Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains.
Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains.
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DOI：
10.1016/s0021-9258(18)69309-3
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发表时间：
1986-10
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影响因子：
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通讯作者：
Heather;Simmerman;John H. Collins;Janet L. Theiberts;Adam;WegenerS;Larry;JonesST
中科院分区：
文献类型：
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作者：
Heather;Simmerman;John H. Collins;Janet L. Theiberts;Adam;WegenerS;Larry;JonesST
磷蛋白是心肌肌浆网中的一种调节蛋白，被cAMP和钙/钙调蛋白依赖的蛋白激酶活性所磷酸化。在本报告中，我们给出了犬心脏磷蛋白的部分氨基酸序列，并对这两种蛋白激酶的磷酸化位点进行了鉴定。用气相蛋白质测序鉴定了20个NH2末端残基。由胰酶或木瓜酶消化产生的重叠多肽延长了序列16个残基，得到以下一级结构：Ser-Ala-Ile-Arg-Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-Ala-Arg-Gln-Asn-Leu-Gln-Asn-Leu-Phe-Ile-Asn-Phe-(Cys)-Leu-Ile-Leu-Ile-(Cys)-亮氨酸-Ile-。用胰酶切割cAMP依赖的或钙/钙调素依赖的蛋白激酶磷酸化的磷蛋白，并用反相高效液相色谱法纯化主要的磷酸化多肽(占结合的32P标记的70%以上)。从任何一种激酶磷酸化的磷蛋白得到的放射性多肽都有相同的序列：Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-。磷蛋白的相邻残基Ser7和Thr8分别被cAMP依赖和钙/钙调蛋白依赖的蛋白激酶磷酸化。这些结果表明，磷蛋白是一种小的、相同的多肽链的寡聚体。每个单体上的一个亲水性的、面向细胞质的NH2末端结构域包含唯一的相邻残基，这些残基被cAMP和钙/钙调蛋白依赖的蛋白激酶活性所磷酸化。通过亲水性分析和二级结构预测表明，磷蛋白单体还含有一个疏水结构域，它可以形成足够长的两亲性螺旋来穿越肌浆网膜。提出了磷蛋白作为五聚体的模型，其中每个单体的两亲性α-螺旋是五聚体膜锚定结构域的一个亚单位，该结构域由一个外部疏水表面和一个含有极性侧链的内部亲水性区域组成。
Phospholamban is a regulatory protein in cardiac sarcoplasmic reticulum that is phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. In this report, we present the partial amino acid sequence of canine cardiac phospholamban and the identification of the sites phosphorylated by these two protein kinases. Gas-phase protein sequencing was used to identify 20 NH2-terminal residues. Overlap peptides produced by trypsin or papain digestion extended the sequence 16 residues to give the following primary structure: Ser-Ala-Ile-Arg-Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-Ala- Arg-Gln-Asn-Leu-Gln-Asn-Leu-Phe-Ile-Asn-Phe-(Cys)-Leu-Ile-Leu-Ile-(Cys)- Leu-Leu-Leu-Ile-. Phospholamban phosphorylated by either cAMP-dependent or Ca2+/calmodulin-dependent protein kinase was cleaved with trypsin, and the major phosphorylated peptide (comprising greater than 70% of the incorporated 32P label) was purified by reverse-phase high performance liquid chromatography. The identical sequence was revealed for the radioactive peptide obtained from phospholamban phosphorylated by either kinase: Arg-Ala-Ser-Thr-Ile-Glu-Met-Pro-Gln-Gln-. The adjacent residues Ser7 and Thr8 of phospholamban were identified as the unique sites phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinases, respectively. These results establish that phospholamban is an oligomer of small, identical polypeptide chains. A hydrophilic, cytoplasmically oriented NH2-terminal domain on each monomer contains the unique, adjacent residues phosphorylated by cAMP- and Ca2+/calmodulin-dependent protein kinase activities. Analysis by hydropathic profiling and secondary structure prediction suggests that phospholamban monomers also contain a hydrophobic domain, which could form amphipathic helices sufficiently long to traverse the sarcoplasmic reticulum membrane. A model of phospholamban as a pentamer is presented in which the amphipathic alpha-helix of each monomer is a subunit of the pentameric membrane-anchored domain, which is comprised of an exterior hydrophobic surface and an interior hydrophilic region containing polar side chains.