BIOCHEMICAL-CHARACTERIZATION OF A PALMITOYL ACYLTRANSFERASE ACTIVITY THAT PALMITOYLATES MYRISTOYLATED PROTEINS
BIOCHEMICAL-CHARACTERIZATION OF A PALMITOYL ACYLTRANSFERASE ACTIVITY THAT PALMITOYLATES MYRISTOYLATED PROTEINS
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DOI：
10.1074/jbc.270.38.22399
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发表时间：
1995-09-22
影响因子：
4.8
通讯作者：
RESH, MD
中科院分区：
文献类型：
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作者：
BERTHIAUME, L;RESH, MD
最近，通过可逆的棕榈酸化-脱氨酸化循环对信号转导的动态调节已经被描述，然而，由于我们对涉及的特定的转移酶和硫代酯酶缺乏了解，对脂肪酰化反应的进一步了解一直受到阻碍。在这里，我们描述了一种棕榈酰化‘’myrGlyCys‘’的棕榈酰化‘’myrGlyCys‘’含有蛋白酪氨酸激酶(PTKs)家族成员的分析方法。由于已经证明了Src家族的成员Fyn PTK的N-肉豆蔻酰化是棕榈酰化的先决条件，我们开发了一个新的单一的质粒载体，允许在大肠杆菌中过表达肉豆蔻酰化的Fyn底物，将纯化的肉豆蔻酰化的蛋白底物与[I-125]碘-Almitoyl CoA孵育，在牛脑裂解物存在的情况下，通过SDS-聚丙烯酰胺凝胶和放射自显影检测到放射性转移到Fyn底物上。本实验用于部分纯化和鉴定牛脑中的PAT活性。在这里，我们证明了PAT是一种膜结合酶，它可以棕榈酰化含有半胱氨酸残基的肉豆蔻酰化FYN底物。PAT的活性通过硫酯键将棕榈酸酯结合到Fyn蛋白上，并表现出对长链脂肪酸的脂酰化CoA偏好，这可能是PAT对Fyn和其他Src家族成员的棕榈酰化调节PTK的定位和信号功能。
Dynamic regulation of signal transduction by revers reversible palmitoylation-depalmitoylation cycles has been recently described, However, further understanding of fatty acylation reactions has been hampered by our lack of knowledge about the specific transferases and thio esterases involved. Here, we describe an assay for the palmitoyl acyltransferase (PAT) that palmitoylates ''myrGlyCys'' containing members of the Src family of protein tyrosine kinases (PTKs), Since N-myristoylation of Fyn PTK, a member of the Src family, has been shown to be a prerequisite for palmitoylation, a new single plasmid vector that allows overexpression of myristoylated Fyn substrate in Escherichia coli was developed, Purified myristoylated protein substrates were incubated with [I-125]iodopalmitoyl CoA, a palmitoyl CoA analog, in the presence of bovine brain lysates, Transfer of radiolabel to the Fyn substrate was detected by SDS-polyacrylamide gel electrophoresis and autoradiography. This assay was used to partially purify and characterize PAT activity from bovine brain. Here, we demonstrate that PAT is a membrane-bound enzyme, which palmitoylates myristoylated Fyn substrates containing a cysteine residue in position three. The PAT activity attached palmitate to Fyn proteins via a thioester Linkage and exhibited a fatty acyl CoA preference for long chain fatty acids, It is likely that palmitoylation of Fyn and other Src family members by PAT regulates PTK localization and signaling functions.