Submitted on October 14, 2008
Revised on March 30, 2009
Accepted on March 30, 2009
FXYD1 PHOSPHORYLATION IN VITRO AND IN ADULT RAT CARDIAC MYOCYTES: THREONINE 69 IS A NOVEL SUBSTRATE FOR PROTEIN KINASE C
William Fuller1*, Jacqueline Howie1, Linda McLatchie2, Roberta Weber1, C. James Hastie1, Kerry Burness1, Davor Pavlovic2, and Michael Jonathan Shattock3
1 Dundee University
2 King's College London
3 The Rayne Institute
* To whom correspondence should be addressed. E-mail: w.fuller{at}dundee.ac.uk.
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, and confocal microscopy of myocytes co-stained for total and either S63 or S68 phosphorylated FXYD1 indicates that in unstimulated cells, phosphorylated FXYD1 is more concentrated in t-tubular membranes than surface sarcolemma. 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 50mM sodium in the patch pipette. Activation of PKA or PKC increased pump currents (from 2.1±0.2pA/pF in unstimulated cells to 2.9±0.1pA/pF for PKA and 3.4±0.2pA/pF for PKC). Following kinase activation, phosphorylated FXYD1 was co-immunoprecipitated with sodium pump 
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.
