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Am J Physiol Cell Physiol 296: C1346-C1355, 2009. First published April 1, 2009; doi:10.1152/ajpcell.00523.2008
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C

William Fuller,1 Jacqueline Howie,1 Linda M. McLatchie,2 Roberta J. Weber,1 C. James Hastie,3 Kerry Burness,3 Davor Pavlovic,2 and Michael J. Shattock2

1The Institute of Cardiovascular Research, Department of Medicine, University of Dundee, Ninewells Hospital, Dundee, United Kingdom; 2Cardiovascular Division, King's College London, The Rayne Institute, St. Thomas’ Hospital, London, United Kingdom; and 3Division of Signal Transduction Therapy, School of Life Sciences, University of Dundee, Dundee, United Kingdom

Submitted 14 October 2008 ; accepted in final form 30 March 2009

FXYD1 (phospholemman), the primary sarcolemmal kinase substrate in the heart, is a regulator of the cardiac sodium pump. We investigated phosphorylation of FXYD1 peptides by purified kinases using HPLC, mass spectrometry, and Edman sequencing, and FXYD1 phosphorylation in cultured adult rat ventricular myocytes treated with PKA and PKC agonists by phosphospecific immunoblotting. PKA phosphorylates serines 63 and 68 (S63 and S68) and PKC phosphorylates S63, S68, and a new site, threonine 69 (T69). In unstimulated myocytes, FXYD1 is ~30% phosphorylated at S63 and S68, but barely phosphorylated at T69. S63 and S68 are rapidly dephosphorylated following acute inhibition of PKC in unstimulated cells. Receptor-mediated PKC activation causes sustained phosphorylation of S63 and S68, but transient phosphorylation of T69. To characterize the effect of T69 phosphorylation on sodium pump function, we measured pump currents using whole cell voltage clamping of cultured adult rat ventricular myocytes with 50 mM sodium in the patch pipette. Activation of PKA or PKC increased pump currents (from 2.1 ± 0.2 pA/pF in unstimulated cells to 2.9 ± 0.1 pA/pF for PKA and 3.4 ± 0.2 pA/pF for PKC). Following kinase activation, phosphorylated FXYD1 was coimmunoprecipitated with sodium pump {alpha}1-subunit. We conclude that T69 is a previously undescribed phosphorylation site in FXYD1. Acute T69 phosphorylation elicits stimulation of the sodium pump additional to that induced by S63 and S68 phosphorylation.

Na+-K+-ATPase; Na+-K+ pump; phospholemman; FXYD; protein phosphorylation


Address for reprint requests and other correspondence: W. Fuller, Inst. of Cardiovascular Research, Vascular and Inflammatory Diseases Research Unit, Univ. of Dundee, Dept. of Medicine, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK (e-mail: w.fuller{at}dundee.ac.uk)






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