Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na+-K+-ATPase
Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na+-K+-ATPase
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DOI：
10.1152/ajpcell.00027.2010
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发表时间：
2010-12-01
影响因子：
5.5
通讯作者：
Bers, Donald M.
中科院分区：
文献类型：
--
作者：
Han, Fei;Bossuyt, Julie;Bers, Donald M.
Han F，Bossuyt J，Martin JL，Despa S，Bers DM.磷膜蛋白磷酸化位点在介导Na +-K +-ATP酶激酶依赖性调节中的作用。美国生理学杂志细胞生理学299：C1363-C1369，2010年。首次发表于2010年9月22日; doi：10.1152/ajpcell.00027.2010。Phospholemman（PLM）是心脏中PKA（Ser68）和PKC（Ser63和Ser68）介导的磷酸化的主要靶标。在完整的心肌细胞中，PLM与Na +-K +-ATP酶（NKA）结合并抑制NKA，主要是通过降低其对内部Na+的亲和力。这种抑制作用在PLM被PKA或PKC磷酸化后被解除。本文的目的是区分Ser63和Ser68 PLM磷酸化位点在介导激酶诱导的NKA功能调节中的作用。我们在稳定表达大鼠NKA-α（1）的HeLa细胞中表达野生型（WT）PLM和S63A、S68A和AA（Ser63和Ser68到丙氨酸的双突变体）PLM突变体，并测量PKA和PKC激活对NKA介导的细胞内Na+浓度下降的影响。PLM表达（WT或突变体）显著降低了NKA对内部Na+的表观亲和力，并且对最大泵速率（V-max）没有显著影响。用毛喉素（20 μ M）激活PKA恢复了表达WT但不表达AA PLM的细胞中的NKA Na+亲和力，并且在两种情况下都不影响V-max。类似地，在表达WT、S63A和S68A PLM的细胞中，用300 nM佛波醇12，13-二丁酸酯激活PKC增加NKA Na+亲和力，而在表达AA PLM的细胞中没有影响。在没有PLM的情况下，毛喉素和佛波醇12，13-二丁酸酯都不影响NKA功能。我们的结论是，PLM的Ser63或Ser68磷酸化是必要的和足够的完全缓解PLM诱导的NKA抑制。
Han F, Bossuyt J, Martin JL, Despa S, Bers DM. Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na+-K+-ATPase. Am J Physiol Cell Physiol 299: C1363-C1369, 2010. First published September 22, 2010; doi:10.1152/ajpcell.00027.2010.-Phospholemman (PLM) is a major target for phosphorylation mediated by both PKA (at Ser68) and PKC (at both Ser63 and Ser68) in the heart. In intact cardiac myocytes, PLM associates with and inhibits Na+-K+-ATPase (NKA), mainly by reducing its affinity for internal Na+. The inhibition is relieved upon PLM phosphorylation by PKA or PKC. The aim here was to distinguish the role of the Ser63 and Ser68 PLM phosphorylation sites in mediating kinase-induced modulation of NKA function. We expressed wild-type (WT) PLM and S63A, S68A, and AA (Ser63 and Ser68 to alanine double mutant) PLM mutants in HeLa cells that stably express rat NKA-alpha(1) and we measured the effect of PKA and PKC activation on NKA-mediated intracellular Na+ concentration decline. PLM expression (WT or mutant) significantly decreased the apparent NKA affinity for internal Na+ and had no significant effect on the maximum pump rate (V-max). PKA activation with forskolin (20 mu M) restored NKA Na+ affinity in cells expressing WT but not AA PLM and did not affect V-max in either case. Similarly, PKC activation with 300 nM phorbol 12,13-dibutyrate increased NKA Na+ affinity in cells expressing WT, S63A, and S68A PLM and had no effect in cells expressing AA PLM. Neither forskolin nor phorbol 12,13-dibutyrate affected NKA function in the absence of PLM. We conclude that PLM phosphorylation at either Ser63 or Ser68 is both necessary and sufficient for completely relieving the PLM-induced NKA inhibition.