Wegener A D, Simmerman H K, Lindemann J P, Jones L R
Department of Medicine, Indiana University School of Medicine, Indianapolis.
J Biol Chem. 1989 Jul 5;264(19):11468-74.
Phospholamban is the major membrane protein of the heart phosphorylated in response to beta-adrenergic stimulation. In cell-free systems, cAMP-dependent protein kinase catalyzes exclusive phosphorylation of serine 16 of phospholamban, whereas Ca2+/calmodulin-dependent protein kinase gives exclusive phosphorylation of threonine 17 (Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). In this work we have localized the sites of phospholamban phosphorylation in intact ventricles treated with the beta-adrenergic agonist isoproterenol. Isolation of phosphorylated phospholamban from 32P-perfused guinea pig ventricles, followed by partial acid hydrolysis and phosphoamino acid analysis, revealed phosphorylation of both serine and threonine residues. At steady state after isoproterenol exposure, phospholamban contained approximately equimolar amounts of these two phosphoamino acids. Two major tryptic phosphopeptides containing greater than 90% of the incorporated radioactivity were obtained from phospholamban labeled in intact ventricles. The amino acid sequences of these two tryptic peptides corresponded exactly to residues 14-25 and 15-25 of canine cardiac phospholamban, thus localizing the sites of in situ phosphorylation to serine 16 and threonine 17. Phosphorylation of phospholamban at two sites in heart perfused with isoproterenol was supported by detection of 11 distinct mobility forms of the pentameric protein by use of the Western blotting method, consistent with each phospholamban monomer containing two phosphorylation sites, and with each pentamer containing from 0 to 10 incorporated phosphates. Our results localize the sites of in situ phospholamban phosphorylation to serine 16 and threonine 17 and, furthermore, are consistent with the phosphorylations of these 2 residues being catalyzed by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively.
受磷蛋白是心脏中主要的膜蛋白，可响应β - 肾上腺素能刺激而发生磷酸化。在无细胞体系中，cAMP依赖性蛋白激酶催化受磷蛋白丝氨酸16的特异性磷酸化，而Ca2 + /钙调蛋白依赖性蛋白激酶则催化苏氨酸17的特异性磷酸化（西默曼，H.K.B.，柯林斯，J.H.，泰伯特，J.L.，韦格纳，A.D.，以及琼斯，L.R.（1986年）《生物化学杂志》261卷，13333 - 13341页）。在本研究中，我们确定了用β - 肾上腺素能激动剂异丙肾上腺素处理的完整心室中受磷蛋白的磷酸化位点。从用32P灌注的豚鼠心室中分离出磷酸化的受磷蛋白，随后进行部分酸水解和磷酸氨基酸分析，结果显示丝氨酸和苏氨酸残基均发生了磷酸化。在暴露于异丙肾上腺素后的稳态下，受磷蛋白中这两种磷酸氨基酸的含量大致相等。从完整心室中标记的受磷蛋白获得了两个主要的胰蛋白酶磷酸肽，其包含超过90%的掺入放射性。这两个胰蛋白酶肽的氨基酸序列与犬心脏受磷蛋白的14 - 25位和15 - 25位残基完全对应，从而将原位磷酸化位点定位到丝氨酸16和苏氨酸17。通过蛋白质印迹法检测到五聚体蛋白有11种不同的迁移形式，这支持了在灌注异丙肾上腺素的心脏中受磷蛋白在两个位点发生磷酸化，这与每个受磷蛋白单体含有两个磷酸化位点且每个五聚体含有0至10个掺入的磷酸盐一致。我们的结果将原位受磷蛋白磷酸化位点定位到丝氨酸16和苏氨酸17，此外，还与这两个残基的磷酸化分别由cAMP依赖性和Ca2 + /钙调蛋白依赖性蛋白激酶催化相一致。
