PALMITOYLATION OF MULTIPLE SRC-FAMILY KINASES AT A HOMOLOGOUS N-TERMINAL MOTIF
PALMITOYLATION OF MULTIPLE SRC-FAMILY KINASES AT A HOMOLOGOUS N-TERMINAL MOTIF
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DOI：
10.1042/bj3030749
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发表时间：
1994-11-01
影响因子：
4.1
通讯作者：
MAGEE, AI
中科院分区：
文献类型：
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作者：
KOEGL, M;ZLATKINE, P;MAGEE, AI
我们最近发现了一个新的 N 末端含有半胱氨酸的基序，它指定了几个 G 蛋白 α 亚基的棕榈酰化 [Parenti, Vigano, Newman, Milligan 和 Magee (1993) Biochem. J. 291, 349-353]。相关基序出现在蛋白酪氨酸激酶 Src 家族成员（Src 本身和 Blk 除外）的 N 末端。我们研究了Src、Fyn、Yes 和Lck 基因产物是否被棕榈酰化。当Src在COS细胞中内源表达时，未用[H-3]棕榈酸酯标记。相反，从 COS 细胞中免疫沉淀的内源 Yes 是棕榈酰化的。 Fyn 在感染重组杆状病毒的昆虫细胞中被棕榈酰化，并且棕榈酰化与蛋白质合成无关，表明这种脂质的动态周转。脂肪酸分析表明大部分标签是以棕榈酸酯的形式掺入的。 Lck 在 COS 细胞中转染表达时被棕榈酰化。在每个测试的系统中，所有这些蛋白酪氨酸激酶也被检测到肉豆蔻酰化。用转染COS细胞表达的Lck突变体进行的实验表明，位置3和位置5的半胱氨酸都是棕榈酰化位点，并且肉豆蔻酰化是棕榈酰化所必需的。为了确认 Fyn N 末端区域的半胱氨酸发生棕榈酰化，使用定点诱变将位置 3 和 6 的半胱氨酸替换为丙氨酸。所得蛋白质未被棕榈酰化，但在 COS 细胞中表达时仍被肉豆蔻酰化。位点 2 处的甘氨酸至丙氨酸突变体也未被棕榈酰化，表明肉豆蔻酰化是棕榈酰化的先决条件。我们的数据表明，含有 N 末端半胱氨酸基序的 Src 家族成员确实是棕榈酰化的。与 Ras 类比，棕榈酰化可能在 Src 家族蛋白酪氨酸激酶的定位和功能中发挥重要作用。
We have recently identified a novel N-terminal cysteine-containing motif which specifies the palmitoylation of several G-protein alpha-subunits [Parenti, Vigano, Newman, Milligan and Magee (1993) Biochem. J. 291, 349-353]. A related motif occurs at the N-terminus of members of the Src family of protein tyrosine kinases except for Src itself and Blk. We have investigated whether the Src, Fyn, Yes and Lck gene products are palmitoylated. Src was not labelled with [H-3]palmitate when endogenously expressed in COS cells. In contrast, endogenous Yes immunoprecipitated from COS cells was palmitoylated. Fyn was palmitoylated in insect cells infected with a recombinant baculovirus and the palmitoylation was independent of protein synthesis, suggesting a dynamic turnover of this lipid. Fatty acid analysis indicated that most of the label was incorporated as palmitate. Lck was palmitoylated when expressed by transfection in COS cells. All of these protein tyrosine kinases were also detectably myristoylated in each of the systems tested. Experiments performed with mutants of Lck expressed by transfection in COS cells indicated that cysteines at positions 3 and 5 were both palmitoylation sites and that myristoylation was required for palmitoylation. To confirm that palmitoylation was occurring on cysteines in the N-terminal region of Fyn, site-directed mutagenesis was used to replace the cysteines at positions 3 and 6 with alanine. The resulting protein was not palmitoylated but was still myristoylated when expressed in COS cells. A glycine to alanine mutant at position 2 was also not palmitoylated, showing that myristoylation is a prerequisite for palmitoylation. Our data indicate that Src family members containing the N-terminal cysteine motif are indeed palmitoylated. By analogy with Ras, it is possible that palmitoylation may play an important role in the localization and function of Src family protein tyrosine kinases.