FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C
FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C
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DOI：
10.1152/ajpcell.00523.2008
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发表时间：
2009-06-01
影响因子：
5.5
通讯作者：
Shattock, Michael J.
中科院分区：
文献类型：
--
作者：
Fuller, William;Howie, Jacqueline;Shattock, Michael J.
Fuller W，Howie J，McLatchie LM，Weber RJ，Hastie CJ，Burness K，Pavlovic D，Shattock MJ. FXYD 1在体外和成年大鼠心肌细胞中的磷酸化：苏氨酸69是蛋白激酶C的新底物。美国生理学杂志细胞生理学296：C1346-C1355，2009年。首次发表于2009年4月1日; doi：10.1152/ajpcell.00523.2008。FXYD 1（磷脂酶原）是心脏中的主要肌膜激酶底物，是心脏钠泵的调节剂。我们研究了FXYD 1肽的磷酸化纯化的激酶，使用HPLC，质谱，Edman测序，和FXYD 1磷酸化培养的成年大鼠心室肌细胞PKA和PKC激动剂处理磷酸化免疫印迹。PKA磷酸化丝氨酸63和68（S63和S68），PKC磷酸化S63、S68和一个新位点，苏氨酸69（T69）。在未受刺激的肌细胞中，FXYD 1在S63和S68处类似于30%磷酸化，但在T69处几乎没有磷酸化。S63和S68在未受刺激的细胞中急性抑制PKC后迅速去磷酸化。受体介导的PKC激活导致S63和S68的持续磷酸化，但T69的瞬时磷酸化。为了表征T69磷酸化对钠泵功能的影响，我们使用全细胞电压钳技术在贴片移液管中用50 mM钠培养的成年大鼠心室肌细胞测量泵电流。PKA或PKC的激活增加了泵电流（从未刺激细胞的2.1 +/- 0.2 pA/pF增加到PKA的2.9 +/- 0.1 pA/pF和PKC的3.4 +/- 0.2 pA/pF）。激酶激活后，磷酸化FXYD 1与钠泵α（1）亚基共免疫沉淀。我们的结论是，T69是一个以前未描述的磷酸化位点FXYD 1。除了S63和S68磷酸化诱导的刺激外，急性T69磷酸化还刺激钠泵。
Fuller W, Howie J, McLatchie LM, Weber RJ, Hastie CJ, Burness K, Pavlovic D, Shattock MJ. FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C. Am J Physiol Cell Physiol 296: C1346-C1355, 2009. First published April 1, 2009; doi:10.1152/ajpcell.00523.2008.-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 similar to 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.