Liu Y, Fisher D A, Storm D R
Department of Pharmacology, University of Washington School of Medicine, Seattle 98195.
Biochemistry. 1993 Oct 12;32(40):10714-9. doi: 10.1021/bi00091a023.
Neuromodulin (GAP-43) is a neurospecific calmodulin binding protein which is implicated in neuronal growth and regeneration. It is concentrated in neuronal growth cones and associates with membranes through the palmitoylation of the N-terminal peptide MLCCMRRTK at Cys-3 and Cys-4. In the present study, we have identified critical amino acid residues required for palmitoylation and membrane association of neuromodulin in vivo. Several neuromodulin constructs with point mutations were tested for membrane association and palmitoylation. Wild-type neuromodulin expressed in COS-7 cells incorporated [3H]palmitic acid, whereas a mutant in which both Cys-3 and Cys-4 were substituted with glycine was not palmitoylated in vivo. Mutant proteins in which either Cys-3 or Cys-4 was substituted with leucine incorporated 75% and 25% of [3H]palmitic acid, respectively, compared to wild-type neuromodulin. The relative distribution of mutant neuromodulins expressed in COS-7 cells was quantitated by immunoblot analysis of the membrane and cytosolic fractions. There was a general correlation between membrane association of mutant neuromodulins and the extent to which they were palmitoylated in vivo. Additional point mutations in the acylation domain of neuromodulin indicated that a short hydrophobic amino acid sequence N-terminal to Cys-4 may be required for optimal palmitoylation and membrane association. We conclude that Cys-4 is critical for the palmitoylation and membrane association of neuromodulin.
神经调节蛋白（GAP - 43）是一种神经特异性钙调蛋白结合蛋白，与神经元的生长和再生有关。它集中在神经元生长锥中，并通过N端肽MLCCMRRTK的半胱氨酸-3和半胱氨酸-4的棕榈酰化与膜结合。在本研究中，我们确定了神经调节蛋白在体内进行棕榈酰化和膜结合所需的关键氨基酸残基。对几种具有点突变的神经调节蛋白构建体进行了膜结合和棕榈酰化测试。在COS - 7细胞中表达的野生型神经调节蛋白掺入了[3H]棕榈酸，而半胱氨酸-3和半胱氨酸-4都被甘氨酸取代的突变体在体内未发生棕榈酰化。与野生型神经调节蛋白相比，半胱氨酸-3或半胱氨酸-4被亮氨酸取代的突变蛋白分别掺入了75%和25%的[3H]棕榈酸。通过对膜和胞质部分的免疫印迹分析，对COS - 7细胞中表达的突变神经调节蛋白的相对分布进行了定量。突变神经调节蛋白的膜结合与其在体内的棕榈酰化程度之间存在普遍相关性。神经调节蛋白酰化结构域中的其他点突变表明，半胱氨酸-4 N端的一段短疏水氨基酸序列可能是最佳棕榈酰化和膜结合所必需的。我们得出结论，半胱氨酸-4对神经调节蛋白的棕榈酰化和膜结合至关重要。
