Abstract
The RAS family includes three RAS genes, which encode four highly homologous proteins: H-, N-, and KRAS4A and 4B, the latter two being alternatively spliced forms differing only at the carboxyl terminus. Hyperactivation of RAS is common in human cancer, including hematological malignancies. Since RAS proteins are difficult to target, identification of alternative means to block RAS oncogenic signaling is critical for developing therapies against RAS-driven cancers. The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Among RAS PTMs, palmitoylation is required for the high affinity plasma membrane binding of NRAS, HRAS and KRAS4A. We have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for NRAS related cancers. In addition, it has been shown that KRAS4A plays an essential role in the development of KRAS-driven lung cancer, suggesting that targeting palmitoylation may impact on cancers with KRAS mutations as well. However, the exact role of palmitoylation in KRAS4A oncogenic activity is not known. In this study we compared the effect of palmitoylation on leukemogenic potential of oncogenic NRAS and KRAS4A whose high expression level is detected in some KRAS mutated leukemia cell lines. We found that palmitoylation of oncogenic NRAS and KRAS4A is both critical for their plasma membrane attachment and their activity to activate the MEK-ERK signaling pathway. Mutation at the palmitoylation site of the oncogenic KRAS4A significantly abrogates its leukemogenic potential. However, unlike NRAS, palmitoylation defective KRAS4A still induces leukemia in mice, albeit with a long latency. Using NRAS/KRAS4A chimeric constructs, we found that the KIKK (Lys-Ile-Lys-Lys) motif between amino acids 181 and 189 in the hypervariable region of KRAS4A effects on the oncogenic activity of KRAS4A, though its contribution is less than that of palmitoylation. After replacing the three lysine residues in the KIKK motif with glutamine residues, the leukemogenic potential of oncogenic KRAS4A is partially inhibited, while inactivating mutations of both palmitoylation site and KIKK motif completely suppress the ability of oncogenic KRAS4A to induce leukemia in mice. On the other hand, replacement of lysine residues in KIKK motif with arginine residues does not change the oncogenic activity of KRAS4A, indicating that the KIKK motif functions by providing positive charged residues. These studies demonstrate that KRAS4A contains an additional membrane association motif comparing to NRAS and suggest that though therapies targeting RAS palmitoylation would inhibit KRAS4A tumorigenesis, interfering the KIKK motif of KRAS4A would enhance the therapeutic effectiveness.
