or CaMKII of SR, that stoichiometric phosphorylation of RYR2 on Ser-2809 is in vitro by PKA or These data a description of stoichiometric phosphorylation of Ser-2809 by PKA (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar) and this through the description of stoichiometric phosphorylation by of Ser-2809 phosphorylation of RYR2 is accompanied by dissociation of a regulatory protein FKBP12.6 (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar) and a in channel activity. The phosphorylation status of Ser-2809 has been to as the in the binding and dissociation of The present that the of with RYR2 CaMKII would be expected to stoichiometric phosphorylation of Ser-2809 and be to affect FKBP12.6 was not observed in et al. (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar), which might be in one of the CaMKII phosphorylation of RYR2 might have been in the study of et al. (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar), the on the present that phosphorylation of RYR2 is observed of CaMKII are CaMKII phosphorylates a of sites on RYR2 the binding of FKBP12.6 might be by the phosphorylation status of such that dissociation Ser-2809 is phosphorylated but not Ser-2809 and be site are Phosphorylation of Ser-2809 the corresponding residue, in appears elevated in heart failure (14Reiken S. Gaburjakova M. Guatimosim S. Gomez A.M. D'Armiento J. Burkhoff D. Wang J. Vassort G. Lederer W.J. Marks A.R. J. Biol. Chem. 2003; 278: 444-453Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, A.R. S.O. Reiken S. Trends 2002; PubMed Scopus Google Scholar, S.O. Gaburjakova J. Gaburjakova M. C. K. Marks A.R. Circ. Res. PubMed Scopus Google Scholar). To date this has been to PKA activity the of this study suggest that CaMKII would be capable of to this Both kinases and are likely to be in situations of (e.g. heart as has been shown with other SR D. Trends 2002; PubMed Scopus Google Scholar), and be to establish the of kinase to Ser-2809 phosphorylation in vivo and in in response to and Phosphorylation for the description of RYR2 phosphorylation, et al. (9Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar) described a phosphorylation site on RYR2 which was heavily phosphorylated by CaMKII and phosphorylated by et al. (7Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar) that this site was phosphorylated to by PKA, and we have demonstrated in this study that be phosphorylated to by PKA or These of phosphate into the receptor, in which both stoichiometric phosphorylation of These data that CaMKII phosphorylates four sites in addition to Ser-2809 or than four sites these sites are not phosphorylated to full The sequence of RYR2 a of CaMKII phosphorylation and be a to identify which of these are phosphorylated in vitro and in We are to Schulman for CaMKII, to for to for with and to Jones for SR