Critical evaluation of cardiac Ca2+-ATPase phosphorylation on serine 38 using a phosphorylation site-specific antibody.
The phosphorylation of the cardiac muscle isoform of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) on serine 38 has been described as a regulatory event capable of very significant enhancement of enzyme activity (Hawkins, C., Xu, A., and Narayanan, N. (1994) J. Biol. Chem. 269, 31198-31206). Independent confirmation of these observations has not been forthcoming. This study has utilized a polyclonal antibody specific for the phosphorylated serine 38 epitope on the Ca(2+)-ATPase to evaluate the phosphorylation of SERCA2a in isolated sarcoplasmic reticulum vesicles and isolated rat ventricular myocytes. A quantitative Western blot approach failed to detect serine 38-phosphorylated Ca(2+)-ATPase in either kinase-treated sarcoplasmic reticulum vesicles or suitably stimulated cardiac myocytes. Calibration standards confirmed that the detection sensitivity of assays was adequate to detect Ser-38 phosphorylation if it occurred on at least 1% of Ca(2+)-ATPase molecules in SR vesicle experiments or on at least 0.1% of Ca(2+)-ATPase molecules in cardiac myocytes. The failure to detect a phosphorylated form of the Ca(2+)-ATPase in either preparation (isolated myocyte, purified sarcoplasmic reticulum vesicles) suggests that Ser-38 phosphorylation of the Ca(2+)-ATPase is not a significant regulatory feature of cardiac Ca(2+) homeostasis.
Rodriguez
Patricia
P
School of Biochemistry & Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Jackson
Wayne A
WA
Colyer
John
J
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000378:metabolism* / Q000502:physiology*; D002138:Calibration; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D015151:Immunoblotting; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D032342:Muscle Cells / Q000166:cytology / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D000939:Epitopes; D010455:Peptides; D012694:Serine; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases