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J. Biol. Chem., Vol. 279, Issue 17, 17111-17119, April 23, 2004

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Critical Evaluation of Cardiac Ca²+-ATPase Phosphorylation on Serine 38 Using a Phosphorylation Site-specific Antibody^*

Patricia Rodriguez, Wayne A. Jackson, and John Colyer¶

From the School of Biochemistry & Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom and Badrilla Ltd., P. O. Box 251, Leeds LS17 9WA, United Kingdom

The phosphorylation of the cardiac muscle isoform of the sarcoplasmic reticulum (SR) Ca²⁺-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 Ca²⁺-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 Ca²⁺-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 Ca²⁺-ATPase molecules in SR vesicle experiments or on at least 0.1% of Ca²⁺-ATPase molecules in cardiac myocytes. The failure to detect a phosphorylated form of the Ca²⁺-ATPase in either preparation (isolated myocyte, purified sarcoplasmic reticulum vesicles) suggests that Ser-38 phosphorylation of the Ca²⁺-ATPase is not a significant regulatory feature of cardiac Ca²⁺ homeostasis.

Received for publication, January 15, 2004

^* This work was supported by British Heart Foundation Grant PG/99186. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of BHF Ph.D. fellowship FS/92014. Present address: Cyclacel Ltd., Dundee Technopole, James Lindsay Place, Dundee DD1 5JJ, United Kingdom.

^¶ To whom correspondence should be addressed. E-mail: j.colyer{at}leeds.ac.uk.

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