Brede Gaute, Solheim Jorun, Stang Espen, Prydz Hans
Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, N-0349 Oslo, Norway.
Exp Cell Res. 2003 Dec 10;291(2):299-312. doi: 10.1016/j.yexcr.2003.07.009.
We have dissected the molecular determinants involved in targeting the protein serine kinase PSKH1 to the endoplasmic reticulum (ER), the Golgi apparatus, and the plasma membrane (PM). Given this intracellular localization pattern, a potential role of PSKH1 in the secretory pathway was explored. The amino-terminal of PSKH1 revealed a striking similarity to the often acylated Src homology domain 4 (SH4)-harboring nonreceptor tyrosine kinases. Biochemical studies demonstrated that PSKH1 is myristoylated on glycine 2 and palmitoylated on cysteine 3. Dual amino-terminal acylation targets PSKH1 to Golgi as shown by colocalization with beta-COP and GM130, while nonpalmitoylated (myristoylated only) PSKH1 targets intracellular membranes colocalizing with protein disulphide isomerase (PDI, a marker for ER). Immunoelectron microscopy revealed that the dually acylated amino-terminal domain (in fusion with EGFP) was targeted to Golgi membranes as well as to the plasma membrane (PM), suggesting that the amino-terminal domain provides PSKH1 with membrane specificity dependent on its fatty acylation status. Subcellular fractionation by sucrose gradient analysis confirmed the impact of dual fatty acylation on endomembrane targeting, while cytosol and membrane fractioning revealed that myristoylation but not palmitoylation was required for general membrane association. A minimal region required for proper Golgi targeting of PSKH1 was identified within the first 29 amino acids. Expression of a PSKH1 mutant where the COOH-terminal kinase domain was swapped with green fluorescent protein and cysteine 3 was exchanged with serine resulted in disassembly of the Golgi apparatus as visualized by redistribution of beta-COP and GM130 to a diffuse cytoplasmic pattern, while leaving the tubulin skeleton intact. Our results suggest a structural and regulatory role of PSKH1 in maintenance of the Golgi apparatus, a key organelle within the secretory pathway.
我们剖析了蛋白质丝氨酸激酶PSKH1靶向内质网（ER）、高尔基体和质膜（PM）所涉及的分子决定因素。鉴于这种细胞内定位模式，我们探讨了PSKH1在分泌途径中的潜在作用。PSKH1的氨基末端与通常酰化的含有Src同源结构域4（SH4）的非受体酪氨酸激酶具有显著相似性。生化研究表明，PSKH1在甘氨酸2处发生肉豆蔻酰化，在半胱氨酸3处发生棕榈酰化。双氨基末端酰化将PSKH1靶向高尔基体，这通过与β-COP和GM130共定位得以证明，而非棕榈酰化（仅肉豆蔻酰化）的PSKH1靶向与蛋白质二硫键异构酶（PDI，内质网的标志物）共定位的细胞内膜。免疫电子显微镜显示，双酰化的氨基末端结构域（与EGFP融合）靶向高尔基体膜以及质膜，这表明氨基末端结构域根据其脂肪酰化状态为PSKH1提供膜特异性。通过蔗糖梯度分析进行的亚细胞分级分离证实了双脂肪酰化对内膜靶向的影响，而胞质溶胶和膜分级分离表明，一般膜结合需要肉豆蔻酰化而非棕榈酰化。在最初的29个氨基酸内确定了PSKH1正确靶向高尔基体所需的最小区域。将COOH末端激酶结构域与绿色荧光蛋白交换且将半胱氨酸3与丝氨酸交换的PSKH1突变体的表达导致高尔基体解体，这通过β-COP和GM130重新分布到弥漫性细胞质模式得以可视化，而微管骨架保持完整。我们的结果表明PSKH1在维持高尔基体（分泌途径中的关键细胞器）方面具有结构和调节作用。
