Serine phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase in the intact beating rabbit heart.
Recent studies have demonstrated that Ca(2+)/calmodulin-dependent protein kinase phosphorylates the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum (SR) in vitro. Also, evidence from in vitro studies suggested that this phosphorylation, occurring at Ser(38), results in stimulation of Ca(2+) transport. In the present study, we investigated whether serine phosphorylation of the SR Ca(2+)-ATPase occurs in the intact functioning heart. Hearts removed from anesthetized rabbits were subjected to retrograde aortic perfusion of the coronary arteries with oxygenated mammalian Ringer solution containing (32)P(i) and contractions were monitored by recording systolic left ventricular pressure development. Following 45-50 min of (32)P perfusion, the hearts were freeze-clamped, SR isolated, and analyzed for protein phosphorylation. SDS-polyacrylamide gel electrophoresis and autoradiography showed phosphorylation of several peptides including the Ca(2+)-ATPase and Ca(2+) release channel (ryanodine receptor). The identity of Ca(2+)-ATPase as a phosphorylated substrate was confirmed by Western immunoblotting as well as immunoprecipitation using a cardiac SR Ca(2+)-ATPase-specific monoclonal antibody. The Ca(2+)-ATPase showed immunoreactivity with a phosphoserine monoclonal antibody indicating that the in situ phosphorylation occurred at the serine residue. Quantification of Ca(2+)-ATPase phosphorylation in situ yielded a value of 208 +/- 12 pmol (32)P/mg SR protein which corresponded to the phosphorylation of approximately 20% of the Ca(2+) pump units in the SR membrane. Since this phosphorylation occurred under basal conditions (i.e., in the absence of any inotropic intervention), a considerable steady-state pool of serine-phosphorylated Ca(2+)-ATPase likely exists in the normally beating heart. These findings demonstrate that serine phosphorylation of the Ca(2+)-ATPase is a physiological event which may be important in the regulation of SR function.
Xu
A
A
Department of Physiology, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
Netticadan
T
T
Jones
D L
DL
Narayanan
N
N
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D009132:Muscles / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D010477:Perfusion; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012694:Serine / Q000378:metabolism*
D010748:Phosphopeptides; D012694:Serine; D000252:Calcium-Transporting ATPases