Jackson J H, Cochrane C G, Bourne J R, Solski P A, Buss J E, Der C J
Immunology Department, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 1990 Apr;87(8):3042-6. doi: 10.1073/pnas.87.8.3042.
Oncogenic forms of ras proteins are synthesized in the cytosol and must become membrane associated to cause malignant transformation. Palmitic acid and an isoprenoid (farnesol) intermediate in cholesterol biosynthesis are attached to separate cysteine residues near the C termini of H-ras, N-ras, and Kirsten-ras (K-ras) exon 4A-encoded proteins. These lipid modifications have been suggested to promote or stabilize the association of ras proteins with membranes. Because preventing isoprenylation also prevents palmitoylation, examining the importance of isoprenylation alone has not been possible. However, the oncogenic human [Val12]K-ras 4B protein is not palmitoylated but is isoprenylated, membrane associated, and fully transforming. We therefore constructed mutant [Val12]K-ras 4B proteins that were not isoprenylated to examine the effects of isoprenylation in the absence of palmitoylation. The nonisoprenylated mutant proteins both failed to associate with membranes and did not transform NIH 3T3 cells. In addition, inhibition of isoprenoid and cholesterol synthesis with the drug compactin also decreased [Val12]K-ras 4B protein isoprenylation and membrane association. These results unequivocally demonstrate that isoprenylation, rather than palmitoylation, is essential for ras membrane binding and ras transforming activity. These findings clearly indicate the biological significance of ras protein modification by farnesol and suggest that this modification may be important for facilitating the processing, trafficking, and biological activity of other isoprenylated proteins. Because K-ras is the most frequently activated oncogene in a wide spectrum of human malignancies, study of this pathway could lead to important therapeutic treatments.
致癌形式的Ras蛋白在胞质溶胶中合成，必须与膜结合才能导致恶性转化。棕榈酸和胆固醇生物合成中的类异戊二烯（法呢醇）中间体分别连接到H-Ras、N-Ras和Kirsten-Ras（K-Ras）外显子4A编码蛋白C末端附近的不同半胱氨酸残基上。这些脂质修饰被认为可促进或稳定Ras蛋白与膜的结合。由于阻止异戊二烯化也会阻止棕榈酰化，因此无法单独研究异戊二烯化的重要性。然而，致癌性人类[Val12]K-Ras 4B蛋白未被棕榈酰化，但被异戊二烯化、与膜结合且具有完全转化活性。因此，我们构建了未被异戊二烯化的突变型[Val12]K-Ras 4B蛋白，以研究在不存在棕榈酰化的情况下异戊二烯化的作用。未被异戊二烯化的突变蛋白均无法与膜结合，也不能转化NIH 3T3细胞。此外，用药物洛伐他汀抑制类异戊二烯和胆固醇合成也会降低[Val12]K-Ras 4B蛋白的异戊二烯化和膜结合。这些结果明确表明，异戊二烯化而非棕榈酰化对于Ras膜结合和Ras转化活性至关重要。这些发现清楚地表明了法呢醇对Ras蛋白修饰的生物学意义，并表明这种修饰可能对促进其他异戊二烯化蛋白的加工、运输和生物学活性很重要。由于K-Ras是多种人类恶性肿瘤中最常被激活的致癌基因，对该途径的研究可能会带来重要的治疗方法。
