The N-terminal SH4 region of the Src family kinase Fyn is modified by methylation and heterogeneous fatty acylation - Role in membrane targeting, cell adhesion, and spreading
The N-terminal SH4 region of the Src family kinase Fyn is modified by methylation and heterogeneous fatty acylation - Role in membrane targeting, cell adhesion, and spreading
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DOI：
10.1074/jbc.m311180200
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发表时间：
2004-02-27
影响因子：
4.8
通讯作者：
Resh, MD
中科院分区：
文献类型：
--
作者：
Liang, XQ;Lu, Y;Resh, MD
Src家族激酶的N-末端SH 4结构域负责促进膜结合和质膜靶向。大多数Src家族激酶含有N-末端Met-Gly-Cys共有序列，其在去除甲硫氨酸后经历肉豆蔻酸酯和棕榈酸酯的双重酰化。以前的研究Src家族激酶脂肪酰化依赖于放射性脂肪酸的放射性标记的细胞。虽然这种方法可用于验证给定的脂肪酸与蛋白质连接，但它不能揭示其他脂肪酸或其他修饰基团是否与蛋白质连接。在这里，我们使用基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱法，以确定脂肪酰化物种的Src家族激酶Fyn。我们的研究结果表明，Fyn是有效的肉豆蔻酰化和一些肉豆蔻酰化的蛋白质也不均匀的S-酰化与棕榈酸酯，棕榈油酸酯，硬脂酸酯，或油酸。此外，我们首次表明，Fyn是三甲基化的赖氨酸残基7和/或9内的N-末端区域。肉豆蔻酰化和棕榈酰化都是Fyn甲基化所必需的。然而，一般的甲基化抑制剂对Fyn的肉豆蔻酰化和棕榈酰化没有抑制作用，这表明甲基化发生在肉豆蔻酰化和棕榈酰化之后。不能被甲基化的Fyn的赖氨酸突变体不能促进细胞粘附和铺展，这表明甲基化对Fyn功能是重要的。
The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function.